Protective role of extracts of neem seeds in diabetes causedby streptozotocin in rats
S. Guptaa, M. Katariab, P.K. Guptaa,∗, S. Murganandana, R.C. Yashroyca Division of Pharmacology and Toxicology, Indian Veterinary Research Institute, Izatnagar 243 122, U.P., India
b Division of Biochemistry, Indian Veterinary Research Institute, Izatnagar 243 122, U.P., Indiac Division of Biophysics, Indian Veterinary Research Institute, Izatnagar 243 122, U.P., India
Received 25 September 2002; received in revised form 16 August 2003; accepted 2 September 2003
Neem (Azadirachta indica A. Juss, Meliaceae) is anindigenous tree grown all over India and Burma and isattributed to have many medicinal properties. Despite con-siderable progress in the management of diabetes mellitusby synthetic drugs, the search for indigenous antidiabeticagents still continues. Antihyperglycemic/hypoglycemicactivity of neem leaves in dogs (Murty and Rao, 1978) andneem seed oil (Dixit et al., 1986) was reported. Neem seedoil is also reported to have spermicidal activity (Sinha et al.,1984). Previously, biochemical and hematological changesin rats and effect on cattle tickBoophilus microplus havebeen reported (Gupta et al., 2000, 2001a,b).
Hyperglycemia can cause oxidative stress, which in turnmay result in cellular tissue damage. The harmful influ-ence of diabetes on metabolism of tissues and organs is
well known. Likewise, uncontrolled hyperglycemia can leadto disturbances in the structure and functions of organs(Kuyvenhoven and Meinders, 1999; West, 2000).
Streptozotocin (STZ) causes diabetes mellitus. Diabetesis associated with the generation of reactive oxygen species(ROS) causing oxidative damage particularly to heart andkidney (Mohamed et al., 1999). Glucose level increased theproduction of free radicals cell damage markers, such asmalonaldehyde and conjugated dienes (Cuncio et al., 1995).
The present investigation was undertaken to evaluatethe protective effect of petroleum ether extracts of neem(Azadirachta indica) seed husk and kernel on oxidativedamage induced by STZ in tissues.
2. Material and methods
2.1. Animal
Male adult albino rats (150–200 g) were procured fromthe Laboratory Animal Resource Section of the Indian
186 S. Gupta et al. / Journal of Ethnopharmacology 90 (2004) 185–189
Veterinary Research Institute, Izatnagar. The animals weremaintained on standard ration and provided with clean drink-ing water ad lib. The animals were kept in air-conditionedroom (temperature 20± 2 ◦C) and acclimatized for a periodof 7 days.
2.2. Plant material
Seeds of neem (Azadirachta indica) were procured fromthe local market and were dried in shade. From the seeds,husks, and kernel were separated. Petroleum ether extracts(60–80◦C) were obtained using Soxhlet apparatus.
2.3. Induction of diabetes
To induce diabetes, STZ (Sigma), prepared freshly in cit-rate buffer, pH 4.5, was immediately injected intravenously(55 mg/kg) through tail vein (Tomlinson et al., 1992). Therats were monitored for plasma glucose levels at weekly in-tervals. The rats with fasting glucose value of >250 mg/dlwere considered diabetic. Blood samples were drawn byretro-orbital venepuncture technique. Plasma was separatedby centrifugation at 2000 rpm for 15 min. Glucose levelswere measured byo-toludine method using standard kitsfrom Qualigens India Ltd., India. Based on the plasma glu-cose levels, uniformly diabetic rats were selected on day 30after the injection of STZ.
2.4. Treatment of animal
Animals were divided into five groups of six animals andtreated as follows:
Group-I was given citrate buffer and served as control(without STZ). STZ induced diabetic rats were dividedin four groups (Groups II–V). Group-II diabetic control.Group-III positive control (insulin 6 U/kg, i.p.). Group-IVNSK (2 gm/kg, b.wt., LD50 < 18 mg/kg). Group-V NSH(0.9 gm/kg, b.wt., 1/20 LD50) (Gupta et al., 2001a,b). Treat-ments were given orally for 28 days.
2.5. Collection of samples
The blood samples were collected at the start and 7th,14th, 21st, and 28th day of experiment in two aliquots fromretro-orbital plexus using micro-capillary technique (Sorgand Buckner, 1964). In one of the aliquot, no anticoagu-lant was used. Serum was separated. In the second aliquots,heparin was used as anticoagulant. Plasma was separated.The erythrocytes from heparinized blood were employedfor membrane preparation by the method ofHanahan andEkhlom (1974)using 1 mM EDTA in hypotonic buffer. Theerythrocytes were washed thrice with ice-cold saline andsuspended in 50% (v/v) of saline. After blood collection, allthe rats were sacrificed by euthanasia. The organs such asliver, heart, and kidney were excised immediately and kepton ice, homogenized, and homogenates were centrifuged at
10,000×g for 10 min in Sorvall refrigerated centrifuge. Su-pernatants were collected and stored at 4◦C.
2.6. Estimation of antioxidant enzymes
2.6.1. Superoxide dismutaseSuperoxide dismutase (SOD) activity was determined
in the homogenates and erythrocytes according toMadeshand Balasubramanium (1998). A colorimetric assay involv-ing generation of superoxide by pyrogallol auto-oxidationand the inhibition of superoxide-dependent reduction ofthe tetrazolium dye, MTT (3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide) to its formazan by SODwas measured at 570 nm. Amount of MTT formazan wascalculated by using molar extinction coefficientE570 of17,000 M−1 cm−1. One unit of SOD was defined as theamount of protein required to inhibit the MTT reduction by50%.
2.6.2. CatalaseCatalase (CAT) activity was measured in homogenates
and erythrocyte by the method ofMaehly and Chance(1954). The utilization of H2O2 by CAT in the sampleswas measured spectrophotometrically as decrease in opticaldensity at 254 nm. The substrate (H2O2) concentration was20 mM for erythrocyte and cardiac tissue CAT measure-ment, while 2 mM for the renal CAT.
2.6.3. Lipid peroxidationLipid peroxidation (LPO) in tissue homogenates and
erythrocytes was studied by measuring thiobarbituric acidreactive substances (TBARS) according toOhkawa et al.(1979). The results are expressed as nmol malonaldehyde(MDA) formed per ml packed erythrocyte and nmol MDAformed per g tissue per 30 min in heart, liver, and kidneyhomogenates.
2.6.4. Serum creatine phosphokinaseSerum from animals was evaluated for creatine phospho-
kinase (CPK) activity using the kit obtained from QualigensIndia Ltd.
2.7. Statistical analysis
Statistical analysis of data was done using Student’ ‘t’-test(Snecdecor and Cochran, 1976).
3. Results
3.1. Protection against cardiac injury by petroleum etherextract of neem seed kernel and husk
Serum CPK activity is increased significantly in STZ-induced diabetic rats as compared to normal rats (Table 1).Treatment with insulin (6 U/kg, i.p.), petroleum ether extract
S. Gupta et al. / Journal of Ethnopharmacology 90 (2004) 185–189 187
Table 1Effect of NSK and NSH given orally daily for 28 days on serum creatinephosphokinase of STZ-induced diabetic rats
Values are mean±S.E. of six animals.∗P < 0.05,∗∗P < 0.01 as comparedto diabetic control.
of kernel (NSK, 2.0 g/kg) and husk (NSH, 0.09 g/kg) re-sulted in a significant reduction of serum phosphokinaseactivity of diabetic rats. The efficiency of decrease in CPKwas in order of insulin> NSK > NSH.
3.2. Antioxidant activity
STZ administration resulted in a significant elevation ofcardiac SOD, CAT, and LPO. With repeated administrationof insulin, NSK, and NSH for 28 days, the cardiac LPOwas significantly reduced comparable to normal control rats(Table 2).
Erythrocyte LPO was significantly increased in diabeticcontrol and after repeated administration of NSK and NSH,the LPO was significantly reduced comparable to the nor-mal control rats. Erythrocyte CAT and SOD was decreasedin diabetic rats as compared to normal control which wasregained to some extent by insulin and NSH (Table 3).
STZ administration resulted in a significant elevation inhepatic SOD, CAT, and LPO in diabetic control as com-pared to normal control (Table 4). In spite of the rise in theantioxidant enzyme activity in hepatic tissue, there was sig-nificant elevation of LPO. Repeated administration of NSKand NSH were ineffective in inhibiting the hepatic LPO.Hepatic CAT activity after NSH treatment was significantlyinhibited as compared to diabetic control and comparable tocontrol.
SOD and CAT enzyme activities were significantly de-creased in kidney as compared control (Table 5) along with
Table 2Effect of NSK and NSH orally for 28 days on SOD, CAT, and LPO ofheart in STZ-induced diabetic rats
Values are mean±S.E. of six animals.∗P < 0.05,∗∗P < 0.01 as comparedto diabetic control. Ua: velocity constant per second, Ub: amount ofhemoglobin (mg) inhibiting MTT by 50%, c: nM of MDA produced/gtissue per minute.
Table 3Effect of NSK and NSH orally for 28 days on SOD, CAT, and LPO oferythrocytes in STZ-induced diabetic rats
Values are mean±S.E. of six animals.∗P < 0.05,∗∗P < 0.01 as comparedto diabetic control. Ua: velocity constant per second, Ub: amount ofhemoglobin (mg) inhibiting MTT by 50%, c: nM of MDA produced/gtissue per minute.
Table 4Effect of NSK and NSH orally for 28 days on SOD, CAT, and LPO ofliver in STZ-induced diabetic rats
Values are mean±S.E. of six animals.∗P < 0.05,∗∗P < 0.01 as comparedto diabetic control. Ua: velocity constant per second, Ub: amount ofhemoglobin (mg) inhibiting MTT by 50%, c: nM of MDA produced/gtissue per minute.
Table 5Effect of NSK and NSH orally for 28 days on SOD, CAT, and LPO ofkidney in STZ-induced diabetic rats
Values are mean±S.E. of six animals.∗P < 0.05,∗∗P < 0.01 as comparedto diabetic control. Ua: velocity constant per second, Ub: amount ofhemoglobin (mg) inhibiting MTT by 50%, c: nM of MDA produced/gtissue per minute.
the significant elevation of LPO. With repeated administra-tion of NSK and NSH, the activity was regained to someextent but not comparable to control.
4. Discussion
STZ is a commonly employed compound for inductionof type-1 diabetes (Tomlinson et al., 1992). STZ causes dia-betes by rapid depletion of�-cells which leads to reduction
188 S. Gupta et al. / Journal of Ethnopharmacology 90 (2004) 185–189
in insulin release. Hyperglycemia causes oxidative damageby generation of ROS (Mohamed et al., 1999) and develop-ment of diabetic complications (Donnini et al., 1996; Baynesand Thorpe, 1999). Further, the STZ diabetic animals mayexhibit most of the diabetic complications, namely, myocar-dial cardiovascular, gastrointestinal, nervous, vas deferens,kidney, and urinary bladder dysfunctions (Ozturk et al.,1996).
Increased serum CPK level in diabetic rats indicate car-diac muscular damage. Elevated concentration of CPK wererecovered by treatment with insulin, NSK, or NSH suggest-ing their cardioprotective effect. Insulin was more effectiveas compared to NSK and NSH.
SOD and CAT are considered primary antioxidant en-zymes, since they are involved in direct elimination ofROMs (Halliwell and Cutteridge, 1985). Effect of STZ inLPO, CAT and SOD activities was found to be tissue depen-dent. In spite of increased cardiac CAT and SOD activitiesin diabetic rats, increase in LPO was observed. Higher LPOand low SOD and CAT activity indicates an oxidative stresscondition. The effect on LPO, CAT, and SOD was reversedby insulin, NSK, and NSH treatments. The observationsuggests that in order to overcome the oxidative damagein heart, some other compensatory mechanisms exist inheart in addition to antioxidant enzymes. Reversal of in-creased enzymes and inhibition of LPO appears to be dueto free radical scavenger activity of petroleum ether extractof neem seed kernel (NSK) and husk (NSH) in heart. Theerythrocyte LPO was significantly increased in diabeticcontrols with reduction in antioxidant enzyme activities ofSOD and CAT. Treatment with insulin and NSK stimulatedSOD and CAT to reverse oxidative damage to erythrocytemembrane.
The study revealed that CAT and SOD activities weresignificantly inhibited along with an elevation of LPO inkidney of STZ-treated diabetic animals which is not reversedby different treatments. The results are in agreement withearlier data (Bryzewska et al., 1995; Parthiban et al., 1995).Significant increase in LPO, SOD, and CAT in pancreas,heart, and blood, and increase in glutathione peroxidase inkidney and pancreas in diabetes was reported byKakar et al.(1995). Increase in heart LPO in diabetic rats was observedby Krishna Kumar et al. (1999).
The experimental results indicated that LPO played a rolein tissue injury in STZ-induced diabetic rats. Petroleum etherextract of neem seed kernel (NSK) and husk (NSH) reducedthe LPO in heart and erythrocytes, thus effectively protectedcell functions and structure. STZ-induced diabetic oxidativechanges of cardiac and erythrocyte toxicity as observed wasreversed by the significant stimulation of antioxidant defensemechanism in erythrocytes or compensatory elevation of an-tioxidant defense mechanism in cardiac tissue by NSK andNSH. In STZ-induced diabetes, however, renal and hepatictoxicity was not prevented by NSK and NSH.
The results indicate that petroleum ether extract of neemseed kernel (NSK) and husk (NSH) showed significant pro-
tection against the oxidative damage induced by STZ in heartand erythrocytes of rats. NSK and NSH may act as cardio-protective and free radical scavenger agent.
References
Baynes, J.W., Thorpe, S.R., 1999. Role of oxidative stress in diabeticcomplications. A new perspective on an old paradigm. Diabetes 48,1–9.
Bryzewska, M., Zavodnik, I.B., Niekurzak, A., Szosland, K., 1995. Ox-idative processes in red blood cells from normal and diabetic indi-viduals. Biochemistry and Molecular Biology International 37, 345–354.
Cuncio, F., Pegoraro, I., Dello-Russo, P., Falleti, F., Perrella, G., Ceriello,A., 1995. SOD and GSH inhibit the high glucose induced oxidativedamage and the PDGF increased secretion in cultured human endothe-lial cells. Thrombosis and Haemostasis 74, 963–973.
Dixit, V.P., Shina, R., Tank. Effect of neem seed oil on blood glucose con-centration of normal and allaxon diabetic rats. Journal of Ethnophar-macology 17, 95–98.
Donnini, D., Zambito, A.M., Perella, G., Ambesi-Impiombat, O., Curcio,F., 1996. Glucose may induce cell death through a free radical-mediatedmechanism. Biochemical and Biophysical Research Communications219, 412–417.
Gupta, P.K., Gupta, S., Khan, M.H., 2000. In vitro evaluation of petroleumfractions of different parts of neem seed (Azadirachta indica) againstcattle ticks,Bophilus microphos. Indian Journal of Environmental Tox-icology 10, 38–39.
Gupta, S., Gupta, P.K., Kataria, M., Vijjan, V.K., 2001a. Toxicological andbiological evaluation of different extracts of neem seed (Azadirachtaindica) in rats. Advances in Pharmacology and Toxicology 2, 1–7.
Gupta, S., Kataria, M., Gupta, P.K., Vijjan, V.K., 2001b. Effect ofpetroleum ether extracts of different parts of neem (Azadirachta indica)seed on haematological and biochemical parameters in rats. IndianJournal of Animal Research 359, 21–26.
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Kakar, R., Kalra, J., Mantha, S.V., Prasad, K., 1995. Lipid peroxidationand activity of antioxidant enzymes in diabetic rats. Molecular andCellular Biochemistry 18, 151, 113–119.
Krishna Kumar, K.T., Augusti, Vijaymnal, P.L., 1999. Hypoglycemicand antioxidant activity of Salacia oblonga wall extract instreptozotocin-induced diabetic rats. Indian Journal of Physiology andPharmacology 43, 510–514.
Kuyvenhoven, J.P., Meinders, A.E., 1999. Oxidative stress and diabeticmellitus pathogenesis of long-term complications. European Journal ofInternational Medicine 10, 9–19.
Madesh, M., Balasubramanium, K.A., 1998. Microtitre plate assay forsuperoxide dismutase using MTT reduction by superoxide. IndianJournal of Biochemistry and Biophysics 35, 184–188.
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Mohamed, A.K., Bierhaus, A., Schiekofer, S., Tritschler, H., Ziegler, H.,Nawroth, P.P., 1999. The role of oxidative stress and NF (B) activationin late diabetic complications. Biofactors 10, 175–179.
Murty, K.S., Rao, D.K., 1978. A preliminary study on hypoglycemicand antihyperglycemic effects ofAzadirachta indica. Indian Journal ofPharmacology 10, 247–250.
Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides inanimal tissues by thiobarbituric acid reaction. Analytical Biochemistry95, 351–358.
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Journal of Ethnopharmacology 90 (2004) 191–194
Influence of aqueous extract fromNeurada procumbens L.on blood pressure of rats
H.B. Chen∗, M.W. Islam, R. Radhakrishnan, S.A. Wahab, M.A. NajiDepartment of Pharmacology and Toxicology, Zayed Complex for Herbal Research and Traditional Medicine,
Ministry of Health, Post Box 29300, Abu Dhabi, Saudi Arabia
Received 20 May 2002; received in revised form 11 September 2003; accepted 12 September 2003
Neurada procumbens L. (Rosaceae), called sa‘ada by Ara-bian people, is a desert plant widespread on shallow and deepsands in the Arabian Peninsula. It is a grey-green, annualherb with numerous prostrate branches radiating to 20 cmfrom the roots. The leaves are relatively thick with clearmidrib, wavy margins, and bluntly rounded tip on its stalk.Its flowers, blooming from January to May, are about 1 cmdiameter, solitary in leaf axils with small petals. The fruit isa distinctive disc 1–1.5 cm across, rough and spiny above,smooth beneath and this usually remains as collar aroundbase stem of new plants (Western, 1989; Hepper and Friis,1994).
Neurada procumbens is one kind of food of camels, but ithas been considered safe and edible and Bedouin eat it some-times (James, 1990). In traditional Arabian medicine, theplant has been used to treat diarrhea and dysentery; as well,it has been used as a tonic to ‘increase heart and respirationfunctions’. During a screening test of Arabian plants, theaqueous extract ofNeurada procumbens caused increasedblood pressure in anaesthetized normotensive rats. This led
∗ Corresponding author. Fax:+971-2-6313-742.
to the present study, to investigate and prove its effects onthe blood pressure and vascular smooth muscles of rats.
2. Methodology
2.1. Plant and its extract
Neurada procumbens L. was collected from Umm AlQuwein of the United Arab Emirates in April, 2001 andwas authenticated by the botanists in the Zayed Complex forHerbal Research and Traditional Medicine, where a voucherspecimen (w139) has been deposited. The whole plant wasdried under shade and powdered. The powder was exhaus-tively extracted with distilled water. The solvent of the liquidextract was removed completely by evaporation under vac-uum at 40◦C, using a Buchi Rotary Evaporator. The driedextract contained 31/100 g of the starting crude material.
2.2. Animals
Wistar rats of either sex weighing 300–350 g were usedfor the study. Their diastolic and systolic blood pressureunder anesthesia was 63±4 mmHg and 110±5 mmHg. Malespontaneous hypertensive rats (SHR) weighing 350–400 gwere used for experiment and their blood pressure was 116±
192 H.B. Chen et al. / Journal of Ethnopharmacology 90 (2004) 191–194
9 mmHg and 150± 5 mmHg. The animals were bred in theAnimal Holding Unit of the complex under the standardenvironment condition.
2.3. Blood pressure in anaesthetized normotensive rats
The rats were anaesthetized with urethane (1.2 g/kg, i.p.)and the carotid artery was cannulated (Shuyun et al., 1991).The blood pressure was measured directly from the can-nula using a transducer–amplifier–recorder assembly (LEC-TROMED, UK) and the chart recorder was calibrated inmmHg to measure the blood pressure. For administration ofthe extract, a 1 cm abdominal incision was made to exposethe stomach. After a stabilization period of 30 min, the ini-tial value of the blood pressure was recorded. The extract ofNeurada procumbens (1 g/kg) or normal saline was injectedinto the stomach through the abdominal incision and the in-cision was then closed. The blood pressure was recordedat 30, 60, 90, 120, 150, and 180 min after the administra-tion. The heart rate was later manually computed from therecording.
2.4. Blood pressure in conscious SHR
A system of Dataquest ARTTM 2.1 PhysioTelemetrywas used for the experiment (Data Sciences International,2000). A Data Exchange Matrix connected the PhysioTelReceivers (Model RPC-1) and Ambient Pressure reference(Model APR-1) to the PCI card. A pressure transmitter(TA11PA-C40) was implanted into the abdominal cavityof SHR with its catheter connected to the dorsal aorta(Waynforth and Flecknell, 1994). The rats were used forthe experiment three weeks after the implantation.
At the first day of the experiment, the SHR were admin-istered orally with distilled water at 1 ml/100 g body weight.At the second day, the same SHR were administered orallywith the extract ofNeurada procumbens at 1 g/kg. Bloodpressures and heart rates were detected automatically byPhysioTelemetry system for 6 h, from 1 h before and 5 h afterthe administration. The samples of blood pressure and heart
Table 1Influence of the aqueous extract ofNeurada procumbens (NP) on the blood pressure and heart rate of the anaesthetized normotensive rats (n = 6),expressed as percentage of the initial value
0 min 30 min 60 min 90 min 120 min 150 min 180 min
DBP: diastolic blood pressure; SBP: systolic blood pressure; HR: heart rate.∗ Significantly different from control value atP < 0.05.
rate were collected continuously for 10 s in every 5 min. Themean values of the blood pressure and heart rate in 1 h wereused to present the blood pressure and heart rate in this pe-riod.
2.5. Rat aortic strips
The direct effect of the aqueous extract fromNueradaprocumbens on the vascular smooth muscles was evaluatedusing isolated rat aortic strips (Shuyun et al., 1991). Aor-tas were isolated from Wistar rats, spirally cut into stripsof 2 cm length and 3 mm width and suspended in an or-gan bath containing Kreb’s solution, bubbled with Carbogenand maintained at 37◦C. One gram resting tension was ap-plied to the tissue which was then allowed to equilibrate for30 min. The extract was added at concentrations of 0, 0.2,0.5, 1.0, and 2.0 mg/ml and the responses were recorded for10 min. Phentolamine at 150�M was added 10 min beforethe extract to check its influence.
2.6. Statistical analysis
All data are expressed as mean±S.E.M. The significanceof the difference between the means of data was evaluated bythe Student’st-test.P-values less than 0.05 were consideredsignificant.
3. Result
3.1. Blood pressure in anaesthetized normotensive rats
The aqueous extract ofNeurada procumbens, at 1 g/kgorally, significantly increased both diastolic and systolicblood pressure of the anaesthetized normotensive rats with-out significantly affecting their heart rate. The effect startedfrom 60 min after the administration and was maintaineduntil the end of the experiment, 180 min after the adminis-tration. The increases at 150 and 180 min was significant(Table 1).
H.B. Chen et al. / Journal of Ethnopharmacology 90 (2004) 191–194 193
Table 2Influence of the aqueous extract ofNeurada procumbens (NP) on the blood pressure and heart rate of conscious SHR (n = 7), expressed as percentageof the initial value
Zero hour First hour Second hour Third hour Fourth hour Fifth hour
DBP: diastolic blood pressure; SBP: systolic blood pressure; HR: heart rate.∗ Significantly different from the value of water treatment atP < 0.05.
3.2. Blood pressure in conscious SHR
The aqueous extract ofNeurada procumbens, at 1 g/kgorally, increased both diastolic and systolic blood pressure,from the first hour till the third hour after the administra-tion. The increasing during the second hour was significant(P < 0.05). The heart rate of the second hour also increasedthough not significantly (Table 2).
3.3. Rat aortic strips
The aqueous extract ofNeurada procumbens, at theconcentrations of 0.2, 0.5, 1.0, and 2.0 mg/kg, produced adose-dependent increase in the resting tension of the iso-lated rat aortic strips (Fig. 1). The vasoconstrictive effectwas reduced partially by phentolamine (150�M).
0
0.05
0.1
0.15
0.2
0.25
0.3
0 2 4 6 8 10 12
Minutes
g
control
NP0.2mg/ml
NP0.5mg/ml
NP1.0mg/ml
NP2.0mg/ml
Fig. 1. Influence of the aqueous extract ofNeurada procumbense on resting tension of rat aortic strips (n = 6); NP: Neurada procumbense.
4. Discussion
The present study revealed that the aqueous extract ofNeurada procumbens, oral administration, significantly in-creased the diastolic and systolic blood pressure in anaes-thetized normotensive rats and conscious SHR. In vitro, theextract produced a significant vasoconstrictive effect on aor-tic strips, which was reduced by pre-treatment with phento-lamine. This suggests that the vasoconstrictive effect causedby Neurada procumbens is, at least in part, mediated throughalpha-adrenergic receptors.
There is only one research paper aboutNeurada procum-bens, which reported that the plant contained alkaloids,flavoniods, saponins, sterols/triterpenes, coumarins, volatileoils, and tannins (Mossa et al., 1983). The same pa-per also reported that the ethanolic extract ofNeurada
194 H.B. Chen et al. / Journal of Ethnopharmacology 90 (2004) 191–194
procumbens reduced the blood pressure in rabbits whenit was administered intravenously. The different results ofNeurada procumbens on blood pressure from the presentstudy and the previous paper are considered here to bedue to the different extractive solvents and/or differentadministrations.
Bedouin and Arabs usually eatNeurada procumbens di-rectly or take its decoction as a medicine. Therefore theuse of an aqueous extract of the plant and its oral ad-ministration used in the present study are similar to folkusage.
Accordingly, Neurada procumbens might not be sosafe as it has been considered. Its effects on blood pres-sure and vascular smooth muscles exhibited in this studyimply that the plant might have similar effects in hu-man beings though more investigations are needed. Thisstudy also warns people, especially those suffering fromcardiovascular diseases, to be careful when they use theplant.
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Western, A.R., 1989. The Flora of the United Arab Emirates—An Intro-duction. The United Arab Emirates University, Abu Dhabi, p. 68.
Journal of Ethnopharmacology 90 (2004) 195–204
Effect of polyherbal formulation on experimentalmodels of inflammatory bowel diseases
A.G. Jagtapa, S.S. Shirkea,∗, A.S. Phadkeba Department of Pharmacology, Bombay College of Pharmacy, Kalina, Santacruz (E), Mumbai 400 098, India
b Department of Dravyaguna (Herbal Pharmacology), YMT Ayurvedic Medical College, Kharghar, Navi Mumbai, India
Received 3 September 2002; accepted 22 September 2003
Abstract
A polyherbal ayurvedic formulation from an ancient authentic classical text of ayurveda was evaluated for its activity against inflammatorybowel disease (IBD). The polyherbal formulation contained four different drugs viz., Bilwa (Aegle marmeloes), Dhanyak (Coriandrumsativum), Musta (Cyperus rotundus) and Vala (Vetiveria zinzanioids).
The formulation has been tried before in clinical practice and was found to be useful in certain number of cases of IBD (ulcerative colitis),so was tried in the same form i.e., decoction (aqueous extract) in experimental animals to revalidate the claims of the same.
The formulation was tried on two different experimental animal models of inflammatory bowel disease, which are acetic acid-inducedcolitis in mice and indomethacin-induced enterocolitis in rats. Prednisolone was used as the standard drug for comparison.
Inflammatory bowel disease is the general term used todescribe two different chronic nonspecific disorders of thegastrointestinal tract i.e., ulcerative colitis (UC) and Crohn’sdisease (CD). Certain differences in the disease pattern jus-tify a distinction between the two. However, the diseases areconsidered together because of similarities in their presenta-tion, pathology, investigations, complications and treatment.
Both diseases are more common in western populationsand in urban rather than rural areas. UC is most prevalent inNorth America, Northern Europe and Australia. The preva-lence is roughly 10 times lower in southern and Eastern Eu-rope, Africa, Asia and South America. Some reports haveindicated that disease is now seen with increased frequencyin parts of Asia such as India, Bangladesh and Japan. Thedistribution of CD is similar. It has its highest incidence inNorthern Europe. This disorder is rarely seen in Asian coun-tries such as India and Bangladesh (Whelan, 1990).
UC typically presents as a persistent bloody diarrhea andabdominal pain. It is a diffuse mucosal and submucosal dis-ease involving only the colon (Green and Haris, 1997).
Crohn’s disease is a chronic transmural disease causinginflammation in any segment of the alimentary tract. Ap-proximate 75% of the patients have small bowel involve-ment and 90% of these patients have disease in the terminalileum. CD presents usually with pain and diarrhea (Dipiroand Schade, 1999).
The etiology of IBD is still not clear but different factorshave been postulated as possible etiologic agents for IBD.They are genetic factors, infective agents, immunologicalbasis, smoking, medications and pathological factors (Dipiroand Schade, 1999; Berardi, 2000).
There is evidence that pathogenesis of IBD involves ge-netic factors. Relatives of IBD patients are at 5–40 timesgreater risk of developing the disease (Hellers and Bernell,1990). Many infective agents like special strains ofEs-cherichia coli, Aerobactor aerogens, Proteus andStaphylo-coccussp. have shown to be involved in IBD either directlyor indirectly. Alterations in the mucosal immune system arecentral to the pathogenesis of IBD. Smoking increases therisk for CD and decreases the risk for UC. Medications like
196 A.G. Jagtap et al. / Journal of Ethnopharmacology 90 (2004) 195–204
non steroidal anti-inflammatory drugs (NSAIDs) can causevariety of effects in patients with IBD, including asymp-tomatic mucosal inflammation, strictures, obstruction andhemorrhage. Emotional and psychological factors have beenimplicated in the etiology of IBD but there is no evidencethat stress is causative and psychotherapy is effective. Noneof the above stated factors can be considered as primarycause of the disease, but it is clear that they do influence itsdevelopment (Berardi, 2000).
The mechanism as well as the exact etiology and patho-genesis of the disease development is unclear. This presentsthe major obstacle in the treatment of IBD and so there isno permanent cure available for the disease. Many drugs,which are used in IBD, offer symptomatic or temporaryrelief. Commonly used medications include aminosali-cylates, glucocorticoids, antibiotics and immunomodula-tors.
The ayurvedic polyherbal formulation in the form of de-coction, mentioned in one of the standard ancient authentictext of Ayurveda namely ‘Sharangdhara Samhita’, was cho-sen.
The same formulation in the abovesaid reference has beenspecifically indicated in Annapachana where ‘Anna’ is con-sidered to be the technical term in Ayurveda, which repre-sents macromolecules formed due to improper digestion ofthe diet due to gastrointestinal upset. This ‘Anna’ is consid-ered to be toxic in nature and the basic reason for the variousdisorders.
This formulation has been tried by a few of Ayurvedicphysicians to treat IBD and encouraging results were re-ported. This formulation contains a mixture of four differentherbal drugs namely:
Unripe fruits of Bilwa (Aegle marmeloes)Ripe fruits of Dhanyak/Dhania (Coriandrum sativum)Rhizomes of Musta (Cyperus rotundus)Roots of Vala/Usheer (Vetiveria zinzanioids)
The drugs were shade dried, finely ground and then mixedin 1:1:1: proportion on weight basis. This mixture was usedto prepare the decoction.
The formulation has also been reported in Ayurvedic com-pendium as follows:
This in translation says that Dhanyak, Balak (Vala), Bilwa,Abda (Musta) are to be boiled in water to get a decoction,which is useful in pain in the abdomen, also considered asantidiarrheal (Grahi) and is best amongst digestive prepara-tions to correct digestion and metabolism.
Individually these herbal drugs are used in classicalayurvedic therapeutics in gastrointestinal complaints, butthere is no scientific proof regarding their activity in IBDeither singly or in combination.
The unripe fruit of Bilwa is used against diarrhea andintestinal conditions. It contains marmelosin as a chief con-stituent, which is a furocoumarin. It also contains carbohy-drate pectin, volatile oil and tannins and possess astringent,digestive and stomachic actions and is useful in chronicdiarrheas (Chopra et al., 1958). Its activity is also reportedin ancient ayurvedic literature.
This means Bilwa is known as an effective antidiarrhealand an appetizer (deepan).
Rhizomes of Musta contain carbohydrates and volatileoil made up of mainly sesquiterpenes (Ohira et al., 1998).It stimulates thyroid, increases metabolism, acts as carmi-native, astringent, antispasmodic (Ayurvedic encyclopedia,1998). The petroleum extract of roots has shown anti-inflam-matory activity against carrageenan induced paw edema inrats (Kim et al., 1998). It has shown cytoprotective effectagainst ethanol induced ulceration in rats (Zhu et al., 1997).It has also given in Ayurvedic literature as:
This means Musta (cyperus rotundus) is antidiarrheal,increases the appetite (deepan) and helps in digestion(pachan).
This means it controls the severe form of diarrhea.Ripe fruits of Dhanyak contain fixed oil, volatile oil and
proteins, which gives carminative, aromatic, stimulant ac-tions (Kokate et al., 2001). It is reported in ayurvedic liter-ature as
Oil of Dhania is known for its antidiarrheal, diuretic, an-tipyretic activity.
Volatile oil obtained by Vala contains mainly vetiveroland vetivones and present good antibacterial, antifungal,stimulant, refrigerant and stomachic actions (Ayurvedicencyclopedia, 1998).
Usheer is known to be useful inPachan i.e. useful incorrecting digestive metabolism.
From these reported activities it can be seen that thesedrugs may have an effect on inflammatory bowel diseases.Therefore, we decided to try out this polyherbal formu-lation on experimental models of IBD. The experimental
A.G. Jagtap et al. / Journal of Ethnopharmacology 90 (2004) 195–204 197
models used for the study include indomethacin-inducedenterocolitis in rats and Acetic acid-induced colitis inmice.
We have used prednisolone as the standard drug forcomparison. Aminosalicylates and glucocorticoids are verycommonly used drugs for IBD. Glucocorticoids are givenin acute attacks of the disease and offer immediate relief.Aminosalicylates like sulphasalazine, mesalamine are givenchronically to maintain remission and prevent relapses. Aswe are using acute experimental animal models, we decidedto use glucocorticoid as the standard drug.
Glucocorticoids act as anti-inflammatories by decreasingthe recruitment of macrophages in the affected area. Theysuppress the synthesis of many inflammatory mediators e.g.,production of IL-1 from monocytes, production of IL-2 andtumor necrosis factor from lymphocytes. They also inhibitthe enzyme phospholipase A2 and thus decrease availabilityof prostaglandins and leukotrienes (Schimmer and Parker,1996).
We have used a myeloperoxidase assay to quantify theinflammation. Myeloperoxidase (MPO) is an enzyme foundin neutrophils and in much lower concentrations in mono-cytes and macrophages. This enzyme catalyses the oxidationof electron donors (e.g., halides) by hydrogen peroxides.In case of inflammatory conditions like IBD, the levels ofneutrophils in inflamed tissues, and consequently MPO en-zyme, increase. Therefore, assessment of MPO activity canbe used for quantitation of intestinal inflammation.
2. Materials and methods
2.1. Crude drugs
A mixture of crude drugs containing the following drugsin finely ground state in the proportion of 1:1:1:1 was made.
20 g of mixture of drugs with 150 ml of distilled waterwere macerated at ambient temperature for 24 h. After 24 hthe drug macerate was boiled for 45 min and filtered throughmuslin cloth to get a decoction. The volume of the decoctionwas adjusted such that 20 g of mixture gave 50 ml of thedecoction.
2.3. Animals used
Wistar albino rats weighting between 200 and 250 g ofeither sex and Swiss albino mice weighting between 20 and30 g were used for the study.
Animals were maintained under standard conditions oftemperature (23± 1◦C), relative humidity (55± 10%), and12 h/12 h light/dark cycle, and fed with a standard pelletdiet with water ad libitum. They were housed in standardpolypropylene cages with wire mesh top. All studies werecarried out using six rats or six mice in each group.
2.4. Dosages of polyherbal formulation and standarddrugs used
Polyherbal formulation (freshly prepared decoction) wasadministered to animals orally for 7 days. On 8th day, thedisease was induced by irritants indomethacin or acetic acid.The drug treatment was continued even after administrationof irritants.
A fresh decoction was used every time to simulate theconditions in which it is given to humans.
Doses of the formulation were used as follows:30 ml/kg in two divided doses in rats and40 ml/kg in two divided doses in mice.Standard drug used for comparison was prednisolone.
Prednisolone was not given as pretreatment. It was given onthe day of irritant administration.
Prednisolone was given in a dose of 2 mg/kg per day,orally in rats and 1.14 mg/kg per day, orally in mice, assuspension containing 0.5% of sodium CMC.
2.5. Indomethacin-induced enterocolitis in rats
The study comprised four different groups of six animalseach as follows (Elson et al., 1998):
• Normal or untreated animals which did not receive anytreatment;
• Control animals received only indomethacin (7.5 mg/kg,s.c.), on two consecutive days;
• Drug treated animals received 7 days pretreatment withpolyherbal formulation and indomethacin (7.5 mg/kg, s.c.)on 8th and 9th day. Drug treatment was continued till 11thday;
• Prednisolone treated group, which received prednisolone(2 mg/kg, p.o., for 4 days) and indomethacin (7.5 mg/kg,s.c., for 2 days). Prednisolone and indomethacin treatmentwas started on the same day.
Rats of either sex (200–250 g) were given indomethacin7.5 mg/kg subcutaneously on two consecutive days. On thefourth day animals were sacrificed by cervical dislocationand dissected open to remove GIT (from stomach to anus).GIT was flushed gently with saline and cut open.
Caecum, 10 cm long pieces of ileum and colon werescored for inflammation based on their macroscopic features.Tissues were fixed in 10% formalin saline and examinedhistopathologically.
Quantification of inflammation was done using myeloper-oxidase assay.
198 A.G. Jagtap et al. / Journal of Ethnopharmacology 90 (2004) 195–204
2.6. Acetic acid-induced colitis in mice
Study comprised of four different groups as follows (Elsonet al., 1998):
• Normal or untreated animals which did not receive anytreatment;
• Control animals received 0.1 ml of 6% acetic acid solution(once, intrarectally);
• Drug treated animals received 7 days pretreatment withpolyherbal formulation and 0.1 ml of 6% acetic acid so-lution, intrarectally on 8th day. Drug treatment was con-tinued till 10th day;
• Prednisolone treated group, which received prednisolone(1.14 mg/kg, for 3 days) and acetic acid (0.1 ml, 6% so-lution, once, intrarectally). Prednisolone treatment wasstarted on the day of acetic acid treatment.
Mice of either sex (20–30 g) were used for the study.Overnight fasted animals were anaesthetized using pento-barbitone sodium (40 mg/kg, i.p.). 0.1 ml of 6% acetic acidsolution was then instilled into the rectum of a mouse. An-imals were allowed to hang in air by holding their tails for1–2 min. This prevented spillage of the solution from therectum.
After 48 h animals were sacrificed by cervical dislocationand dissected open to remove colon. Colon was flushed gen-tly with saline, cut open and scored for inflammation basedon the macroscopic features. Tissues were fixed in 10% for-malin saline and examined histopathologically.
Quantification of inflammation was done using myeloper-oxidase assay.
2.7. Evaluation of the disease
The disease induced in experimental animals was eval-uated based on its macroscopic and microscopic charac-teristics. Evaluation pattern for macroscopic characteristics,given byMorris et al. (1989)was used after some modifi-cations. The inflammation was quantitated using myeloper-oxidase assay.
2.7.1. Evaluation based on macroscopic characters
2.7.1.1. Scoring for rat ileum and colon.Pieces of ratileum and colon (10 cm long each) were scored for macro-scopic features using following scoring pattern.
Score Macroscopic changes
0 No visible change1 Hyperemia at sites2 Lesions having diameter l mm or less3 Lesions having diameter 2 mm or less
(number< 5)4 Lesions having diameter 2 mm or less
(number 5–10)5 Lesions having diameter 2 mm or less
(number> 10)
Score Macroscopic changes
6 Lesions having diameter more than2 mm (number< 5)
7 Lesions having diameter more than2 mm (number 5–10)
8 Lesions having diameter more than2 mm (number> 10)
2.7.1.2. Scoring for rat caecum and mice colon.Rat cae-cum and mice colon (5 cm long) was scored for macroscopicfeatures using following scoring pattern.
Score Percentage area affected
0 01 1–52 5–103 10–254 25–505 50–756 75–100
Score for an individual rat is calculated as the combinedscore of ileum, colon, and caecum.
2.7.2. Evaluation based on microscopic (histologic)characters
To process for microscopic studies, 5�m thick paraf-fin sections were stained in haematoxyline and eosin.The stained sections were examined for any inflammatorychanges like infiltration of the cells, necrotic foci, damageto tissue structures like payers patches, damage to nucleus,etc.
2.7.3. Myeloperoxidase assay for quantification ofinflammation (Krawisz et al., 1984; Bradley et al., 1982):
Pieces of inflamed tissues (rat ileum-2 cm, micecolon-4 cm) were taken. The tissue was then rinsed withice-cold saline, blotted dry, weighed and excised. Mincedtissue was homogenized in 10 volumes of ice-cold potas-sium phosphate buffer (pH 7.4), using Remi tissue ho-mogenizer (RQ-127A). The homogenate was centrifugedat 3500 rpm for 30 min at 4◦C (Remi centrifuge C23).The supernatant was discarded. 10 ml of ice-cold 50 mMpotassium phosphate buffer (pH 6.0), containing 0.5% hex-adecyl trimethyl ammonium bromide (HETAB) and l0 mMEDTA was then added to the pellet. It was then subjectedto one cycle of freezing and thawing and brief period (l5 s)of sonication. After sonication solution was centrifuged at15,000 rpm for 20 min (Remi centrifuge, R24). Myeloper-oxidase activity was measured spectrophotometrically asfollows.
0.1 ml of supernatant was combined with 2.9 ml of 50 mMphosphate buffer containing 0.167 mg/mlO-dianisidine hy-drochloride and 0.0005% H2O2. The change in absorbance
A.G. Jagtap et al. / Journal of Ethnopharmacology 90 (2004) 195–204 199
was measured spectrophotometrically (Shimadzu UV 1 60AUV-VIS spectrophotometer), at 460 nm.
One unit of MPO activity is defined as the change inabsorbance per minute by 1.0 at room temperature, in thefinal reaction.
Calculation of MPO activity:
MPO activity(U/g) = X
wt of the piece of tissue taken
whereX
= 10× change in absorbance per minute
Volume of supernatant taken in the final reaction
3. Results
3.1. Indomethacin-induced enterocolitis in rats
Two days treatment with indomethacin (7.5 mg/kg, s.c.),produced severe inflammation in rat intestine. The middleportion of the small intestine i.e., jejunum and proximalileum showed more inflammation compared to proximal por-tion of the small intestine. Caecum was the most severely af-fected part, showing hemorrhagic spots. The ileum showedmany lesions, which were transmural. In between there wereskip areas of normal tissue. In some animals the large intes-tine was also found to be affected with hemorrhagic lesions.
Evaluation based on macroscopic features showed signif-icantly lower score values for drug treated and prednisolonetreated group compared to the control group. Score valuesof the drug treated group were comparable with the scoresobtained in prednisolone treated group. (Table 1).
Histological examination of control group showed ad-vanced lesions as necrosis of even payers patches and frag-mentation of nuclei. The drug treated group showed reduced
Fig. 1. L.S. of normal caecum of rat (indomethacin induced enterocolitis in rats).
Table 1Evaluation based on macroscopic features in indomethacin-induced ente-rocolitis in rats
Group Treatment Mean of macroscopicscores± S.E.M.
Control Indomethacin (7.5 mg/kg, s.c.) 10.8± 0.6Drug treated Polyherbal formulation
(30 ml/kg per day, orally),indomethacin (7.5 mg/kg, s.c.)
3.5 ± 0.4a,b
Prednisolonetreated
Prednisolone (2 mg/kg per day,orally), indomethacin(7.5 mg/kg, s.c.)
2.5 ± 0.5a
Each value represents mean of macroscopic scores± S.E.M. (n = 6).a Significant decrease in macroscopic score values according to
Student’st-test (two-tailed) atP < 0.01 w.r.t. control.b n.s. difference in macroscopic score values of drug treated group
and prednisolone treated group in not significant, according to Student’st-test (two-tailed).
intensity of lesions without any evidence of necrosis, re-generation or inflammatory reaction. Prednisolone treatmentshowed suppressed inflammatory reaction (Figs. 1–4).
The myeloperoxidase assay showed significant increasein MPO activity of control group compared to normal un-treated group. The drug treated and prednisolone treatedgroup showed significant reduction in MPO activity com-pared to the control group. MPO activity of the drug treatedgroup was comparable with the prednisolone treated group(Table 2).
3.2. Acetic acid-induced colitis in mice
Intrarectal instillation of acetic acid caused inflammatoryreaction in the colon. The inflammation covered rectum anddistal colon portion. The visible changes included severeepithelial necrosis and ulcerated mucosa.
Drug treated and prednisolone treated group showed sig-nificantly lower score values of macroscopic evaluation as
200 A.G. Jagtap et al. / Journal of Ethnopharmacology 90 (2004) 195–204
Fig. 2. L.S. of inflamed caecum of rat showing submucosa withsevere infiltrationand edema (indomethacin induced enterocolitis in rats). Expression initalics is shown by an arrow.
Fig. 3. L.S. of caecum of drug treated rat showingsubmucosal edemaand few inflammatory cells (indomethacin induced enterocolitis in rats).
compared to the control group and values obtained for thedrug treated group were comparable with the prednisolonetreated group (Table 3).
Histological examination of control group showedmassive necrosis of the mucosa and submucosa. Payerspatches appeared distorted with karyohexis and karyol-ysis. Drug treated group showed mild lesions, regenera-tion and inflammatory reaction. The prednisolone treatedgroup showed suppressed inflammatory reaction. (Figs. 5and 6).
The myeloperoxidase assay showed significant increasein MPO activity of control group compared to normalgroup. The drug treated and prednisolone treated groupsshowed significant decrease in MPO activity compared tothe control group (Table 4).
4. Discussion
We have studied two experimental animal models for IBD.Intrarectal instillation of acetic acid in mice affected only thedistal colon portion. The inflammation was not transmural.Massive necrosis of mucosal and submucosal layers was ob-served. This model shares many of the histologic features ofulcerative colitis in human beings including mucosal edema,neutrophil infiltration of the mucosa and submucosal ulcer-ation (Sharon and Stenson, 1985).
The mechanisms by which acetic acid produces inflam-mation appear to involve the entry of the protonated formof the acid into epithelium, where it dissociates to liber-ate protons within intracellular acidification that most likelyaccounts for the epithelial injury observed (Yamada et al.,
A.G. Jagtap et al. / Journal of Ethnopharmacology 90 (2004) 195–204 201
Fig. 4. L.S. of caecum of Prednisolone treated rat showingsubmucosal edema(indomethacin induced enterocolitis in rats).
Table 2Evaluation of MPO activity in indomethacin-induced enterocolitis
Group Treatment MPO activity(U/g) ± S.E.M.
Normal None 11.2± 0.6Control Indomethacin (7.5 mg/kg, s.c.) 32.8± 1.1a
Drug treated Polyherbal formulation(30 ml/kg per day, orally),indomethacin (7.5 mg/kg, s.c.)
16.5 ± 1.0b,c
Prednisolonetreated
Prednisolone (2 mg/kg per day,orally), indomethacin(7.5 mg/kg, s.c.)
12.8 ± 0.6b
Each value represents mean of MPO activity (U/g) ± S.E.M. (n = 6).a Significant increase in MPO activity according to Student’st-test
(two-tailed) atP < 0.01 w.r.t. normal.b Significant decrease in MPO activity according to Student’st-test
(two-tailed) atP < 0.01 w.r.t. control.c Significantly higher MPO activity according to Student’st-test
(two-tailed) atP < 0.01 w.r.t. prednisolone treated group.
Table 3Evaluation based on macroscopic features in acetic acid-induced colitis
Group Treatment Mean of macroscopicscores± S.E.M.
Control Acetic acid (6%, 0.1 ml,once, intrarectally)
2.8 ± 0.16
Drug treated Polyherbal formulation(40 ml/kg per day, orally),acetic acid (6%, 0.1 ml,once, intrarectally)
Each value represents mean of macroscopic scores± S.E.M. (n = 6).a Significant decrease in macroscopic score values according to
Student’st-test (two-tailed) atP < 0.01 w.r.t. control.b n.s.: difference in macroscopic score values of drug treated group
and prednisolone treated group in not significant, according to Student’st-test (two-tailed).
1992). The inflammatory response initiated by acetic acidincludes activation of cyclooxygenase and lipooxygenasepathways (Sharon and Stenson, 1985).
Indomethacin given subcutaneously in rats affected themiddle portion of small intestine (jejunum and proximalileum) and most severely the caecum. The inflammationwas not continuous. It showed some patches of normal tis-sue (skip areas). Also the necrotic foci were transmural.These findings suggest that this experimental model resem-ble Crohn’s disease (Banerjee and Peters, 1989).
The pathogenesis of the lesions produced by indomethacinis not clear. Pathogenic mechanism was thought to beprostaglandin-related in indomethacin enteropathy in rats.Local changes in intestinal microflora were also thought tobe important and this is confirmed by reports that germ free
Table 4Evaluation of MPO activity in acetic acid-induced colitis
Group Treatment MPO activity(U/g) ± S.E.M.
Normal None 0.5± 0.1Control Acetic acid (6%, 0.1 ml,
once, intrarectally)7.9 ± 0.1a
Drug treated Polyherbal formulation(40 ml/kg per day, orally),acetic acid (6%, 0.1 ml,once, intrarectally)
Each value represents mean of MPO activity (U/g) ± S.E.M. (n = 6).a Significant increase in MPO activity according to Student’st-test
(two-tailed) atP < 0.01 w.r.t. normal.b Significant decrease in MPO activity according to Student’st-test
(two-tailed) atP < 0.01 w.r.t. control.c Significantly higher MPO activity according to Student’st-test
(two-tailed) atP < 0.05 w.r.t. prednisolone treated group.
202 A.G. Jagtap et al. / Journal of Ethnopharmacology 90 (2004) 195–204
Fig. 5. L.S. inflamed colon of a mouse showingsevere infiltrationof submucosa (acetic acid induced colitis in mice).
animals do not develop lesions readily (Kent et al., 1969).Thus, the postulated mechanism is that altered mucosalprostaglandin synthesis compromises intestinal integrity,resulting in mucosal response to the bacterial products.(Banerjee and Peters, 1989).
The treatment with polyherbal formulation has shown adecrease in the macroscopic scores for the inflammation.Histopathology examination of drug treated group revealedless damage compared to control group. A significant de-crease in MPO activity was also observed. All these obser-vations support the findings that the polyherbal formulationwas able to offer significant protection in both the modelsstudied.
Acetic acid-induced colitis and indomethacin-induced en-terocolitis simulate two different disease conditions, whichare ulcerative colitis and Crohn’s disease respectively. On
Fig. 6. L.S. of normal colon of a mouse (acetic acid induced colitis in mice).
this basis we can say that the polyherbal formulation understudy may be useful in treating UC as well as CD in humans.
The prednisolone treatment has shown significant pro-tection in both the animal models under our study. Thepolyherbal formulation was found comparable with pred-nisolone. The formulation at doses 30 and 40 ml/kg was asgood as prednisolone at the doses 2 and 1.14 mg/kg for ratsand mice, respectively. It is possible that the formulation actsby the same mechanism as the prednisolone i.e., by decreas-ing the number of neutrophils and reduction in the synthesisof inflammatory mediators.
The formulation consists of four different drugs. All thesedrugs have effect on GIT.
Unripe fruit of Aegle marmeloescontains marmelosin asa chief constituent, which is a furocoumarin. It also con-tains carbohydrate pectin, volatile oil and tannins. Due to
A.G. Jagtap et al. / Journal of Ethnopharmacology 90 (2004) 195–204 203
its high pectin content it is useful in diarrhea, dysentery.Volatile oil and tannins present astringent properties. Un-ripe fruit showed activity against some intestinal parasites(Das and Das, 1995; Indian Herbal Pharmacopoeia, vol. II.,1999). The plant also has antibacterial and antifungal activ-ity (Valsaraj et al., 1997; Sasidharan et al., 1998).
Ripe fruits of Coriandrum sativumcontain fixed oil,volatile oil and proteins. It gives carminative and stomachicactions. Volatile oil of Coriander contains mainly alcohol,linolol (Kokate et al., 2001). The oil has strong antifungalactivity at very low concentrations (Pandey and Pant, 1997).
Rhizomes ofCyperus rotunduscontain carbohydrates &volatile oil made up of mainly sesquiterpenes (Ohira et al.,1998). The plant has been used for its anti-inflammatory,analgesic, diuretic properties in folklore remedies. (Kimet al., 1998). It has shown cytoprotective effect againstethanol-induced ulceration in rats (Zhu et al, 1997).
Oil of roots ofVetiveria zinzanioidsmainly contains alco-hols like vetiverol, and ketones-� and� vetivones. It presentsstimulant, stomachic actions (Ayurvedic encyclopedia,1998). Vetiver oil has good antifungal (Dubey and Mishra,1990; Chaumont and Bardey, 1989) and antibacterial prop-erties (Gangrade et al., 1990).
The polyherbal formulation, which is a combina-tion of all these drugs have shown to be effective inindomethacin-induced enterocolitis in rats as well as Aceticacid-induced colitis in mice.Cyperus rotundusmay be themain active constituent of the formulation, which has pro-tected the animals against experimentally induced diseasebecause of its cytoprotective and anti-inflammatory activi-ties. The remaining three drugs may be responsible for giv-ing symptomatic relief and just the supportive treatments.The drugsAegle marmeloesandVetiveria zinzanioidspos-sess good antibacterial properties; therefore, they may beuseful in combating intestinal or other pathogens, whichplay important role in the pathogenesis of IBD.Corian-drum sativumandVetiveria zinzanioidshas carminative andstomachic actions, which might be useful in gastrointestinaldisturbances occurring in inflammatory bowel, conditions.Aegle marmeloeshas good antidiarrheal activity and maybe useful in disturbed GI motility.
The mechanism of development of disease in indome-thacin-induced enterocolitis involves role of protectiveprostaglandins and intestinal pathogens. The formulationmight be active due to its anti-inflammatory, cytoprotectiveand antimicrobial properties. Acetic acid-induced coli-tis involves inflammatory response initiated by the injurycaused by acid. It involves stimulation of cyclooxygenaseand lipooxygenase pathways and generation of inflamma-tory mediators like prostaglandins and leukotrienes. Thepolyherbal formulation may have an effect on synthesis orrelease of these inflammatory mediators.
So it can be concluded from the present study that thepolyherbal formulation may prove useful in the treatmentof IBD. However, more detailed phytochemical studies arenecessary to identify the active principles and exact mecha-
nisms of action. Proper clinical investigation should also becarried out to confirm the activity in human disease.
Acknowledgements
The authors are grateful to University Grant Commission,for providing funds required to carry out the research work.We express our sincere thanks to Dr. Lonkar, Head of Pathol-ogy department, Bombay Veterinary College, Mumbai andDr. Moodbidri, Head of Biochemistry department, Instituteof Research in Reproduction, Mumbai for their help in ourhistopathological studies.
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Journal of Ethnopharmacology 90 (2004) 205–215
Antioxidant, antimicrobial, antiulcer and analgesicactivities of nettle (Urtica dioicaL.)
Ilhami Gülçina, Ö. Irfan Küfrevioglua,∗, Münir Oktayb, Mehmet Emin Büyükokurogluc
a Department of Chemistry, Faculty of Science and Arts, Atatürk University, 25240 Erzurum, Turkeyb Department of Chemistry Education, Kazım Karabekir Education Faculty, Atatürk University, 25240 Erzurum, Turkey
c Department of Pharmacology, Medical Faculty, Atatürk University, 25240 Erzurum, Turkey
Received 27 January 2003; received in revised form 30 July 2003; accepted 22 September 2003
Lipid peroxidation is an important deteriorate reaction infood during storage and processing. It not only causes a lossin food quality but also is believed to be associated with somediseases such as carcinogenesis, mutagenesis, ageing, andarteriosclerosis (Yagi, 1987). The role of active oxygen andfree radicals in tissue damage in such diseases, are becomingincreasingly recognized (Halliwell and Gutteridge, 1985).Cancer, emphysema, cirrhosis, arteriosclerosis, and arthritishave all been correlated with oxidative damage. Active oxy-gen, either in the form of superoxide (O2
•−), hydrogen per-oxide (H2O2), hydroxyl radical (OH•), or singled oxygen(1O2), is a product of normal metabolism and attacks bio-logical molecules, leading to cell or tissue injury. When themechanism of antioxidant protection becomes unbalancedby exogenous factors such as smoking, ionising radiation,certain pollutants, organic solvents and pesticides and en-dogenous factors such as normal aerobic respiration, stimu-
lated polymorphonuclear leukocytes and macrophages, andperoxisomes may occur, resulting in above-mentioned dis-eases and accelerating ageing (Büyükokuroglu et al., 2001).However, antioxidant supplements or foods rich in antioxi-dants may be used to help the human body in reducing ox-idative damage by free radicals and active oxygen (Halliwelland Gutteridge, 1984; Mau et al., 2001; Gülçin et al., 2002b).Recently, various phytochemicals and their effects on health,especially the suppression of active oxygen species by nat-ural antioxidants from teas, spices and herbs, have been in-tensively studied (Ho et al., 1994). The most commonlyused antioxidants at the present time are butylated hydrox-yanisole (BHA), butylated hydroxytoluene (BHT), propylgallate (PG), andtert-butylhydroquinone (TBHQ) (Sherwin,1990). However, they are suspected of being responsiblefor liver damage and carcinogenesis in laboratory animals(Grice, 1986; Wichi, 1988). Therefore, the development andutilization of more effective antioxidants of natural originare desired (Gülçin et al., 2002a; Oktay et al., 2003).
Aqueous infusions of Mediterranean herbs includ-ing Urtica dioica, exhibit antioxidant activity towardsiron-promoted oxidation of phospholipids, linoleic acid, anddeoxyribose (Matsingou et al., 2001). Also, the electrogen-
206 I. Gülçin et al. / Journal of Ethnopharmacology 90 (2004) 205–215
erated bromine method was used for estimating the antiox-idant capacity of plant materials such asUrtica dioica andplant-based medicinal preparations (Abdullin et al., 2002).It was reported thatUrtica dioica prevent the damage of ratliver tissue structure (Lebedev et al., 2001).
Urtica dioicaherbs are used to treat stomachache in Turk-ish folk medicine (Yesilada et al., 2001). In addition, thisherb is used to treat rheumatic pain and for colds and cough(Sezik et al., 1997) and is used against liver insufficiency(Yesilada et al., 1993).
The aim of the present study was to investigate antioxidantactivity by using different antioxidant tests including reduc-ing power, free radical scavenging, superoxide anion rad-ical scavenging, hydrogen peroxide scavenging, and metalchelating activities. An important goal of this research wasto examine antimicrobial, antiulcer, and analgesic activityof WEN.
2. Materials and methods
2.1. Antioxidant activities
2.1.1. ChemicalsAmmonium thiocyanate was purchased from E. Merck.
2.1.2. Plant material and extractionNettle was collected in May, in Dumlu area in Erzurum,
Turkey, and authenticated by Prof. Dr.Ismet Hasenekoglu,Department of Biology Education, Kazım Karabekir Edu-cation Faculty, Atatürk University. Then, nettle was left ona bench to dry. The dried sample was chopped into smallparts with a blender. For water extraction, 20 g dried aerialparts of nettle ground into a fine powder in a mill and wasmixed with 400 ml boiling water by magnetic stirrer duringfifteen minutes. Then the extract was filtered over What-man No.1 paper. The filtrate was frozen and lyophilized ina lyophilizator at 5�mHg pressure at−50◦C (Labconco,Freezone 1L). The extract of nettle was placed in a plasticbottle, and then stored at−20◦C until used.
2.1.3. Total antioxidant activity determinationThe antioxidant activity of WEN was determined ac-
cording to the thiocyanate method (Mitsuda et al., 1996).For stock solution; 20 mg lyophilized WEN was dissolvedin 20 ml water. Then the solution, which contains differ-ent amount of stock WEN solution or standards samples(50, 100 and 250�g) in 2.5 ml of potassium phosphate
buffer (0.04 M, pH 7.0) was added to 2.5 ml of linoleicacid emulsion in potassium phosphate buffer (0.04 M, pH7.0). Each solution was then incubated at 37◦C in a glassflask in the dark. At intervals during incubation, each so-lution was stirred for 3 min, 0.1�l this incubation solution,0.1 ml FeCl2 and 0.1 ml thiocyanate were transferred tothe test tube, which containing 4.7 ml ethanol. Then thissolution incubated for 5 min. Finally, the peroxide valuewas determined by reading the absorbance at 500 nm ina spectrophotometer (8500 II, Bio-Crom Gmbh, Zurich,Switzerland). During the linoleic acid oxidation, perox-ides formed and these compounds oxidize Fe2+ to Fe3+.The latter Fe3+ ions form complex with SCN−, whichhas a maximum absorbance at 500 nm. Therefore higherabsorbance values indicate higher linoleic acid oxidation.The solutions without added WEN or standards were usedas blank samples. Five millilitres linoleic acid emulsion isconsisting of 17.5�g Tween-20, 15.5�l linoleic acid and0.04 M potassium phosphate buffer (pH 7.0). On the otherhand, 5 ml control composed of 2.5 ml linoleic acid emul-sion and 2.5 ml potassium phosphate buffer (0.04 M, pH7.0). All data about total antioxidant activity are the averageof duplicate analyses. The inhibition of lipid peroxidationin percentage was calculated by the following equation:
Percent inhibition=[A0 − A1
A0
]× 100
whereA0 was the absorbance of the control reaction andA1was the absorbance in the presence of the sample of WEN(Duh et al., 1999).
2.1.4. Reducing powerThe reducing power of WEN was determined accord-
ing to the method ofOyaizu (1986). The different doses ofWEN (50, 100 and 250�g) in 1 ml of distilled water weremixed with phosphate buffer (2.5 ml, 0.2 M, pH 6.6) andpotassium ferricyanide [K3Fe(CN)6] (2.5 ml, 1%). The mix-ture was incubated at 50◦C for 20 min. A portion (2.5 ml)of TCA (10%) was added to the mixture, which was thencentrifuged for 10 min at 1000× g (MSE Mistral 2000,UK, Serial No.: S693/02/444). The upper layer of solution(2.5 ml) was mixed with distilled water (2.5 ml) and FeCl3(0.5 ml, 0.1%), and the absorbance was measured at 700 nmin a spectrophotometer (8500 II, Bio-Crom GmbH, Zurich,Switzerland). Higher absorbance of the reaction mixture in-dicated greater reducing power.
2.1.5. Superoxide anion scavenging activityMeasurement of superoxide anion scavenging activity of
WEN was based on the method described byLiu et al.(1997)with slight modifications (Gülçin et al., 2003c). Su-peroxide radicals are generated in phenazine methosulphate(PMS)–nicotinamide adenine dinucleotide (NADH) systemsby oxidation of NADH and assayed by the reduction of ni-troblue tetrazolium (NBT). In this experiments, the super-oxide radicals were generated in 3 ml of Tris–HCl buffer
I. Gülçin et al. / Journal of Ethnopharmacology 90 (2004) 205–215 207
(16 mM, pH 8.0) containing 1 ml of NBT (50�M) solution,1 ml NADH (78�M) solution and 1 ml sample solution ofWEN (100�g/ml) were mixed. The reaction was started byadding 1 ml of PMS solution (10�M) to the mixture. Thereaction mixture was incubated at 25◦C for 5 min, and theabsorbance at 560 nm in a spectrophotometer (8500 II, Bio-Crom GmbH, Zurich, Switzerland) was measured againstblank samples.l-Ascorbic acid was used as a control. De-crease in absorbance of the reaction mixture indicated in-creased superoxide anion scavenging activity. The percent-age inhibition of superoxide anion generation was calculatedusing the following formula
Percent inhibition=[A0 − A1
A0
]× 100
where A0 was the absorbance of the control (l-Ascorbicacid), andA1 was the absorbance of WEN or standards (Yeet al., 2000).
2.1.6. Free radical scavenging activityThe free radical scavenging activity of WEN was mea-
sured by 1,1-diphenyl-2-picryl-hydrazil (DPPH•) using themethod ofShimada et al. (1992). Briefly, 0.1 mM solutionof DPPH• in ethanol was prepared. Then, 1 ml of this solu-tion was added to 3 ml of WEN solution at different doses(50–250�g). The mixture was shaken vigorously and al-lowed to stand at room temperature for 30 min. Then theabsorbance was measured at 517 nm in a spectrophotome-ter (8500 II, Bio-Crom GmbH, Zurich, Switzerland). Lowerabsorbance of the reaction mixture indicated higher free rad-ical scavenging activity. The DPPH• concentration (mM) inthe reaction medium was calculated from the following cali-bration curve, determined by linear regression (R2: 0.9769):
Absorbance= 104.09× [DPPH•]
The DPPH radical concentration was calculated using thefollowing equation:
DPPH• scavenging effect(%) = 100−[A0 − A1
A0× 100
]
whereA0 was the absorbance of the control reaction andA1was the absorbance in the presence of the sample of WEN(Oktay et al., 2003).
2.1.7. Metal chelating activityThe chelating of ferrous ions by the WEN and standards
was estimated by the method of Dinis (Dinis et al., 1994).Briefly, extracts (50–250�g) were added to a solution of2 mM FeCl2 (0.05 ml). The reaction was initiated by theaddition of 5 mM ferrozine (0.2 ml) and the mixture wasshaken vigorously and left standing at room temperature forten minutes. After the mixture had reached equilibrium, theabsorbance of the solution was then measured spectrophoto-metrically at 562 nm in a spectrophotometer (8500 II, Bio-Crom GmbH, Zurich, Switzerland). The percentage of in-
hibition of ferrozine-Fe2+ complex formation was given bythe formula:
Percent inhibition=[A0 − A1
A0
]× 100
whereA0 was the absorbance of the control, andA1 wasthe absorbance in the presence of the sample of WEN andstandards. The control contains FeCl2 and ferrozine (Gülçinet al., 2003a).
2.1.8. Scavenging of hydrogen peroxideThe ability of the WEN to scavenge hydrogen peroxide
was determined according to the method ofRuch et al.(1989). A solution of hydrogen peroxide (40 mM) wasprepared in phosphate buffer (pH 7.4). Hydrogen peroxideconcentration was determined spectrophotometrically fromabsorption at 230 nm in a spectrophotometer (8500 II, Bio-Crom GmbH, Zurich, Switzerland). Extracts (50–250�g)in distilled water were added to a hydrogen peroxide so-lution (0.6 ml, 40 mM). Absorbance of hydrogen peroxideat 230 nm was determined after ten minute against a blanksolution containing in phosphate buffer without hydrogenperoxide. The percentage of scavenging of hydrogen perox-ide of WEN and standard compounds was calculated usingthe following equation:
Percent scavenged [H2O2] =[A0 − A1
A0
]× 100
whereA0 was the absorbance of the control, andA1 wasthe absorbance in the presence of the sample of WEN andstandards (Gülçin et al., 2003b).
2.1.9. Determination of total phenolic compoundsTotal soluble phenolic compounds in the WEN were deter-
mined with Folin–Ciocalteu reagent according to the methodof Slinkard (Slinkard and Singleton, 1977) using pyrocate-chol as a standard phenolic compound. Briefly, 1 ml of theWEN solution (contains 1000�g extract) in a volumetricflask diluted with distilled water (46 ml). One milliliter ofFolin–Ciocalteu reagent was added and the content of theflask was mixed thoroughly. After 3 min 3 ml of Na2CO3(2%) was added and then was allowed to stand for 2 hwith intermittent shaking. The absorbance was measured at760 nm in a spectrophotometer (8500 II, Bio-Crom GmbH,Zurich, Switzerland). The total concentration of phenoliccompounds in the WEN determined as microgram of pyro-catechol equivalent by using an equation that was obtainedfrom standard pyrocatechol graph (Gülçin et al., 2002b):
Absorbance= 0.0053
× total phenols [pyrocatechol equivalent(�g)]
− 0.0059.
208 I. Gülçin et al. / Journal of Ethnopharmacology 90 (2004) 205–215
2.2. Antimicrobial activities
2.2.1. Preparation of test microorganismsFor the purpose of antimicrobial evaluation ten mi-
croorganisms were used.Pseudomonas aeruginosa(ATCC9027, Gram-negative),Escherichia coli (ATCC 9837,Gram-negative), Proteus mirabilis (Clinical isolate,Gram-negative), Citrobacter koseri (Clinical isolate,Gram-negative), Enterobacter aerogenes(Clinical iso-late, Gram-negative),Staphylococcus aureus(ATCC 6538,Gram-positive),Streptococcus pneumoniae(ATCC 49619,Gram-positive),Micrococcus luteus(Clinical isolate, Gram-positive), Staphylococcus epidermidis(clinical isolate,Gram-positive), andCandida albicans(ATCC 10231) mi-croorganism strains were employed for determination ofantimicrobial activity. Clinical isolates of microorganismswere defined by Dr. Ekrem Kireçci, Department of Micro-biology, Medical Faculty, Atatürk University, Erzurum.
Bacteria and yeast were obtained from the stock culturesof Microbiology Laboratory, Department of Microbiology,Medical Faculty, Atatürk University, Erzurum. The bacterialand yeast stock cultures were maintained on Muller HintonAgar (Oxoid CM 337, Basingstoke, Hampshire, UK) slants,respectively, which were stored at 4◦C. These bacteria weremaintained on Blood agar base (Oxoid CM55, Basingstoke,Hampshire, UK). The yeast was maintained on Sabouraud-dextrose agar (Oxoid CM41, Basingstoke, Hampshire, UK).
2.2.2. Antimicrobial activity determinationAgar cultures of the test microorganisms were prepared
as described byMackeen et al. (1997). Three to five sim-ilar colonies were selected and transferred with loop into5 ml of Tryptone Soya broth (Oxoid CM 129, Basingstoke,Hampshire, UK). The broth cultures were incubated for24 h at 37◦C. The WEN was dissolved in sterile water forthe assay by magnetic stirrer. For screening, sterile, 6 mmdiameter filter paper disc were impregnated with 250�g ofthe WEN. Then the paper discs were placed onto MuellerHinton agar (Oxoid CM337, Basingstoke, Hampshire, UK).The inoculum for each organism was prepared from brothcultures. The concentration of cultures was adjusted to 108
colony forming units (1× 108 CFU/ml). The results wererecorded by measuring the zones of growth inhibition sur-rounding the disc. Clear inhibition zones around the discsindicated the presence of antimicrobial activity. All data onantimicrobial activity are the average of triplicate analyses.Netilmicin (30�g per disc), amoxicillin-clavulanic acid(20–10�g per disc), ofloxacin (5�g per disc, BBLTM SensidiscTM), and antifungal miconazole nitrate (40�g per disc,DRG International) were used as reference standards, whichas recommended by the National Committee for ClinicalLaboratory Standards (NCCLS).
2.3. Antiulcer activities
Forty albino Sprague–Dawley male rats with a weight of190–225 g were used for the experiment. The rats were fed
with standard laboratory chow and water before the experi-ment. The laboratory was windowless with automatic tem-perature (22± 1◦C) and lighting controls (14 h light/10 hdark). Rats were divided into five equal groups (n = 8) andhoused in cages. Twenty-four hours before the experiment,the rats were fasted and allowed access to water ad libitum.
Anti-ulcerogenic effect of WEN was investigated by us-ing the ethanol-induced ulcer model (Büyükokuroglu et al.,2002). On the day of the experiment, groups 1, 2 and 3 wereinjected with 10 mg/kg WEN, while group 4 was injectedwith 20 mg/kg famotidine and group 5 with saline solution.All of drugs were administered intraperitoneally in 0.5 mlvehicle. Following a 30-min-period, all the animals weregiven 1 ml of ethanol (70%) by oral gavages. One hour af-ter the administration of ethanol, animals were sacrificed bydecapitation. The stomach of each was removed and openedalong the greater curvature and washed in physiologicalsaline solution. For the measurement of the gross gastric mu-cosal lesions, freshly excised stomach was laid flat and themucosal lesions were traced on clear acetate paper. Grossmucosal lesions were recognized as haemorrhage or linearbreaks (erosions) with damage to the mucosal surface. Thearea of stomach and gross lesions were approximately cal-culated by planimetry using a simple magnifier. The resultswere translated to the term of “total ulcer area/total gastricarea” and these were expressed as an ulcer index (%).
2.4. Writhing test
All experiments were performed on no-fasted male andfemale albino Swiss mice weighing 30–38 g, which were ob-tained from animal house in the Atatürk University, MedicalFaculty. Animals were divided into five equal groups of 6each. Animals were pretreated with 50, 100 and 200 mg/kgdoses of WEN and 200 mg/kg dose of metamizol as ref-erence drug. Control animals received an equal volume of0.9% NaCl in distilled water. Drugs and saline were given60 min before acetic acid injection.
Writhing test was determined according to the method ofZakaria et al. (2001). Writhing was induced by 10 mg/kg ofintraperitoneally acetic acid (0.6%) injection. Ten millime-tres after acetic acid injection, the mice were placed in atransparent box and the number of writhes was counted forperiod of 10 min. Writhing movement was accepted as con-traction of the abdominal muscles accompanied by stretch-ing of the hind limbs. Antinociceptive effect was expressedas the reduction of the number of writhing between controland pretreated mice.
Percentage reduction of the number of writhing(%)
=[A0 − A1
A0
]× 100
whereA0 was the number of writhing of the control, andA1 was the number of writhing of pretreatment with WEN(Gülçin et al., 2003d).
I. Gülçin et al. / Journal of Ethnopharmacology 90 (2004) 205–215 209
2.5. Statistical analysis
Experimental results concerning this study were mean±S.D. of three parallel measurements. Analysis of variancewas performed by ANOVA procedures. Significant differ-ences between means were determined by Duncan’s multi-ple range tests.P values<0.05 were regarded as significantandP values<0.01 very significant.
3. Results and discussion
3.1. Antioxidant capacity
The antioxidant activity of putative antioxidants have beenattributed to various mechanisms, among which are preven-tion of chain initiation, binding of transition metal ion cata-lysts, decomposition of peroxides, prevention of continuedhydrogen abstraction, reductive capacity and radical scav-enging (Diplock, 1997; Oktay et al., 2003). Numerous an-tioxidant methods and modifications have been proposedto evaluate antioxidant activity and to explain how antioxi-dants function. Of these, total antioxidant activity, reducingpower, DPPH assay, metal chelating, active oxygen speciessuch as H2O2, O2
•− and OH• quenching assays are mostcommonly used for the evaluation of antioxidant activitiesof extracts (Duh et al., 1999; Amarowicz et al., 2000; Changet al., 2002).
Total antioxidant activity of WEN was determined bythe thiocyanate method. WEN exhibited effective antioxi-dant activity at all doses. The effects of various amounts ofWEN (from 50 to 250�g) on peroxidation of linoleic acidemulsion are shown inFig. 1. The antioxidant activity ofWEN increased concentration dependently. WEN (50, 100and 250�g) showed higher antioxidant activities than thatof 100�g concentration of�-tocopherol. After incubationtimes the percentage inhibition of peroxidation in linoleic
Fig. 1. Antioxidant activity of different doses of WEN and�-tocopherol in the linoleic acid emulsion was determined by the thiocyanate method. Theindicated amounts of dried extract of WEN were presented in 5 ml of linoleic acid emulsion. The control was the linoleic acid emulsion without WENextract. Results are average of duplicate experiments (WEN: water extract of nettle).
acid emulsion was 39, 66 and 98%, respectively, and greaterthan that of�-tocopherol (30%).
Fig. 2shows the reductive capabilities of WEN comparedto tocopherol. For the measurements of the reductive ability,we investigated the Fe3+–Fe2+ transformation in the pres-ence of WEN samples using the method ofOyaizu (1986).The reducing capacity of a compound may serve as a signifi-cant indicator of its potential antioxidant activity (Meir et al.,1995). Like the antioxidant activity, the reducing power ofWEN increased concentration dependently. All of the con-centrations of WEN showed higher activities than the con-trol in a statistically significant (P < 0.05) manner.
In the PMS–NADH–NBT system, superoxide anion de-rived from dissolved oxygen by PMS–NADH coupling re-action reduces NBT. The decrease of absorbance at 560 nmwith antioxidants indicates the consumption of superoxideanion in the reaction mixture (Oktay et al., 2003). Fig. 3shows the percentage inhibition of superoxide radical gen-eration by 100�g of WEN and comparison with same dosesof BHA, BHT and�-tocopherol. The WEN exhibited highersuperoxide radical scavenging activity than BHA, BHT and�-tocopherol (P < 0.01). The percentage inhibition of su-peroxide generation by 100�g amount of WEN was foundas 97% and greater than that of some doses of BHA, BHT,and tocopherol (95, 83 and 60%), respectively. Superoxideradical scavenging activity of those samples followed theorder: WEN> BHA > BHT > �-tocopherol.
The effect of antioxidants on DPPH radical scavenging isthought to be due to their hydrogen donating ability. DPPHis a stable free radical and accepts an electron or hydro-gen radical to become a stable diamagnetic molecule. Themodel of scavenging the stable DPPH radical is a widelyused method to evaluate antioxidant activities in a relativelyshort time compare to other methods (Soares et al., 1997).The reduction capability on the DPPH radical is determinedby the decrease in its absorbance at 517 nm induced by an-tioxidants. The maximum absorption of a stable DPPH rad-
210 I. Gülçin et al. / Journal of Ethnopharmacology 90 (2004) 205–215
Fig. 2. Reducing power of WEN, and�-tocopherol by spectrophotometric detection of the Fe3+–Fe2+ transformation. Control was test sample withoutextract or�-tocopherol. Higher absorbance indicates higher reducing power (WEN: water extract of nettle).
ical in ethanol is at 517 nm. The decrease in absorbance ofDPPH radical caused by antioxidants is due to the reactionbetween antioxidant molecules and radical, which results inthe scavenging of the radical by hydrogen donation. This isvisualized as a discoloration from purple to yellow. Hence,DPPH is usually used as a substrate to evaluate antioxidantactivity (Duh et al., 1999; Chang et al., 2002; Gülçin et al.,2003c). Fig. 4 illustrates a significant (P < 0.01) decreasein the concentration of DPPH radical due to the scaveng-ing ability of the WEN and standards. WEN and BHAshowed almost equal DPPH scavenging activity, however,significantly are lower than that of quercetin. The scaveng-ing effect of WEN and standards on the DPPH radical de-creased in the order of quercetin> WEN > BHA andwere 93, 37 and 32% at the concentration of 60�g/ml,respectively.
It was reported that oxidative stress, which occurs whenfree radical formation exceeds the body’s ability to protectitself, forms the biological basis of chronic conditions suchas arteriosclerosis (Fatimah et al., 1998). Based on the data
Fig. 3. Superoxide anion radical scavenging activity of 100�g of WEN, BHA, BHT, and�-tocopherol by the PMS–NADH–NBT method (BHA: butylatedhydroxyanisole, BHT: butylated hydroxytoluene, WEN: water extract of nettle).
obtained from this study, WEN exhibits free radical inhibitoror scavenger activity as well as a primary antioxidant thatreacts with free radicals, which may limit free radical dam-age occurring in the human body.
The chelating of ferrous ions by WEN was estimatedwith the method ofDinis et al. (1994). Ferrozine can quan-titatively form complexes with Fe2+. In the presence ofchelating agents, the complex formation is disrupted andeventually that the red colour of the complex fades. Mea-surement of colour reduction therefore allows estimation ofthe chelating activity of the co-existing chelator (Yamaguchiet al., 2000). In this assay WEN and standard antioxidantcompound interfered with the formation of ferrous and fer-rozine complex, suggesting that they have chelating activityand capture ferrous ion before ferrozine. Iron can stimulatelipid peroxidation by the Fenton reaction, and also accel-erates peroxidation by decomposing lipid hydroperoxidesinto peroxyl and alkoxyl radicals that can themselves ab-stract hydrogen and perpetuate the chain reaction of lipidperoxidation (Chang et al., 2002; Halliwell, 1991).
I. Gülçin et al. / Journal of Ethnopharmacology 90 (2004) 205–215 211
Fig. 4. Comparison of free radical scavenging activity of quercetin, BHA, and WEN on 1,1-diphenyl-2-picrylhydrazyl radical (BHA: butylated hydrox-yanisole, WEN: water extract of nettle).
As shown inFig. 5, the formation of the Fe2+-ferrozinecomplex was not completed in the presence of WEN, indi-cating that WEN chelates the iron. The absorbance of Fe2+-ferrozine complex was linearly decreased dose-dependently(from 50 to 250�g). The difference between WEN and thecontrol was statistically significant (P < 0.01). The percent-ages of metal chelating capacity of 250�g concentrationof WEN, �-tocopherol, BHA, and BHT were found as 92,43, 66 and 41%, respectively. The metal scavenging effectof WEN and standards decreased in the order of WEN>
BHA > �−tocopherol> BHT.Metal chelating capacity is important since it reduced the
concentration of the catalysing transition metal in lipid per-oxidation (Duh et al., 1999). It was reported that chelatingagents, which form bonds with a metal, are effective as sec-ondary antioxidants because they reduce the redox poten-tial thereby stabilizing the oxidized form of the metal ion(Gordon, 1990). The data obtained fromFig. 5revealed thatWEN demonstrate a marked capacity for iron binding, sug-
Fig. 5. Metal chelating effect of different amount of water extract of nettle, BHA, BHT, and�-tocopherol on ferrous ions (BHA: butylated hydroxyanisole,BHT: butylated hydroxytoluene, WEN: water extract of nettle).
gesting that their action as peroxidation protector may berelated to its iron binding capacity.
Scavenging of H2O2 by WEN may be attributed to theirphenolics, which could donate electrons to H2O2, thus neu-tralizing it to water The H2O2 scavenging capacity of anextract may be attributed to the structural features of theiractive components, which determine their electron donatingabilities (Wettasinghe and Shahidi, 2000).
The ability of WEN to scavenge H2O2 was determined ac-cording to the method ofRuch et al. (1989). The scavengingability of WEN on H2O2 is shown inFig. 6 and comparedwith BHA, BHT and�-tocopherol as standards. WEN wascapable of scavenging H2O2 in a dose-dependent manner.Two-hundred and fifty micrograms of WEN exhibited 23%scavenging activity on H2O2. On the other hand, at the sameconcentration; BHA, BHT and�-tocopherol showed 38, 86and 57% activity respectively. These results indicated thatWEN posses effective H2O2 scavenging activity but lowerthan BHA, BHT and�-tocopherol. However, there was sta-
212 I. Gülçin et al. / Journal of Ethnopharmacology 90 (2004) 205–215
Fig. 6. Hydrogen peroxide scavenging activity of different amount of WEN, BHA, BHT, and�-tocopherol (BHA: butylated hydroxyanisole, BHT:butylated hydroxytoluene, WEN: water extract of nettle).
tistically a very significant correlation between those val-ues and control (P < 0.01). The H2O2 scavenging effect ofsame dose (250�g) of WEN and standards decreased in theorder of BHT > �−tocopherol> BHA > WEN. Hydro-gen peroxide itself is not very reactive, but it may be toxicto cell since it may give rise to hydroxyl radicals in cells(Halliwell, 1991).
Phenols are very important plant constituents because oftheir scavenging ability due to their hydroxyl groups (Hatanoet al., 1989). According to the recent reports, a highly pos-itive relationship between total phenols and antioxidant ac-tivity was found in many plant species (Vinson et al., 1998;Velioglu et al., 1998; Gülçin et al., 2002b; Oktay et al.,2003). 25.3�g pyrocatechol equivalent of phenols was de-tected in 1 mg of WEN.
The phenolic compounds may contribute directly to theantioxidative action (Duh et al., 1999). It is suggested thatpolyphenolic compounds may have inhibitory effects on mu-tagenesis and carcinogenesis in humans, when up to 1.0 gdaily are ingested from a diet rich in fruits and vegetables(Tanaka et al., 1998). In addition, it was reported that phe-nolic compounds were associated with antioxidant activityand play an important role in stabilizing lipid peroxidation(Yen et al., 1993).
3.2. Antimicrobial activity
In this study, nine different microbial and one yeastspecies were used to screen the possible antimicrobial activ-ity of WEN. WEN exhibited antimicrobial activity againstall tested microorganisms. Of the species used,Staphylo-coccus aureusis one of the most common Gram-positivebacteria causing food poisoning. Its source is not the fooditself, but the humans who contaminate food after if hasbeen processed (Rauha et al., 2000). Interestingly WENshowed antibacterial activity against this bacterium. As it isshown inTable 1, the generation of most bacterial and the
yeast species was inhibited by WEN.Escherichia coli, be-longing to the normal flora of humans, is a Gram-negativebacterium. However, an enterohemmoragic strain ofEs-cherichia coli has caused serious cases of food poisoningand preservatives to eliminate its growth are needed.Can-dida albicansis the microbe responsible for most clinicalyeast infections, e.g. in mouth infections. Miconazole nitrate(40�g per disc), amoxicillin-clavulanic acid (20–10�g perdisc), ofloxacin (5�g per disc), and netilmicin (30�g perdisc) were used as positive controls for bacteria and yeast.
3.3. Effects on acute gastric mucosal lesions induced byethanol
Ulcer indices (UI) are shown inTable 2. Per-oral admin-istration of 70% ethanol produced multiple mucosal lesionsin the rat stomach. Pre-treatment with WEN and famotidinewere found to inhibit the ethanol-induced gastric mucosal
Table 1Antimicrobial activities of WEN (250�g per disc), and miconazole nitrate,amoxicillin-clavulanic acid, ofloxacin, and netilmicin
WEN: water extract of nettle; MN: miconazole nitrate (40�g per disc);ACA: amoxicillin-clavulanic acid (20–10�g per disc); O: ofloxacin (5�gper disc); N: netilmicin (30�g per disc); ND: not detected activity at thisamount of WEN or standards.
I. Gülçin et al. / Journal of Ethnopharmacology 90 (2004) 205–215 213
Table 2The effects of different doses of WEN and famotidine on the ethanol-induced gastric mucosal injury (WEN: water extract of nettle)
Results are means± S.E.M. and data are evaluated by using one-wayanalysis of variance (Tukey test).
∗ P < 0.01, compared to control,n = 8.
injury in rats. Preventive effects of 50, 100 and 200 mg/kgWEN were in a dose-dependent manner (percent inhibitionswere 67.7, 61.1 and 77.8, respectively, compared to control)and there was a statistically significance between the effectsof used WEN doses (P < 0.005). Famotidine also signifi-cantly inhibited the ethanol-induced gastric lesion (percentdecrease was 34.4, compared to ethanol). There were signif-icant differences between all concentrations of WEN effectsand famotidine effect (P < 0.001).
3.4. Analgesic effect
Writhing numbers are shown inTable 3. Pretreatmentwith WEN and metamizol were found to inhibit the aceticacid-induced writhing in mice. Inhibitor effects of 50, 100and 200 mg/kg WEN were in a dose-dependent manner andsignificant (percent decrease, compare to control: 62.1, 70.4and 89.2%, respectively). As seen inTable 3, metamizolalso inhibited the acetic acid-induced writhing significantly(P < 0.01) (decrease compare to control: 39.4%).
4. Conclusion
It is known in traditional therapy thatUrtica dioicaL. (Ur-ticaceae) or nettle has a hypertensive effect (Garnier et al.,1961). Therewith, some other actions of this plant were re-ported such as anti-inflammatory and antirheumatic effects
(Obertreis et al., 1996; Riehemann et al., 1999), acute di-uretic, natriuretic and hypotensive effects (Tahri et al., 2000),cardiovascular effects (Testai et al., 2002), and stimulationof proliferation of human lymphocytes (Wagner et al., 1989).The effects of the nettle are also evoked in the therapy ofthe prostatic hyperplasia (Krzeski et al., 1993; Hiramo et al.,1994; Lichius and Muth, 1997), but this plant has no hypo-glycemic action, as reported byRaman-Ramos et al. (1992)and Swanston-Flatt et al. (1989). Moreover, this plant hasbeen used in the traditional therapy of hypertension (Ziyyatet al., 1997).
On the basis of the results of this study, it is clearly indi-cated that WEN has a powerful antioxidant activity againstvarious oxidative systems in vitro; moreover, WEN can beused as accessible source of natural antioxidants and as apossible food supplement or in pharmaceutical industry. Thevarious antioxidant mechanisms of WEN may be attributedto strong hydrogen donating ability, a metal chelating ability,and their effectiveness as scavengers of hydrogen peroxide,superoxide, and free radicals. Phenolic compounds appearto be responsible for the antioxidant activity of WEN. Inaddition, free radicals have been demonstrated to be a con-tributing factor in the tissue injury and modulation of thepain (Khalil et al., 1999; Van der Laan et al., 1997). Somestudies have revealed that the antioxidants melatonin and�-carotene potentiate the antinociceptive responses (Penn,1995; Pang et al., 2001). It was indicated that vitamin E hasbeneficial effects in improvement of rheumatic disease, in-termittent claudication or angina pectoris due to its antioxi-dant activity (Rapola et al., 1996; Sangha and Stucki, 1998;Kleijnen and Mackerras, 2000). According to the above in-formation, it is said that there is a relationship between an-tioxidant and analgesic activities. Analgesic activities maybe related to antioxidant activity.
Finally, all concentrations of WEN possessed noticeableantimicrobial activity against Gram-positive and -negativebacteria when compared with standard and strong antimi-crobial compounds such as miconazole nitrate, amoxicillin-clavulanic acid, ofloxacin, and netilmicin. At the same timeWEN has effective antiulcer activity against ethanol-inducedulcerogenesis and analgesic effect on acetic acid-inducedstretching and it can be used for therapy of ulcerogenesisand gastric mucosal injury.
Acknowledgements
This study was supported by Atatürk University ResearchFoundation (Project no: 2001/35).
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Journal of Ethnopharmacology 90 (2004) 217–220
Hypoglycemic effect ofAcosmium panamensebark on streptozotocin diabetic rats
Adolfo Andrade-Cettoa,∗, Helmut Wiedenfeldba Departamento de Biolog´ıa Celular, Fac. Ciencias, Universidad Nacional Autónoma de México,
School of Science of Mexico, UNAM, Apartado Postal 70-359, Coyoacan 04510, Mexicob Pharmaceutical Institute, University of Bonn, An der Immenburg 4, D-53121 Bonn, Germany
Received 20 February 2003; accepted 22 September 2003
Abstract
The hypoglycemic effects of water and butanolic extracts prepared from the bark ofAcosmium panamense(Fabaceae) were studied indiabetic rats (streptozotocin (STZ)-induced). Oral application of water extracts at doses of 20 and 200 mg/kg and of butanol extracts at dosesof 20 and 100 mg/kg significantly lowered the plasma glucose levels in diabetic rats within 3 h. Glibenclamide was used as reference andshowed similar hypoglycemic effect like the extracts.
Acosmium panamense(Benth.) Yacolev, syn,SweetiapanamensisBenth., traditional names “Guayacán” and“Bálsamo amarillo” is a tree up to 40 m high, growingin form of codominant species in the tropical rain forest.The main characteristic of the tree is a tall, straight trunkpyramidal treetop with ascendant branches. The externalcortex is plain, scuamosus, and dark gray, the inner cortexis yellow and bitter (Pennington and Sarukhán, 1998).
In Oaxaca State the plant is traditionally used for thetreatment of stomach pain, respiratory problems, diarrhea,malaria, and “marsh fever”. The plant medicine is preparedas an infusion of the bark and it is taken orally 1–2 times perday. In addition,Acosmium panamenseis utilized to treatdiabetes in the village of Soteapan, Veracruz (Leonti, 2002).
Phytochemical studies of the plant yielded to the isola-tion of several quinolizidine alkaloids like acosmine andacosminine, hydroxysparteine, as well as lupinane alkaloids(Balandrin and Kinghorn, 1982; Argueta, 1994; Veitch et al.,1997; Nuzillard et al., 1999).
As the described compounds were isolated from alco-holic plant extracts, the aim of our study was to investigate
the hypoglycemic effect of hydrophilic extracts fromAcos-mium panamensein streptozotocin (STZ)-induced diabeticrats and to identify the main chemical constituents in theseextracts.
2. Materials and methods
According to our previous studies (Andrade-Cetto et al.,2000; Andrade-Cetto and Wiedenfeld, 2001) and related def-initions (Holmsted, 1991), we performed an ethnopharma-cological study focuses on ethnobotanical, phytochemical,and pharmacological methodologies.
2.1. Ethnobotany
Ethnobotanical studies were conducted during severalvisits to the community of San Felipe Usila, Oaxaca,in the period 1997–1999. We followed the method al-ready described (Andrade-Cetto, 1999; Andrade-Cetto andWiedenfeld, 2001). Diabetic people were identified by thelocal health services and local healers. All ethnobotanicaldata were collected through structured and unstructuredinterviews with the traditional healers and the diabetic peo-ple, respectively. The data were referred to plant samples(mini-herbarium) collected at its natural habitats, and thenstored as herbarium vouchers for exact identification.
218 A. Andrade-Cetto, H. Wiedenfeld / Journal of Ethnopharmacology 90 (2004) 217–220
2.2. Materials
With the help of traditional healers and diabetic people,samples ofAcosmium panamensewere collected in San Fe-lipe Usila, Oaxaca, Mexico. Their identity was confirmedand voucher specimen (IMSS14649) was deposited at theIMSS herbarium in Mexico City.
2.3. Preparation of the extracts and isolationof compounds
Plant extracts were prepared from bark samples (300 g)as already described (Andrade-Cetto et al., 2000), re-sulting in 36 g of aqueous extract (WE) and 3.12 g ofbutanolic extract (BE). The latter was used for the phyto-chemical identification of the main components. The BEwas applied on a 100 cm× 2 cm Polygoprep 60–30 C18(Macherey & Nagel, Düren, Germany) flash-column andeluted with H2O/MeOH/AcCN 80:10:10, 4 ml/min (10 mlfractions; monitored by UV-detection and controlled byHPLC).
2.4. Animals
Male Wistar rats were used; 8 weeks old (weighting200–250 g) obtained from the Bioterium of the ScienceSchool, UNAM, and acclimatized with free access to foodand water for at least 1 week in an air conditioned room(25◦C with 55% humidity) under a 12-h light:12-h darkcycle prior to the experiments.
2.5. Induction of experimental diabetes
Diabetes was induced by a single intraperitoneal injectionof a freshly prepared streptozotocin solution (Sigma, no.242-646-8) (50 mg/kg in acetate buffer 0.1 M, pH 4.5) toovernight-fasted rats. Control rats received only the buffer.
Diabetes was identified by polydipsia, polyuria and bymeasuring non-fasting plasma glucose levels 48 h after in-jection of STZ. Animals which did not develop more than250 mg/dl glucose levels were rejected.
2.6. Experimental groups
The diabetic animals were classified into seven groups(1–7) each of them with 11 rats. Group 1 as the controlreceived 1.5 ml of physiological NaCl-solution (vehicle),the rats of group 2 were given the standard oral hypo-glycemic agent glibenclamide (3 mg/kg bodyweight (bw))in the same vehicle, while groups 3 and 4 received WE(20 mg/kg bw) and WE (200 mg/kg bw), groups 5 and 6 re-ceived BE (20 mg/kg bw) and BE (100 mg/kg bw), respec-tively. Group 7 received a mixture of the isolated compounds(M) 20 mg/kg. The extracts were redissolved in 1.5 ml ofphysiological NaCl-solution and administered orally by acanule.
2.7. Collection of blood and determination of blood glucose
Blood samples were taken from the tail vein before oraladministration of the extracts or the vehicle and at times60, 120, and 180 min thereafter. Thirty-two microliters ofblood were used for each assay; the glucose concentrationwas measured in plasma serum with a Reflotron equipmentand confirmed by a Accutrend GCequipment (Roche).
2.8. Statistical analysis
The data were statistically analyzed by unpairedt-test.The plasma glucose levels were expressed as the mean(S.E.M.).
3. Results
3.1. Ethnobotany
The results of our field study in San Felipe Usila con-firmed thatAcosmium panamenseis mainly used as a hypo-glycemic agent against diabetes type II. The tree is locallynamed by his Spanish name “Guayacán” or by his Chinantecname Amac nain. In general the people drink the infusion ofthe bark after boiling 20–30 g in 1 l water. This tea is takenorally during the day as “agua de uso”. Those results con-firm the previously reported use ofAcosmium panamensefor the treatment of diabetes type II.
3.2. Compounds
Besides caffeic acid we found three pyrones. The struc-tures of the isolated compounds1, 2, and3 (Fig. 1), weredetermined by GC-mass and by homo- and hetero-nuclear2D-NMR correlated spectroscopy. Besides the alreadyknown 1; desmethylyangonine (= 6[(E)-2-(4-hydroxy-phenyl)vinyl]-4-methoxy-2H-pyron-2-one) we isolated asnew compounds its;�-d-O-glucoside2, and its;�-d-O-di(1-6)glucoside3. (Wiedenfled and Andrade-Cetto, 2003).
3.3. Activity in diabetic rats
STZ administration at a dosage of 50 mg/kg bw to normalrats significantly elevated the blood glucose levels comparedwith rats injected citrate buffer alone as reported for albinorats (El-Fiky et al., 1996).
In our diabetic rats, the extracts as well as the isolatedcompounds both showed significant hypoglycemic effects(Table 1andFig. 2).
The water extract at doses of 20 mg/kg bw showed activityat 180 min, with a significant reduction (P < 0.01) of plasmaglucose levels. The water extract at a dosage of 200 mg/kgbw showed the same activity at 180 min, but the significancewas higher (P < 0.001). The maximum effect of both waterextracts was observed after 180 min of treatment.
A. Andrade-Cetto, H. Wiedenfeld / Journal of Ethnopharmacology 90 (2004) 217–220 219
1 : R = OH
2 : R =
3 : R =
O
HOHO
OH
CH2OH
O
O O
O
CH3
R
O
HOHO
OH
CH2OH
O
CH2
OOHHO
HO
O
2
3
4
5
6
7
81´
2´
3´
4´
5´
6´
Fig. 1. Structures of isolated compounds 1 to 3.
Table 1Effect of oral administration of aqueous and butanolic extracts of Acosmium panamensebark on plasma glucose concentration in diabetic rats
The number of rats was 11 in all cases. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001 as compared with control time intervals.
Fig. 2. Effect of oral administration of water and butanolic extract of the bark of Acosmium panamensein diabetic rats. ∗P < 0.05, ∗∗P < 0.01, and∗∗∗P < 0.001 as compared with control time intervals.
220 A. Andrade-Cetto, H. Wiedenfeld / Journal of Ethnopharmacology 90 (2004) 217–220
The butanolic extract led to a significant decrease inplasma glucose level compared with the control, at doses of20 mg/kg bw. The effect was significant after 120 min withP < 0.01 ongoing with P < 0.001 at 180 min. At dosesof 100 mg/kg bw the activity was significant from 120 to180 min with P < 0.01 and P < 0.001, respectively. Themaximum activity was observed after 180 min comparableto the water extract.
The mixture of 2 and 3 showed a similar activity withP < 0.01 at 120 min ongoing to 180 min. The gliben-clamide group (3 mg/kg) produced a significant decreasewith P < 0.05 at 120 min ongoing to P < 0.01 at180 min.
Those results indicate that there is no significant differ-ence between the tested plant preparations in comparison toglibenclamide (standard hypoglycemic drug) and a mixtureof the isolated compounds 2 and 3.
4. Discussion
Our ethnopharmacological studies as well as our experi-mental pharmacological data confirm that Acosmium pana-menseis traditionally used in San Felipe Usila as an infu-sion of the bark for the treatment of diabetes type II andthere exist a clear hypoglycaemic activity in our animalstudies.
The diabetes induction by STZ and the use of gliben-clamide in this animal model were discussed previously(Andrade-Cetto et al., 2000; Andrade-Cetto and Wiedenfeld,2001).
Both, water and butanolic extracts of Acosmium pana-mensebark produce a hypoglycemic effect in rats. The wa-ter extracts show a significant activity after 180 min; thehigher dosage shows a higher activity (P < 0.001) com-pared with the lower one (P < 0.01). The butanolic ex-tracts lead to a similar activity after 120 min up to 180 minat P < 0.001. A similar effect can be observed by giv-ing a mixture of isolated compounds. Those data lead tothe assumption that the mixture of compounds 2 and 3witch are the main constituents in the water as well as inthe butanolic extract could be responsible for the measuredactivity.
Further studies will be done to determine the kind of ac-tion of the whole extract as well as for the isolated com-pounds.
Acknowledgements
We are thankful to the people of San Felipe Usila for hisgreat help concerning the ethnobotanical and ethnopharma-cological aspects and to M.V.Z. Mario Soriano-Bautista forhousing the animals.
References
Argueta, V. A., (Coord.). 1994. Atlas of the Traditional Mexican MedicinalPlants, II. National Indigenous Institute, Mexico, p. 713.
Andrade-Cetto, A. 1999. Ethnopharmacological study of Cecropia obtusi-folia Schlechtendal & Cham and Cecropia obtusifoliaBertol. Doctoralthesis, Science School. National University of Mexico, p. 97.
Andrade-Cetto, A., Wiedenfeld, H., Revilla, Ma.C., Islas, S., 2000. Hy-poglycemic effect of Equisetum myriochaetum aerial parts on strepto-zotocin diabetic rats. Journal of Ethnopharmacology. 72, 129–133.
Andrade-Cetto, A., Wiedenfeld, H., 2001. Hypoglycemic effect of Ce-cropia obtusifoliaon streptozotocin diabetic rats. Journal of Ethnophar-macology. 78, 145–149.
Balandrin, M.F., Kinghorn, A.D., 1982. (-)-(�-Hydroxysparteine, a newnatural product from Acosmium panamense. Heterocycles 19, 1931–1934.
Holmsted, B., 1991. Historical perspective and future of Ethnopharma-cology. Journal of Ethnopharmacology. 1, 7–24.
El-Fiky, F.K., Abou-Karam, M.A., Afify, A., 1996. Effect of Luffa ae-gyptiaca(seeds) and Carisa edulis(leaves) extracts on blood glucoselevel of normal and streptozotocin diabetic rats. Journal of Ethnophar-macology 60, 43–47.
Leonti M., 2002. Moko / La Rosa Negra, Ethnobotany of the PopolucaVeracruz, México. Dissertation Doctor of Natural Sciences, SwissFederal Institute of Technology (ETH) Zurich.
Nuzillard, J.M., Conolly, J.D., Delande, C.R.B., Zeches-Hanrot, M.,Men-Olivier, L., 1999. Computer-assisted structural elucidation. Al-kaloids with a novel Diaza-adamantane skeleton from the seeds ofAcosmium panamense(Fabaceae). Tetrahedron 55, 11511–11518.
Pennington T. and J. Sarukhán. 1998. Mexican Tropical Trees.UNAM-FCE, Mexico, 244 p.
Wiedenfled H., and A. Andrade-Cetto. 2003. Pyrone Glycosides fromAcosmium panamense(Benth.) Yacovlev. Z Naturforsch. 58, 637–639.
Journal of Ethnopharmacology 90 (2004) 221–227
Antitrypanosomal and antiplasmodial activity ofmedicinal plants from Cote d’Ivoire
K. Kamanzi Atindehoua,b, C. Schmidc, R. Brunc,∗, M.W. Konéa,b, D. Traorea
a Laboratoire de botanique, UFR Biosciences, Université de Cocody, 22 BP 582 Abidjan, Ivory Coastb Centre Suisse de Recherches Scientifiques en Cˆote d’Ivoire, 01 BP 1303 Abidjan, Ivory Coast
c Medical Parasitology and Infection Biology, Swiss Tropical Institute, CH-4002, Basel, Switzerland
The use of traditional medicinal plant preparations istransmitted orally from one generation to another, a proce-dure that involves the risk of loss of essential information.To assure the continuity of this precious cultural heritage,an increasing number of ethnobotanic inventories have beenestablished. For Cote d’Ivoire, we can refer to the workof Adjanohoun and Aké Assi (1970, 1979), Bouquet andDebray (1974), Vangah-Manda (1986), Zirihi (1991), Weiss(1997), Tra-Bi (1997)andKoné (1998).
In Cote d’Ivoire, as in most other Third World coun-tries, the use of medicinal plants is widespread and thereis a real hope of finding an alternative to synthetic drugsin traditional medicine. However, there is still reluc-tance to adopt traditional medicinal preparations basedon plants. One alleged reason is that their properties areoften not known well enough. Exploitation of traditionalAfrican medicine should be based on a better knowledge
of the chemical and biological properties of the plantsused.
A project was initiated with the aim of (i) identifyingplants and the preparations most frequently used in the tra-ditional medicine in Cote d’Ivoire, and (ii) studying thepharmacological properties of such plants. Our attentionwas drawn particularly to the plants used in the treatmentof infectious diseases to which the population is exposed.Those include bacterial and fungal infections, parasitic in-fections such as malaria and sleeping sickness, and varioushelminthiases. Plants were selected which had indicationsfor various infectious diseases (Bouquet and Debray, 1974;Vangah-Manda, 1986; Bizimana, 1994; Lejoly et al., 1994;Neuwinger, 1996; Weiss, 1997; Tra-Bi, 1997, and Koné,1998) and were collected by us. The plants were initiallytested for activities against bacteria and helminthes, andpromising results were obtained (Koné, 1998; Diehl, 1998;Kamanzi Atindehou et al., 2002).
The objective of this study was to investigate medicinalplants from Cote d’Ivoire for their potential for the treat-ment of sleeping sickness, a disease with a fatal outcomeif untreated, and which still affects hundreds of thousandsof people per year (WHO, 1998). The causative agents are
222 K. Kamanzi Atindehou et al. / Journal of Ethnopharmacology 90 (2004) 221–227
Trypanosoma brucei gambiensein West and Central AfricaandTrypanosoma brucei rhodesiensein East Africa. Theseprotozoan parasites are transmitted by the bite of the tsetsefly. The rural population of tsetse-infested areas is poor,and treatment with pentamidine or melarsoprol is expensive,long lasting and complicated. For the affected population itis of importance to exploit the available plants for the devel-opment of new efficient medicines (remedies) that are moreappropriate and affordable than the few existing drugs. Be-cause malaria is another protozoan disease causing severehealth problems in sub-Saharan Africa, we also decided totest the extracts againstPlasmodium falciparumcausing thesevere form of malaria, malaria tropica.
2. Methodology
2.1. Plant material
The plants were collected in Cote d’Ivoire between1996 and 1997 and identified by Tere Henri, a botanist ofthe Centre Suisse de Recherches Scientifiques (CSRS) inAbidjan. Voucher specimens were deposited at the botan-ical laboratory of the CSRS. The plant material (leaves,fruits, seeds, roots and stem) were dried and powdered. Anamount of 10–25 g of the powder were extracted with 100or 250 ml 90% ethanol, respectively. The extraction wasdone at room temperature under constant shaking for 12 h.
Table 1List of plant species (families) and organs tested
Plant species and family name Parts investigated
Acacia polyacanthaWild. SubspCampylacantha(Hochst. ex A. Rich.) Brenan (Mimosaceae) l.Acanthospermum hispidumDC. (Asteraceae) l.Albizia lebbeckL. (Mimosaceae) r. & st. b.Alchornea cordifolia(Schum. & Thonn.) Müll. Arg. (Euphorbiaceae.) r. & st. b.Alstonia booneiDe Wild. (Apocynaceae) st. b.Alternanthera pungensKunt in H. B. & K. (Amaranthaceae) pl.Ampelocissus africana(Lour.) Merril (Vitaceae) r.Anchomanes difformis(Blume) Engl. (Araceae) l.Andira inermis(Wright) DC. (Fabaceae) l.Anthocleista djalonensisA. Chevalier (Loganiaceae) st. b.Apodostigma pallens(Planch. Ex Oliv.) R. Wilzek (Hipocrateaceae) l. & st. b.Asparagus africanusLam. (Asparagaceae) l. & st. b.Baissea multifloraA. DC. (Apocynaceae) r. b.Baphia bancoensisAubrev. (Fabaceae) l. & st. b.Bombax buonopozenseBeauv. (Bombacaceae) st. b.Bridelia grandisPierre ex Hutch. (Euphorbiaceae) r. & st. b.Bussea occidentalisHutch (Caesalpiniaceae) st. b.Canarium schweinfurthiiEngl. (Burseraceae) r. b.Cassia alataL. (Caesalpiniaceae) l. & r.Cissus populneaGuill. & Perr. (Vitaceae) r.Cnestis ferrugineaDC. (Connaraceae) l.Cochlospermum planchoniHook. f. ex Planch. (Cochlospermaceae) r.Combretum molleR. Br. ex G. Don. (Combretaceae) r. & st. b.Combretum racemosumBeauv. (Combretaceae) l. & r. b.Combretum smeathmanniiG. Don. (Combretaceae) l.Costus lucanuzianusJ. Braun et Schum. (Zingiberraceae) rhiz.Cussonia arboreaHochst. ex A. Rich. (Araliaceae) l.
After filtration, the alcoholic solutions were evaporated andstill liquid residues were then lyophilised and used for thebiological assays.
2.2. In vitro determination of antitrypanosomal activity
The assays were performed according to the proceduresdescribed byFreiburghaus et al. (1996). The extracts weredissolved in 10% DMSO and working stock solutions of1 mg/ml in serum containing culture medium according toBaltz et al. (1985)were prepared. 100�l of the diluted ex-tracts were pipetted in duplicate into the first row of a 96-wellmicrotiter plate (Costar, USA). With complete culturemedium, three-fold serial dilutions were prepared. After theaddition of Trypanosoma brucei rhodesiensebloodstreamform trypanosomes from axenic culture, the concentrationsof the extracts ranged from 500 to 0.07�g/ml. The totalnumber of trypanosomes in each well was 2× 102/100�l.The plate was then incubated for 72 h in a humidified at-mosphere at 37◦C in 5% CO2. Two hours before the end ofthe incubation 10�l of Alamar Blue® solution were added.Fluorescence was measured after 2 h of incubation with thedye Alamar Blue® in a fluorescence plate reader at 530 nmexcitation and 590 nm emission wavelength (Cytofluor2300, Millipore, Bedford, MA) (Räz et al., 1997). The IC50values were calculated by linear interpolation selecting val-ues above and below the 50% mark according toHills et al.(1986). Each assay was done once in triplicate.
K. Kamanzi Atindehou et al. / Journal of Ethnopharmacology 90 (2004) 221–227 223
Table 1 (Continued)
Plant species and family name Parts investigated
Cyathula prostrata(L.) Bl. (Amaranthaceae) pl.Dacryodes klaineana(Pierre) H. J. Lam. (Mimosaceae) st. b.Dichrostachys cinerea(L.) Wight & Arn. (Mimosaceae) l.Dioscorea minutifloraEngl. (Dioscoreaceae) tuber. & l.Distemonanthus benthamianusBaill. (Caesalpiniaceae) r. & st. b.Enantia polycarpaEngl. & Diels (Annonaceae) st. b.Entada abyssinicaSteud. ex Rich. (Mimosaceae) r. b.Entada africanaGuill. & Perr. (Mimosaceae) st. b.Erythrina senegalensisDC. (Fabaceae) st. b.Erythrococca anomala(Juss. ex Poir.) Prain (Euphorbiaceae) l.Erythrophleum ivorenseA. Chev. (Caesalpiniaceae) r. b.Evolvulus alsinoidesL. (Convolvulaceae) pl.Ficus dekdekena(Miq.) A. Rich. (Moracaceae) l.Ficus mucusoWelw. ex Ficalho (Moracaceae) l. & st. b.Ficus surForsk. (Moracaceae) y. l.Ficus vallis-choudaeDel. (Moraceae) st. b.Harrisonia abyssinicaOliver (Simaroubaceae) l.Harungana madagascariensisLam ex Poir. (Clusaceae) l., r.& st. bHibiscus asperHook. f. (Malvaceae) pl.Hymenocardia acidaTul (Hymenocardiaceae) r. & st. b.Irvingia gabonensisBaill. (Irvingiaceae) st. b.Keetia hispida(Benth.) Bridson (Rubiaceae) l.Khaya ivorensisA. Chev. (Meliaceae) st. b.Kigelia africana (Lam.) Benth. (Bignoniaceae) r. b.Landolphia owariensis P. Beauv. (Apocynaceae) l.Lannea acidaA. Rich. (Anacardiaceae) r. & st. b.Lannea welwitschiiEngl. (Anacardiaceae) st. bLeea guineensisRoyen ex L. (Leeaceae) l., r. & st. b.Mallotus oppositifoliusMuell. Arg. (Euphorbiaceae) l.Mansonia altissimaA. Chevalier (Sterculiaceae) st. b.Maranthes floribunda(Bak.) White (Lam.) Exell (Chrysobalanaceae) st. b.Maytenus senegalensis(Lam.) Exell (Celastraceae) l.Microdesmis keayanaJ. Leonard (Pandaceae) l.Millettia zechianaHarms (Fabaceae) st. b.Morinda morindoides(Bak.) Milne-Redh. (Rubiaceae) l.Olax subscorpioideaOliv. (Olacaceae) r. b.Opilia amanthaceaRoxb. (Opiliaceae) l.Pachypodanthium staudtiiEngl. & Diels (Annonaceae) st. b.Parinari excelsaSabine (Chrysobalanaceae) st. b.Paullinia pinnataL. (Sapindaceae) l.Pentaclethra macrophyllaBenth. (Mimosaceae) r. & st. b.Pentadesma butyraceaSabine (Clusiaceae) r. & st. b.Pothomorphe umbellata(L.) Miq. (Piperaceae l.Premna lucensA. Chev. (Verbenaceae) l.Saba senegalensis(A. DC.) Pichon (Apocynaceae) r. b.Sansevieria forskaliana(Schult. f.) Hepper & Wood. (Agavaceae) rhiz.Smilax kraussianaMeisn. (Smilacaceae) rhiz.Strychnos innocuaDel. (Loganiaceae) l.Stylosanthes erectaP. Beauv. (Fabaceae) pl.Terminalia schimperianaHochst. (Combretaceae) y. l.Tetrorchidium didymostemon(Baill.) ex Hoffm. (Euphorbiaceae) l.& r. b.Tiliacora dinklageiEngl. (Menispermaceae) l.Treculia africana Decne(Moraceae) r. & st. b.Trema guineensis(Schumach. & Thonn.) Ficalho (Ulmaceae) st. b.Trichilia emeticaVahl. (Meliaceae) st. b.Trichilia Monadelpha(Thonn.) J. J. De Wilde (Meliaceae) st. b.Uapaca guineensisMuell. Arg. (Euphorbiaceae) r. b.Uapaca togoensisPax. (Euphorbiaceae) l.Waltheria lanceolataR. Br. ex Mast. (Steculiaceae) l. & r. b.Ximenia americanaL. (Olacaceae) r. b.Zanthoxylum gilletii(De Wild) Waterman (Rutaceae) st. b.
224 K. Kamanzi Atindehou et al. / Journal of Ethnopharmacology 90 (2004) 221–227
2.3. In vitro determination of antiplasmodial activity
Antiplasmodial activity was determined using thePlas-modium falciparumstrain K1 (resistant to chloroquine andpyrimethamine). A modification of the [3H]-hypoxanthineincorporation assay was used (Matile and Pink, 1990).Briefly, infected human red blood cells were exposed toserial drug dilutions in microtiter plates for 72 h. Via-bility was assessed by measuring the incorporation of
Table 2Antitrypanosomal and antiplasmodial activity of selected crude plant extracts
Plant species and parts investigated Trypanosoma bruceirhodesienseIC50
nd: not determined.∗ Selectivity index: IC50 L-6 cells/IC50 for Trypanosoma brucei rhodesiense.
[3H]-hypoxanthine during the final 24 h of incubation byliquid scintillation counting. Counts were expressed as per-centage of the control and presented as sigmoidal inhibitioncurves. IC50 values were calculated by linear interpolationselecting values above and below the 50% mark accordingto Hills et al. (1986). Each assay was done once in du-plicate. Only the extracts which resulted in an IC50 valuefor Trypanosoma brucei rhodesiensebelow 25�g/ml weretested for antiplasmodial activity.
K. Kamanzi Atindehou et al. / Journal of Ethnopharmacology 90 (2004) 221–227 225
2.4. Cytotoxicity testing and calculation of the selectivityindex (SI)
The determination of the cytotoxicity was performed withL-6 rat skeletal myoblast cells according to the proceduredescribed byKaminsky et al. (1996). The mammalian cellswere incubated in the presence of various dilutions of ex-tracts. Briefly, L-6 cells were seeded in 96-well microtiterplates at a density of 105 ml−1 in MEM supplemented with10% heat inactivated foetal bovine serum. A three-fold se-rial dilution ranging from 500 to 0.07�g/ml of crude extractin test medium was added. The plates were incubated as de-scribed for the antitrypanosomal assay. After 70 h 10�l ofAlamar Blue® solution were added to each well of the plate.After a further incubation of 2 h, the plates were read in afluorescence reader and the IC50 values calculated.
The selectivity index was determined by dividing theIC50 value for the L-6 cells by the IC50 value for the try-panosomes.
3. Results
Table 1shows the plant species and the parts of the plants,which were used for extract preparation. The IC50 valuesand the selectivity indices (SI) of the most active extractsare presented inTable 2.
Of the 101 tested extracts, 56 were considered inactiveagainstTrypanosoma brucei rhodesiensewith IC50 values>25�g/ml. Thirty-seven showed a weak antitrypanosomalactivity (IC50 values between 25 and 8.1�g/ml) and eightshowed a good activity with IC50 values smaller or equalto 8�g/ml. Three extracts were of particular interest: thoseof stem and leaves ofPothomorphe umbellata(Piperaceae)(IC50 = 2�g/ml and SI= 30.6), that of stembark ofEnantiapolycarpa(Annonaceae) (IC50 = 0.5�g/ml and SI= 636)and that of the rootbark ofTrichilia emetica(Meliaceae)(IC50 = 0.04�g/ml and SI= 209).
Seven of the extracts exhibited an antiplasmodial ac-tivity against K1 strain ofPlasmodium falciparumwithIC50 values below 4�g/ml: Enantia polycarpa stembark (0.126�g/ml), Erythrina senegalensisstem bark(1.82�g/ml), Terminalia schimperiana young leaves(2.37�g/ml), Morinda morindoidesleaves (3.54�g/ml),Trichilia monadelphastem bark (3.61�g/ml), Pothomor-phe umbellatastem and leaves (3.74�g/ml) andTrichiliaemeticaroot bark (3.91�g/ml). All other extracts had IC50values >5�g/ml which was the highest concentration tested.
4. Discussion and conclusions
We have tested 101 crude extracts from 88 medicinalplant species from Cote d’Ivoire. For most of these plants,no specific reports of antitrypanosomal activity exist. Theyare traditionally used as anthelminthics (Aké Assi, 1992;
Bizimana, 1994; Lejoly et al., 1994), against bacteria (Weiss,1997; Koné, 1998) and some of them have antimalarial in-dications (Bouquet and Debray, 1974). This could explainthe high percentage of extracts with no antitrypanosomalactivity (55.5%). However, 36.6% showed a weak activity(IC50 values between 25 and 8.1�g/ml) and 8 had good invitro activities (IC50 ≤ 8�g/ml) againstTrypanosoma bru-cei rhodesiense. Only seven extracts exhibited good in vitroactivity againstPlasmodium falciparum.
Pothomorphe umbellata, Enantia polycarpaandTrichiliaemeticashowed a remarkable selective activity against try-panosomes. These plants are well known and widely usedas medicinal plants in Cote d’Ivoire and other parts of WestAfrica.
Leaves of the Brazilian speciesPothomorphe umbel-lata and Pothomorphe peltataare used in traditionalmedecine for the treatment of malaria. According todeFerreira-da-Cruz et al. (2000), administration ofPotho-morphe umbellataextracts suppressed parasitaemia ofPlasmodium bergheiinfected mice, except when the ad-ministration was intraperitoneal. In that studyPothomorpheumbellataextracts exhibited a good in vitro activity againstPlasmodium falciparumwith an IC50 value of 3.74�g/ml.This plant seems to have a great potential against protozoanparasites since good activities could be observed in vitroand in vivo models when tested againstTrypanosomaandvariousPlasmodiumspecies.
Chemical data of South American specimens ofPotho-morphe umbellataexist. Leave extracts of this plantcontain 4-nerolidylcatecol (Kijjoa et al., 1980) and addi-tional constituents that increase the antioxidant activityof 4-nerolidylcatecol (Desmarchelier et al., 1997). Furtherchemical and pharmacological investigations should allowthe identification of active compounds responsible of theobserved antiprotozoal activity, and the elucidation of theirmode of action.
Enantia polycarpais used in Cote d’Ivoire primarily totreat malaria. According toBouquet and Debray (1974)thejuice of the bark or the decoction is used to treat injuries,leprosy and various eye infections. The chemistry ofEnan-tia polycarpahas been extensively studied byLeboeuf andCave (1972)and byJössang et al. (1977a,b). The bark andthe leaves of the samples from Cote d’Ivoire contain manybiologically active alkaloids that are structurally closely re-lated. The majority of these alkaloids have an isoquinolicstructure, like berberine and the numerous proberberines.Buzas et al. (1959, 1965)found quinolic and isoquinolicalkaloids in the bark of four samples from Cote d’Ivoire:quinine (0.1%) and dihydroquinidine (0.8%). Quinidine, thestereoisomer of quinine is as powerful as quinine againstmalaria, which explains the wide use of the barks ofEnantiapolycarpaas an antimalarial remedy in Cote d’Ivoire.
Regarding the antitrypanosomal activity of the crude ex-tract of the stem bark ofEnantia polycarpawe found virtu-ally the same activity as shown byFreiburghaus et al. (1996)for berberine. Since the antitrypanosomal activity of our
226 K. Kamanzi Atindehou et al. / Journal of Ethnopharmacology 90 (2004) 221–227
extract and that of pure berberine are comparable, it has to beconcluded that it is not the berberine alone which accountsfor the good activity of our extract. Additional constituentsare probably involved such as the numerous proberberinesthat are contained in the plant. Moreover, the selectivity in-dex of the crude extract (SI= 616) is far better than the oneobserved byFreiburghaus et al. (1996)for berberine (SI=4.5). The stem bark extract ofEnantia polycarpawas alsoby far the most active of all extracts forPlasmodium falci-parum. It is interesting to note that another species of thesame genera,Enantia chloranthaOliv. showed in vivo activ-ity againstPlasmodium yoelii(Agbaje and Onabanjo, 1991).
Trichilia emetica is known as a medicinal plant that isused in various ways in West African countries. Accord-ing to Nakatani and Nakanishi (1993), Trichilia emeticais arich source of limonoids (trichilins) and seco-limonoids withantifeedant activity. In our study theTrichilia emeticaex-tract from rootbark was highly active againstTrypanosomabrucei rhodesiensewhereas the antiplasmodial activity was100-fold lower (0.04�g/ml versus 3.91�g/ml). It can bespeculated that the remarkable selectivity for trypanosomesis due to the presence of limonoids which are contained inthat plant. Further investigations have to confirm this. Varia-tions in the chemical contents of a given plant, depending onthe parts studied, the location and the period of collection ofthe samples have to be considered. For example, the leavesof a Tanzanian sample ofTrichilia emeticadid not show anyactivity against trypanosomes (Freiburghaus et al., 1996).
Four other plants species exhibited in vitro antiplamodialactivity against K1 strain ofPlasmodium falciparum. In adecreasing order of activity, these areErythrina senegalen-sis (IC50 = 1.82�g/ml), Terminalia schimperiana(IC50 =2.37�g/ml),Morinda morindoides(IC50 = 3.54�g/ml) andTrichilia monadelpha(IC50 = 3.61�g/ml).
World wide, theErythrina species have a significant his-tory of folkloric medicinal uses and several studies havefocused on the antimicrobial agents found in these species(Mitscher et al., 1987). Antiplasmodial activities are rarelymentioned. A slight in vivo antiplasmodial activity of theaqueous extract ofErythrina senegalensiswas observedwhen tested againstPlasmodium berghei(Saidu et al.,2000). In our work, the ethanolic stem bark extract showeda good activity againstPlasmodium falciparum; thus the an-tiparasitic potential ofErythrina senegalensisis reinforced.
Alkaloid compounds of that plant may be responsiblefor its antiplasmodial activity. Like most species of thegenus,Erythrina senegalensiscontains alkaloids especiallyerysodin, glucoerysodin and hypaphorin (Wandji et al.,1995). Prenylated isoflavones with bioactive properties werealso isolated from that plant (Wandji et al., 1990, 1994,1995).
Ten flavonoids were isolated fromMorinda morindoidesand some of them are biologically active (Cimanga et al.,1995). Moreover, according toTona et al. (2001), Morindamorindoidesextracts produced an in vivo activity againstPlasmodium berghei.
This is the first report on in vitro antitrypanosomal ac-tivity of Enantia polycarpa, Trichilia emeticaand Potho-morphe umbellata. Enantia polycarpaandTrichilia emeticahave to be considered very promising plants for the useagainst malaria and sleeping sickness. Further investigationswith these two plants should be carried out and in vivoexperiments are already in progress. If the pharmacologi-cal properties of the two plants turn out to be favourable,they could become realistic alternatives to modern drugswhich are unaffordable or inaccessible to the poor Africanpopulations.
Acknowledgements
The Centre Suisse de Recherches Scientifiques in Coted’Ivoire and the Swiss Tropical Institute are gratefullyacknowledged for supporting this project.
References
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Journal of Ethnopharmacology 90 (2004) 229–232
Evaluation of hepatoprotective effect of Amalkadi Ghrita againstcarbon tetrachloride-induced hepatic damage in rats
Girish S. Achliya∗, Sudhir G. Wadodkar, Avinash K. DorleDepartment of Pharmaceutical Sciences, Nagpur University Campus, Amravati Road, Nagpur 440033, India
Liver is the most important organ concerned with thebiochemical activities in the human body. It has great ca-pacity to detoxicate toxic substances and synthesize usefulprinciples. Therefore, damage to the liver inflicted by hep-atotoxic agents is of grave consequences. There is an everincreasing need of an agent which could protect it fromsuch damage. In view of severe undesirable side effects ofsynthetic agents, there is growing focus to follow system-atic research methodology and to evaluate scientific basisfor the traditional herbal medicines which are claimed topossess hepatoprotective activity (Shahani, 1999).
Amalkadi Ghrita is one of the Panchagavya Ayurvedicformulation containingEmblica officinalis (Amla), Gly-cyrrhiza glabra (licorice), and cow’s ghee (clarified butterfat). Traditionally, AG has been claimed for its activityagainst liver disorders, and activity on CNS (Tripathi, 1994).Amla is well known for its immunomodulatory (Sai Ramet al., 2002), antioxidant (Bhattacharya et al., 2000), gastro-
protective (Al-Rehaily et al., 2002), hypolipedemic (Augustiet al., 2001), and antimutagenic activity (Sairam et al.,2002; Kaur et al., 2002). Glycyrrhiza had been reported topossess antioxidant (Biondi et al., 2003), antiulcerogenicactivities (Khayyal et al., 2001) and is also claimed to beuseful in liver disorders (Thyagarajan et al., 2002). Cow’sghee is traditionally believed to be rejuvenator, tonic, anduseful in memory loss and CNS disorders (Tripathi, 1994).However, there is lack of scientific data regarding phar-macological evaluation of AG, it was consequently con-sidered worthwhile to screen AG for its hepatoprotectiveactivity.
2. Methodology
2.1. Amalkadi Ghrita (AG)
The formulation was obtained as a gift sample for re-search from Go-Vigyan Anusandhan Kendra, Nagpur,India. The polyherbal formulation AG contains follow-ing herbs—Emblica officinalis (10 g), Glycyrrhiza glabra(10 g), and cow’s ghee (80 g). The formulation was used asreceived for the present investigation. A qualified botanistof Go-Vigyan Anusandhan Kendra, Nagpur, India, authen-
Total protein (g/dl) 6.56± 0.24 2.69± 0.71∗ 6.50 ± 0.54† 4.68 ± 0.73† 5.88 ± 0.36†
Values are expressed as mean± S.D. of six animals in each group. Statistical analysis ANOVA followed by Dunnettt-test.∗ P < 0.05 as compared with group 1.† P < 0.05 as compared with group 2.
ticated all herbal raw materials used in the preparation of aformulation of AG.
Silymarin was obtained as a gift sample from GermanRemedies Ltd., Mumbai for research.
2.2. Animals
Male albino rats weighing 150–200 g were used for thestudy. The animals were housed in clean metabolic cages andmaintained in controlled temperature (23± 2 ◦C) and lightcycle (12 h light and 12 h dark). They were fed with standardpellet diet (Goldmohor brand, Lipton India Ltd., Mumbai,India.) and water ad libitum. The protocol was approved byInstitutional Animal Ethical Committee constituted for thepurpose.
2.3. Experimental
Adult rats of either sex weighing 150–200 g were dividedinto five groups each consisting of six animals. Group 1received liquid paraffin only (3 ml/kg, s.c.) and served ascontrol. Rats of remaining four groups received suspensionof carbon tetrachloride (CCl4) in liquid praraffin (1:2, v/v,1 ml of CCl4/kg, s.c.) to induce hepatic damage 24 h beforestart of treatment. Group 3 received in addition to CCl4 sus-pension, silymarin (100 mg/kg, p.o.) daily. Groups 4 and 5received AG (100 and 300 mg/kg, p.o., respectively) orallyevery day in addition to CCl4 suspension for 8 days. Bloodwithdrawn through retroorbital plexus of rats on 8th day.Serum was separated from blood of each rat by centrifuga-tion for estimation of glutamate oxaloacetate transaminase(GOT) and glutamate pyruvate transaminate (GPT) (Reitmanand Frankel, 1957), alkaline phosphatase (ALP), acid phos-phatase (ACP) (Kind and King, 1954), bilirubin (Malloy andErelyn, 1937), and total protein.
The rats were sacrificed and liver rapidly excised imme-diately after sacrifice. Liver was fixed in formalin (10%),serially sectioned and microscopically examined after stain-ing with hematoxylin and eosin.
2.4. Statistical analysis
The data obtained were analyzed by one-way ANOVAfollowed by Dunnettt-test. The level of significance was setat P < 0.05.
3. Results
Hepatic damage induced by CCl4 caused significant risein marker enzymes SGPT, SGOT, ACP, and ALP and alsoserum bilirubin (Table 1). Oral administration of AG is seento lower significantly the levels of marker enzymes namelySGPT, SGOT, ACP, and ALP. It also lowered serum biliru-bin. The level of serum proteins was significantly (P <
0.05) increased in rats which received AG as comparedto control group. (Table 1). The effect of AG seems dosedependent. However, the protection offered by Silymarinseemed relatively greater.Fig. 1aexhibits the histologicalsection of liver of rats treated with AG. The normalcy ofhepatic cells, central vein, and portal triad can be easilyobserved.
4. Discussion and conclusion
It is well established that hepatotoxicity by CCl4 is dueto enzymatic activation to release CCl3 radical in free state,which in turn disrupts the structure and function of lipidand protein macromolecule in the membrane of the cell or-ganelles (Mujumdar et al., 1998). The increased level ofSGPT, SGOT, ACP, ALP, and bilirubin is conventional indi-cator of liver injury. In the present study, also it was seen thatadministration of CCl4 elevates the levels of serum markerenzymes SGPT, SGOT, ACP, ALP, and serum bilirubin.Level of total protein is lowered. AG- and silymarin-treatedgroups exhibited lower levels of SGPT, SGOT, ACP, ALP,and bilirubin as compared to CCl4-treated group. The treat-ment with AG also significantly elevated total protein levels.
G.S. Achliya et al. / Journal of Ethnopharmacology 90 (2004) 229–232 231
Fig. 1. (a) Section through the liver of CCl4-treated rats showing central vein (CV) and hepatocytes. Note the necrosis of hepatic cells and formationof vacuoles (arrows). A portion of the section is magnified and shown in the window to show vacuoles (arrow heads). (b) Section through the liver ofAG-treated (300 mg/kg, p.o.) rats showing the central vein (CV) and hepatocytes. The liver shows normal histological profile. Arrows indicate normalhepatocytes. A portion of the section is magnified and shown in the window to show normal hepatocytes (arrow heads).
The stabilization of serum bilirubin, SGPT, SGOT, ACP, andALP levels by AG is a clear indication of the improvementof the functional status of the liver cells. The characteristicsfeature of experimental hepatic damage observed is signif-icant decrease in protein level. The rats in a group whichreceived AG showed rectification of lowered protein levels.
These findings can be further corroborated with histopatho-logical studies. The histopathological examination clearlyreveals that the hepatic cells, central vein, and portal triadare almost normal in AG (300 mg/kg, p.o.) group in contrastto group which received CCl4. Thus, AG can be consideredto be an effective hepatoprotective herbal formulation as it
232 G.S. Achliya et al. / Journal of Ethnopharmacology 90 (2004) 229–232
ameliorates almost to normalcy the damage caused by CCl4to hepatic function.
Some of the constituents of AG are known to possesshepatoprotective activity. 18-beta-glycyrrhetinic acid pos-sess such activity (Jeong et al., 2002). Not much is knownabout the other constituents of AG but studies concerningtheir efficacy may prove fruitful. It is difficult at this stage tocomment on the rational of inclusion of such herbs togetherin single formulation for hepatic protection but the possi-bilities of protective role against radicals other than CCl3cannot be overruled. Studies on these lines are presently inprogress.
Acknowledgements
The author express their sincere thanks to Dr. DeepaliPande, M.D. Ayurveda, Go-Vigyan Anusandhan Kendra,Nagpur, India and Mr. Prafulla S. Singru, Research Scholar,Department of Pharmaceutical Sciences, Nagpur, India fortheir generous help.
References
Al-Rehaily, A.J., Al-Howiriny, T.A., Al-Sohaibani, M.O., Rafatullah, S.,2002. Gastroprotective effects of ‘Amla’Emblica officinalis on in vivotest models in rats. Phytomedicine 9, 515–522.
Augusti, K.T., Arathy, S.L., Asha, R., Ramakrishanan, J., Zaira, J., Lekha,V., Smitha, S., Vijayasree, V.M., 2001. A comparative study on thebeneficial effects of garlic (Allium sativum Linn), amla (Emblica Of-ficinalis Gaertn) and onion (Allium cepa Linn) on the hyperlipidemiainduced by butter fat and beef fat in rats. Indian Journal of Experi-mental Biology 39, 760–766.
Bhattacharya, A., Ghoshal, S., Bhattacharya, S.K., 2000. Antioxidantactivity of tannoid principles ofEmblica officinalis (amla) in chronicstress induced changes in rat brain. Indian journal of ExperimentalBiology 38, 877–880.
Biondi, D.M., Rocco, C., Ruberto, G., 2003. New dihydrostilbene deriva-tives from the leaves ofGlycyrrhiza glabra and evaluation of theirantioxidant activity. Journal of Natural Products 66, 477–480.
Jeong, H.G., You, H.J., Park, S.J., Moon, A.R., Chung, Y.C., Kang, S.K.,Chun, H.K., 2002. Hepatoprotective effects of 18-beta-glycyrrhetinicacid on carbon tetrachloride-induced liver injury: inhibition of cy-tochrome P450 2E1 expression. Pharmacological Research 46, 221–227.
Kaur, S., Arora, S., Kaur, K., Kumar, S., 2002. The in vitro antimuta-genic activity of Triphala—an Indian herbal drug. Food Chemistry andToxicology 40, 527–534.
Khayyal, M.T., el-Ghazaly, M.A., Kenawy, S.A., Seif-el-Nasr, M., Mahran,L.G., Kafafi, Y.A., Okpanyi, S.N., 2001. Antiulcerogenic effect ofsome gastrointestinally acting plant extracts and their combination.Arzneimittelforschung 51, 545–553.
Kind, P.R.N., King, A.J., 1954. Elimination of plasma phosphate bydetermination of hydrolysed phenol with aminoantipyrine. Journal ofClinical Pathology 7, 322–326.
Malloy, H.T., Erelyn, E.A., 1937. The determination of bilirubin with thephotoelectric colorimeter. Journal of Biological Chemistry 119, 481–485.
Mujumdar, A.M., Upadhye, A.S., Pradhan, A.M., 1998. Effect ofAzadirachta indica leaf extract on CCl4 induced hepatic damage inalbino rats. Indian Journal of Pharmaceutical Sciences 60, 363–367.
Reitman, S., Frankel, A.S., 1957. A colorimetric method for the determi-nation of serum glutamic oxaloacetic and glutamic pyruvic transami-nase. American Journal of Clinical Pathology 28, 53–56.
Sairam, K., Rao, Ch.V., Babu, M.D., Kumar, K.V., Agrawal, V.K., Goel,R.K., 2002. Antiulcerogenic effect of methanolic extract ofEmblicaofficinalis: an experimental study. Journal of Ethnopharmacology 82,1–9.
Sai Ram, M., Neetu, D., Yogesh, B., Anju, B., Dipti, P., Pauline, T.,Sharma, S.K., Sarada, S.K., Ilavazhagan, G., Kumar, D., Selvamurthy,W., 2002. Cyto-protective and immunomodulating properties of Amla(Emblica officinalis) on lymphocytes: an in-vitro study. Journal ofEthnopharmacology 81, 5–10.
Shahani, S., 1999. Evaluation of hepatoprotective efficacy of APCL-Apolyherbal formulation in vivo in rats. Indian Drugs 36, 628–631.
Thyagarajan, S., Jayaram, S., Gopalakrishnan, V., Hari, R., Jeyakumar,P., Sripathi, M., 2002. Herbal medicines for liver diseases in India.Journal of Gastroenterology and Hepatology 3, S370–S376.
Metastasis is the major problem of treatment failure incancer patients. It is not a passive process but involves strictup and down regulations in the expression of specific genes.Tumour cells after getting detached from the primary tumourenter in to the blood vessels or lymphatics, circulate in thebody fluids and form a new colony at a distant site (Fiddlersand Hart, 1982; Schirmacher, 1985). They also encounterwith immune system in the circulation. Any agent or immunecells itself can interfere with any of these steps and cansignificantly reduce the metastatic potential. Boosting up ofimmune system is also significant in reducing metastasis(Fresa and Murasko, 1986).
Use of immunomodulators in cancer therapy has got abiphasic effect. Many of them inhibit host defense param-eters, which are normal or activated, and some of themtend to stimulate individuals immune system (Hersh, 1982).Several herbal preparations used in indigenous systemsof medicine are known to potentiate the immune system(Davis and Kuttan, 2000). One such plant isTinosporacordifolia Miers, belonging to the family Menispermaceae.It is well documented as an immunostimulant (Sainis et al.,1997). Tinospora cordifolia has been shown to possessanti-allergic, anti-diabetic, anti-hepatotoxic, anti-pyretic andanti-inflammatory properties (Mathew and Kuttan, 1997). It
∗ Corresponding author. Fax:+91-487-307868.E-mail address: [email protected] (G. Kuttan).
is a well-known anti-oxidant as well (Goel and Premkumar,2002). Many of its activities are attributed to the polysac-charide fraction that is isolated from the dried stem of theplant (Chintalwar et al., 1999). In vitro studies using thispolysaccharide have shown that it is a specific mitogen ofB-cells and the T-cells are not affected (Sainis et al., 1997).In this study we extracted the polysaccharide from the driedstem ofTinospora cordifolia by the method of Chintalwaret al. and its anti-metastatic activity was evaluated using anin vivo mice model.
2. Materials and methods
Hydroxyproline, N-acetyl neuraminic acid (NANA),Glucosamine hydrochloride and Glucuronic acid were pur-chased from SISCO Research Laboratory, Mumbai, India.All other reagents were of analytical reagent grade.
2.1. Animals
Six to eight weeks old male C57BL/6 mice were pur-chased from National Institute of Nutrition, Hyderabad, In-dia. The animals were housed in well-ventilated cages keptin air-controlled rooms during the experiment. They werefed with normal mouse chow (Sai Durga Feeds, Banglore,India) and water ad libitum. All the animal experiments wereperformed according to the rules and regulations of AnimalEthics Committee, Government of India.
234 P.V. Leyon, G. Kuttan / Journal of Ethnopharmacology 90 (2004) 233–237
2.2. Cells
B16F-10, a highly metastatic melanoma cell line was pur-chased from National Centre for Cell Sciences, Pune, Indiaand maintained in culture using DMEM (HiMedia, Mumbai)with 10% FCS (Biological Industries, Israel) and antibiotics.
2.3. Drug administration
AuthenticatedTinospora cordifolia was obtained from theAmala Ayurvedic Centre Pharmacy, Thrissur, India and avoucher specimen is kept in the herbarium (Ref. No. ACRCB4/2001). To isolate the polysaccharide we used the proto-col of Chintalwar et al. (1999). In brief the powdered stemwas extracted using methanol by stirring overnight at roomtemperature. The precipitate was then extracted with boil-ing water and the extract was precipitated using acetone.The proteins from this precipitate were removed by treat-ment with TCA. The sample was then dialysed against dis-tilled water and concentrated by lyophilisation. The yieldwas about 0.01%. It was tested for the presence of polysac-charide by Molisch reagent and for uronic acid (Chintalwaret al., 1999). This drug (Pl) was given to the animals at aconcentration of 0.5 mg per dose per animal.
2.4. Determination of the effect of Tinospora cordifoliaadministration on the metastatic potential of B16F-10 cells
Highly metastatic B16F-10 melanoma cells (106 cells/0.05 ml PBS) was injected to each animal via lateral tail vein(day 0). The animals were divided into two groups, compris-ing 14 animals in each group. Group I was receiving onlythe vehicle. In group II drugs were started simultaneouslywith the induction of metastasis and continued for 10 days.
Eight animals from each group were sacrificed after 21days of tumour induction, the lungs were dissected outand blood was collected by heart puncture. The lungs werewashed in PBS and used for morphological examinationusing a dissection microscope, and the blackish metastaticcolonies were counted. Then the tissue was subjected tothe estimation of collagen hydroxyproline (Bergman andLoxley, 1970), hexosamines (Elson and Morgan, 1933) anduronic acid (Bitter and Muir, 1962).
The serum sialic acid levels of all the animals were de-termined according to the procedure ofSkoza and Mohos(1976). The serum�-glutamyltranspeptidase (�-GT) (Szasz,1976) levels were also assayed and expressed as units perlitre.
Rest of six animals in all the groups was observed fortheir survival.
2.5. Histopathology of lungs
Lungs of both treated as well as control metastatictumour-bearing animals were fixed in 10% formalin for 24 h,washed and dehydrated using alcohol series and embedded
in paraffin. Sections (4�m) were stained with hematoxylineand eosin (H&E) mounted in DPX and examined under amicroscope (200×).
2.6. Statistical analyses of the data
All the results are expressed as mean±standard deviation.Student’s ‘t’ test was used to find out the level of co-relation.
3. Results
3.1. Inhibition in lung colonisation of B16F-10 melanomaand increased survival by the treatment
Simultaneous administration of the isolated polysac-charide fraction could reduce the number of pulmonarymetastatic colonies of B16F-10 melanoma cells (Table 1).Vehicle-treated control animals had massive growth of tu-mour and was given an arbitrary-maximum countable num-ber of 250 (Hill et al., 1994), which was reduced to 72± 10by the treatment (Fig. 1). The survival time of this groupwas more than double that of control animals (Table 1).
3.2. Effect of treatment on the biochemical parameters ofmetastases bearing animals
3.2.1. Lung collagen hydroxyprolineEffect of Pl treatment on the lung biochemical parameters
is shown inTable 2. The lung collagen hydroxyproline con-tent was drastically elevated (22.4±0.9�g/mg protein) in thecontrol group compared to the normal level (0.9±0.2�g/mgprotein) indicating the fibrosis of lung tissue. This elevatedlevel was reduced to 8.3±2.0�g/mg proteins by the simul-taneous treatment of Pl.
3.2.2. Lung hexosamine contentThe control animals had a high level of lung hexosamine
content (4.85 ± 0.20 mg/100 mg tissue, dry weight) as
Table 1Effect of polysaccharide fraction fromTinospora cordifolia on lung coloni-sation of B16F-10 melanoma cells and survival of the animalsa
Values are mean± S.D.a The lungs were dissected out and observed for metastases on the
22nd day after induction of B16F-10 melanoma (105 cells). Drug startedsimultaneously with tumour cell induction through the lateral tail vein(10 doses at 24 h interval, i.p).
b Increase in life span= T −C/C×100, whereT andC are the numberof days survived by the treated and control (vehicle-treated) group ofanimals, respectively.
c An arbitrary number of 250 are given for massive number of tumournodules.
∗ P < 0.001.
P.V. Leyon, G. Kuttan / Journal of Ethnopharmacology 90 (2004) 233–237 235
Fig. 1. Morphology of lung of metastasis-induced animals. (A) Normal lung; (B) control; (C) Pl-treated.
Table 2Effect of polysaccharide (Pl) administration on the lung biochemical parameters of metastases bearing animalsa
Values are mean± S.D..a After 21 days of B16F-10 melanoma cells (105 cell/50�l/PBS) implantation through the lateral tail vein, lungs were dissected out and assayed
different biochemical parameters. The vehicle-treated group is the control.∗ P < 0.001.
compared to the normal animals (0.4 ± 0.1 mg/100 mg tis-sue dry weight). Simultaneous administration of the drugcould reduce this level to 0.94± 0.02 mg/100 mg tissue dryweight.
3.2.3. Uronic acid levels of the lungAs in the case of hydroxyproline and hexosamines, uronic
acid level in the lung of control animals was (334.2 ±22.7�g/100 mg tissue wet weight) also very high as com-pared to the normal levels (32.2±2.0�g/100 mg tissue, wetweight). The Pl (94.2±17.4�g/100 mg tissue) could reducethese elevated levels when administered simultaneously withthe tumour cells (Table 2).
3.2.4. Serum sialic acid levelsEffect of drug treatment on the serum biochemical param-
eters is shown inTable 3. The normal serum sialic acid levelof C57BL/6 mice was 21.3 ± 1.5�g/ml that was elevatedto 102.2 ± 8.7�g/ml serum in the metastases bearing ani-
Table 3Effect of polysaccharide fraction fromTinospora cordifolia on serum parameters of metastasis-induced animalsa
Values are mean± S.D.a The animals were sacrificed and blood was collected on the 22nd day after induction of B16F-10 melanoma (105 cells) through the lateral tail vein.
The control group received only the vehicle. Drug started simultaneously with tumour induction (10 doses at 24 h interval, i.p).∗ P < 0.001.
mals. Simultaneous administration of Pl reduced the sialicacid levels to 40.7±7.7�g/ml serum.
3.2.5. Serum γ-glutamyltranspeptidaseThe �-GT activity in the control animals was very high
(35.3± 3.8 U/l) as compared to the�-GT level in the serumof the normal animals (8.2± 1.1 U/l). Simultaneous admin-istration of Pl reduced the elevated levels to 12.5 ± 4.0 U/l.
3.3. Histopathological analysis of lung
The hematoxylin and eosin (H&E)-stained sections oflung tissues are shown inFig. 2 (100×). In the control ani-mals massive tumour growth and fibrosis reduced the alve-olar space thereby reducing the vital capacity of the lung.Same areas were characterised by necrosis around the alve-olar passages and bronchioles. Simultaneous administrationof the compound showed a reduction in tumour mass aroundalveoli and pleura (Fig. 2a–c).
236 P.V. Leyon, G. Kuttan / Journal of Ethnopharmacology 90 (2004) 233–237
Fig. 2. Histopathology of lung of metastatic tumour-bearing animals (100×). (a) Normal lung; (b) control; (c) Pl-treated.
4. Discussion
In this study, the intra-peritoneal administration of thepolysaccharide fraction resulted in a 72% reduction in themetastatic colony formation of B16F-10 melanoma cells. Inone of our previous studies using the hydro-alcohol extract ofTinospora cordifolia, we could obtain significant inhibitionon metastasis (data not shown). Maximum inhibition wasobtained when the animals were pre-treated with the extractprior to tumour induction. The simultaneous administrationcould also reduce the tumour nodule formation by about52%.
Accumulation of extra cellular matrix (ECM) in thelung will reduce the pulmonary function. Estimationof uronic acids and hexosamines—the building materi-als of ‘ground substance’ of ECM—shows an increasedamount in the control animal. Like-wise the estima-tion of hydroxyproline—the major building block ofcollagen—indicates the fibrous accumulation, which againwill reduce the alveolar space and vital capacity of lung.The tumour cells induce the productions of these materialsand reduced levels in the treated animals are indicative ofreduced tumour cell survival in the lung. The lung tumournodules and histopathology of lungs also co-relates withthese results.
Neoplasms often have an increased concentration of sial-glycoproteins on the tumour cell’s surface and are shed orsecreted by these cells, which increase their concentrationin the blood (Gude et al., 2001). Serum�-glutamyltranspep-tidase is also a marker of neoplastic proliferation (Preziosoet al., 1993). A reduction in the levels of these tumour mark-ers in the treated group is also indicative of a reduced tumourgrowth in that group of animals.
The long and short term in vitro experiments have shownthat the polysaccharide fraction was neither directly toxicto the tumour cells or inhibited the proliferation as well(data not shown). These results are indicative of the in-volvement of the immune system in the reduction of themetastatic potential of the drug treated animals. Since thepolysaccharide fraction is a specific mitogen of B-cells(Sainis et al., 1997) the tumour reduction obtained maybe mediated B-cell or non-specific immune cells suchas NK-cell mediated. Further studies have to be con-ducted to find out the exact mechanism of action of thecompound.
Acknowledgements
The authors would like to thank Dr. Ramadasan Kuttan,Director, Amala Cancer Research Centre, for his consistentsupport during the period of study.
References
Bergman, I., Loxley, R., 1970. The determination of hydroxyproline inurinehydrolysate. Clinical Chemica Acta 27, 347–349.
Chintalwar, G., Jain, A., Sipahimalani, A., Banerji, A., Sumariwalla,P., Ramakrishnan, R., Sainis, K., 1999. An immunologically activearabinogalactan fromTinospora cordifolia. Phytochemistry 52, 1089–1093.
Elson, L.A., Morgan, W.T., 1933. A colourimetric method for determina-tion of glucosamine. Journal of Biochemistry 27, 1824–1828.
Fiddler, I.J., Hart, I.R., 1982. Biological diversity in metastatic neoplasm:origins and implications. Science 217, 998–1001.
Fresa, K.L., Murasko, D.M., 1986. Role of natural killer cells in themechanism of the anti-tumour effect of interferon on moloney sarcomavirus-transformed cells. Cancer Research 46, 81–88.
Goel, H.C., Premkumar, I., 2002. Free radical scavenging and metalchelation byTinospora cordifolia, a possible role in radioprotection.Indian Journal of Experimental Biology 40, 727–734.
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Prezioso, J.A., Wang, N., Duty, L., Bloomer, W.D., Go relic,E., 1993. Enhancement of pulmonary metastasis formation andgamma-glutamyltranspeptidase activity in B16 melanoma induced bydifferentiation in vitro. Clinical and Experimental Metastasis 11, 263–274.
Sainis, K.B., Sumariwalla, P.F., Goel, A., Chintalwar, G.J., Sipahimalani,A.T., Banerji, A., 1997. Immunomodulatory properties of stem extractsof Tinospora cordifolia: cell targets and active principles. In: Upadhyay,
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Effects of Aloe preparation on the histamine-inducedgastric secretion in rats
W. Suvitayavata,∗, C. Sumrongkita, S.S. Thirawarapana, N. Bunyapraphatsaraba Department of Physiology, Faculty of Pharmacy, Mahidol University, 447 Sri-Ayudhaya Road, Bangkok 10400, Thailand
b Department of Pharmacognosy, Faculty of Pharmacy, Mahidol University, 447 Sri-Ayudhaya Road, Bangkok 10400, Thailand
Received 1 June 2000; received in revised form 25 September 2003; accepted 29 September 2003
Abstract
The effects of Aloe preparation containing 80% Aloe gel on the gastric acid, pepsin and mucus secretion were evaluated in histamine-inducedgastric fistula model in rats by comparison to the effects of placebo and fresh Aloe gel. Aloe preparation and placebo at a dose of 8 ml/kginhibited gastric acid but stimulated pepsin secretory rates. On the other hand, fresh Aloe gel at a dose of 6.4 ml/kg prolonged histaminestimulatory effects on the gastric acid secretion while it inhibited gastric pepsin secretion. Both Aloe preparation and placebo increased solublemucus secretory rate in a dose-dependent manner whereas fresh Aloe gel had no effect. The Aloe preparation and placebo at a dose of 8 ml/kgincreased the gastric visible mucus content while fresh Aloe gel slightly increased the visible mucus content.
Aloe vera gel have been demonstrated to protect and/orcure gastric ulcer in human (Blitz et al., 1963; Bovik, 1966;Gjerstad and Riner, 1968) and rats (Mahattanadul, 1996;Galal et al., 1975; Kandil and Gobran, 1982; La-ongphanich,1987; Robert et al., 1979; Maze et al., 1997; Suvitayavatet al., 1997). This antiulcer activity has been proposedto be due to anti-inflammatory (Robert et al., 1979), cy-toprotective (Mahattanadul, 1996), healing (Robert et al.,1979; Teradaira et al., 1993) and mucus stimulatory effects(Visuthipanich, 1988). Although several pharmacologi-cally active compounds were isolated fromAloe vera andclaimed to be the healing principles for gastric ulceration,Koo (1994) found that product containing Aloe gel didnot prevent cold-resistant or ethanol-induced gastric lesionformation in rats.
Regulation of gastric secretion could be another antiulcermechanism ofAloe vera gel. Faitel’berg and Stambol’Skii(1969) showed that the Aloe extract increased gastricjuice production in esophagotomized dogs. On the otherhand, Hirata and Suga (1977)found that Aloenin andmagnesium lactate from theAloe arborescens leaf juiceinhibited gastric juice secretion and histamine synthesis.Aloctin A, a glycoprotein inAloe arborescens leaf gel,was also shown to inhibit acid, pepsin output and decreasegastric secretory volume in pylorus-ligated rats (Saitoet al., 1989). Aloctin A also inhibited the developmentof Shay ulcer, indomethacin-induced gastric lesions andwater-immersion stress-induced lesion in pylorus-ligatedrats (Saito et al., 1989; Saito, 1993). However, Kandiland Gobran (1982)reported thatAloe vera gel possesseda prophylactic and curative effect on gastric lesion with-out interfering with gastric pH. The controversy of theprevious reports may be due to various factors such asthe preparing method of Aloe gel, the dose and routeof administration, species of animal and the source ofAloe vera plant. In addition, the stability of the active
240 W. Suvitayavat et al. / Journal of Ethnopharmacology 90 (2004) 239–247
substances may be another factor for this controversialresult.
In order to protect the degradation of the active ingredientsin the Aloe gel, a special Aloe preparation was formulated.This Aloe preparation has been reported to inhibit sponta-neous acid secretion in rats and secretagogue-induced gastricacid secretion in isolated mouse whole stomach, and pro-tect gastric mucosa from HCl-induced gastric lesion in rats(Suvitayavat et al., 1997). However, the effects of this prepa-ration on gastric pepsin and mucus secretion has not beenexamined. In addition, its gastric inhibitory effect should befurther investigated in other models. Thus, this experimentwas set up to study the effects of this Aloe preparation onthe gastric secretions in histamine-stimulated gastric fistulamodel in rats.
2. Materials and methods
2.1. Aloe preparations
2.1.1. Fresh Aloe gelAloe vera (Linn.) Burm. f. is a short-stemmed succulent
herb. The succulent leaves are crowded on the top of theirstems, spreading, grayish green and glaucous; spotted whenyoung, tapering gradually to the point tip, having edgesspiny and bitter latex inside. Flowers borne on the upperpart of the a slender stalk, 50–100 cm high. Each flowerhas six protruding stamens and 3-celled ovary with longstyle (Fransworth and Bunyaprapatsara, 1992). Aloe veragrown in Prachuabkirikun province, Thailand was usedto prepare fresh Aloe gel and Aloe preparation. Aloe gelwas separated from the leaves and blended in food pro-cessor at low speed and squeezed through muslin cloth.One gram of gel yielded approximately 0.9 ml of Aloe geljuice. Fresh Aloe gel was freshly prepared on the day ofexperiment.
2.1.2. Aloe preparationAloe vera L. leaves from the same location as those for
preparing Aloe gel were used to prepare Aloe preparation.Aloe preparation composed of 80% Aloe juice with 0.1%benzoic acid as preservative. Placebo was formulated bythe same procedures as Aloe preparation but Aloe gel wasreplaced with water.
2.2. Animal preparation
Male Wistar rats weighing between 180 and 220 g wereobtained from the National Laboratory Animal Center atSalaya, Mahidol University. Rats were housed in hangingcage in the animal room at Faculty of Pharmacy, MahidolUniversity for at least 2 weeks prior to the experiment. Theanimals were fed with commercial rat diet (FE Zeullic) andtab water ad libitum. The rats were fasted for 48 h beforethe experiment.
Gastric fistula was performed as described byLimlom-wongse et al. (1976). Briefly, the fasted rat was anesthetizedwith Nembutal® (Sodium pentobarbital 50 mg/ml; Abbott,Chicago, USA) at the dose of 50 mg/kg body weight in-traperitoneally. The esophagus was ligated and the tracheawas cannulated by using polyethylene tube No. 206. Afteropening the abdomen, the pyloduodenal junction was care-fully ligated and the stomach was perforated and inserted thepolyethylene tube No. 206 gastric fistula. The instillation ofsolution and collection of sample were performed throughthe gastric fistula.
2.3. Gastric sample collection
The rats were divided into 10 groups (eight rats per group)according to the doses and types of the solution. Group1 was the normal saline control rats at a dose of 8 ml/kg.Groups 2–5 were treated with the Aloe preparation at dosesof 1, 2, 4 and 8 ml/kg, respectively. Groups 6–9 were treatedwith the placebo at doses of 1, 2, 4 and 8 ml/kg, respectively.Group 10 was the 6.4 ml/kg fresh Aloe gel treated rats. Thesample collection was taken in 1-h interval for 4 h and ex-pressed as h1, h2, h3, and h4. After finishing the operation,normal saline (pH 5) at a dose of 8 ml/kg was instilled intothe stomach and the fistula was clamped to allow the expo-sure of the instillate to the gastric mucosa. One hour later,the sample was collected into a tared-collecting tube, sampleh1, which was referred as the basal secretion. Then normalsaline or test solution was instilled into the stomach and his-tamine (Sigma Chemical Co., St Louis, USA) at a dose of10 mg/kg was injected intramuscularly to stimulate the gas-tric secretion. In the case of test solutions at a dose of lowerthan 8 ml/kg, normal saline (pH 5) was added up to 8 ml/kgin order to get the same stomach distention. The gastricsamples were collected at 1-h intervals for three more hours(sample h2, h3 and h4) and the same doses of test solutionswere replaced after each sample collection. At the end of theexperiment, the stomach was removed and opened throughthe lesser curvature. The opened stomach was rinsed withnormal saline pH 7.4 and dried with filter paper. Then theglandular portion was excised and determined the visiblemucus.
2.4. Gastric sample analysis
The gastric sample was weighed and centrifuged (Univer-sal Centrifuge 16A, Hettich) at 5000 rpm for 5 min to pre-cipitate solid residue. The tube with residue was weighedagain. The clear supernatant was separated and kept in icebath. The supernatant was used for determining the gastricacid, pepsin activity, soluble mucus and protein.
2.4.1. Gastric volume determinationThe difference between the weights of tube containing
gastric sample and tube with residue represented the col-lected gastric volume by assuming that the specific gravity
W. Suvitayavat et al. / Journal of Ethnopharmacology 90 (2004) 239–247 241
of gastric sample was 1.00. The gastric secretory rate wascalculated from the collected volume dividing by the weightof stomach glandular part and expressed as ml/g stomach/h.
2.4.2. Gastric acid secretory rate determinationThe collected gastric acid was examined by titration of
gastric sample with 0.01N sodium hydroxide (Carlo Erba,Milano, Italy) to the end point at pH 5.0. The gastric acidsecretory rate was calculated from the collected gastric acidafter subtracting the acidity of the test solution dividing bythe weight of stomach glandular part and expressed as�EqHCl/g stomach/h.
2.4.3. Gastric pepsin secretory rate determinationPepsin secretion was determined by measuring the
proteolytic activity of pepsin in gastric sample by usinghemoglobin (Sigma Chemical Co.) as a substrate and pepsin(480 U/mg solid; Sigma Chemical Co.) as a standard. Theactivity was determined according to the method describedby Miller et al. (1975). The pepsin activity was expressedas U/g stomach/h.
2.4.4. Gastric protein secretory rate determinationThe amount of protein in gastric sample was measured by
colorimetric method (Lowry et al., 1951) using bovine serumalbumin (Sigma Chemical Co.) as a standard. The proteinsecretory rate was determined from the amount of protein incollected gastric sample after subtracting the protein of thetest solution and expressed as mg/g stomach/h.
2.4.5. Gastric soluble mucus secretory rate determinationGastric soluble mucus was determined according to the
method described byWhiteman (1973)and by using chon-droitin sulfate (Sigma Chemical Co.) as a standard. Sincethe mucus in Aloe preparation and fresh Aloe gel can dis-solved in the gastric secretion and adhered on the mucosawith uncalculated amount, gastric soluble and visible mu-cus were determined without subtracting mucus of the testsolution. The gastric soluble mucus secretory rate was cal-culated from the amount of soluble mucus and express as�g chondroitin sulfate/g stomach/h.
2.4.6. Gastric visible mucus determinationVisible mucus on the gastric mucosal surface was deter-
mined according to the modification of the method previ-ously described byCorne et al. (1974). The Alcian blue 8GX (Sigma Chemical Co.) dissolved in MgCl2 was used asa standard. The amount of gastric visible mucus was ex-pressed as�g Alcian blue/g stomach.
2.5. Statistical analysis
One-way ANOVA was used to compare the values foreach of experimental groups. Tukey’s honestly significantdifference (HSD) test was used to differentiate the differencebetween two experimental groups. Paired samplet-test was
to compare the secretory rates at h2, h3 and h4 with thebasal secretion (h1) in each experimental group. TheP-valueof less than 0.05 (P < 0.05) was considered to statisticalsignificant difference.
3. Results
3.1. The amount of acid, pepsin, mucus and protein inAloe preparation, placebo and fresh Aloe gel
The pH of Aloe preparation, placebo and fresh Aloe gelwere 3.28± 0.01, 2.7± 0.01 and 4.85± 0.03, respectively.The concentration of HCl determined from the titration ofAloe preparation and placebo with 0.01N NaOH to pH 5.0were 39.43± 0.55�Eq/ml and 35.3± 0.75�Eq/ml, respec-tively. The amount of acid in fresh Aloe gel was too little tobe determined because its pH was almost equal to the endpoint of the titration. The volume of 0.01N NaOH used forthe titration of fresh Aloe gel to end point at pH 5.0 was lessthan 0.01 ml. All of the test solutions had no pepsin activity.The protein content in Aloe preparation, placebo and freshAloe gel were 0.54± 0.003 mg/ml, 0.31± 0.007 mg/ml and0.30± 0.005 mg/ml, respectively. The amount of mucus inAloe preparation, placebo and fresh Aloe gel were 512.4±20.0�g/ml, 517.2 ± 39.5�g/ml and 34.3 ± 0.5�g/ml, re-spectively.
3.2. Effects of Aloe preparation and placebo onhistamine-induced gastric secretion
trol group was increased after histamine injection and high-est at h2 and then gradually decreased to the basal level at h4(Table 1). The Aloe preparation and placebo-treated groupsat doses of 1, 2 and 4 ml/kg had similar gastric acid secre-tory rate profile pattern as saline control group and showedno significant difference from the control group at the cor-responding period (Table 1). In contrast, the rats treatedwith Aloe preparation and placebo at a dose of 8 ml/kg hadgastric acid secretion less than but not significant differencefrom that of control group. At h2 period, the secretory rateof 8 ml/kg Aloe preparation-treated group was significantlylower than 1, 2 and 4 ml/kg Aloe preparation-treated groupand 1, 2 ml/kg placebo-treated group whereas the secretoryrate of 8 ml/kg placebo-treated rats showed significant dif-ference from those of 1, 2 and 4 ml/kg Aloe preparation- andplacebo-treated groups (Table 1and Fig. 1). It was notedthat the secretory rate at h2 and h3 of these two groupsdid not significantly increase from the basal secretion ofeach group, while the secretory rate of placebo-treatedgroup significantly decreased at h4 (Table 1). There wasno significant difference between the gastric acid secre-tory rate of Aloe preparation and placebo at the samedoses.
242 W. Suvitayavat et al. / Journal of Ethnopharmacology 90 (2004) 239–247
Table 1Effects of Aloe preparation, placebo and fresh Aloe gel on histamine-induced gastric acid secretory rate (�Eq/g stomach/h) at h1, h2, h3 and h4
All values were expressed as mean± S.E.M.∗P < 0.05, ∗∗P < 0.01 vs. basal secretion within group.There was no significant difference between any treated groups and the control group at the same time point.aP < 0.05 vs. 8 ml/kg Aloe gel preparation-treated group at the same time point.bP < 0.05 vs. 8 ml/kg placebo-treated group at the same time point.
3.2.2. Gastric pepsin secretionIn control group, the gastric pepsin secretory rate pro-
file pattern was similar to the gastric acid secretion. Thepepsin secretion pattern of all doses of Aloe preparation- andplacebo-treated groups were similar to that of control group(Table 2). Nevertheless, the pepsin secretory rate of 2, 4and 8 ml/kg Aloe preparation-treated groups and all doses ofplacebo-treated groups at h3 were significantly higher thantheir own basal secretion. In addition, the increase in secre-tory rate of 8 ml/kg Aloe preparation-treated groups and 1,4 and 8 ml/kg placebo-treated groups at h4 were still signif-icantly different from their basal secretory rates (Table 2).
The pepsin secretory rate at h2 of doses 4 and 8 ml/kgof both Aloe preparation and placebo-treated groups weresignificantly higher than that of control group (Fig. 2).There was no significant difference of gastric pepsin secre-tory rate between the same doses of Aloe preparation- andplacebo-treated groups at the same time point. However,
0
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50
60
70
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0 1 2 3 4 5 6 7 8
Dose (ml/kg)
Aci
d (
Eq/
g st
omac
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)
ab
ab
ab
ab
ab
a
Fig. 1. Relationship between the doses of Aloe preparation (�) or placebo(�) and gastric acid secretory rate at h2. aP < 0.05 vs. 8 ml/kg Aloepreparation-treated group. bP < 0.05 vs. 8 ml/kg placebo-treated group.
at h3 and h4, the 4 and 8 ml/kg placebo-treated groupsproduced significantly higher gastric pepsin secretory ratethan the control group whereas those of Aloe preparationdid not.
3.2.3. Gastric protein secretionIn control group, histamine did not increase the gas-
tric protein secretion. On the other hand, the secretoryrate was significantly decreased at h4 (Table 3). Exceptfor the significant increase in protein secretion of 2 ml/kgplacebo-treated group at h2, the secretory rate of 1 and2 ml/kg of Aloe preparation- and placebo-treated groupsremained the same level as basal secretion until h4. In con-trast, the secretory of 4 and 8 ml/kg Aloe preparation- andplacebo-treated groups were significantly increased from thebasal level at h2 (Fig. 3) and remained higher than basal levelthrough h4.
Comparing the gastric protein secretory rate betweengroups showed that the secretory rate of 2 ml/kg Aloepreparation- and placebo-treated group were significantlyhigher than that of control group at h4 and significant lower
0
200400
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0 1 2 3 4 5 6 7 8Dose (ml/Kg)
Pep
sin
(Uni
ts/g
sto
mac
h/hr
)
+
+
+
+
Fig. 2. Relationship between the doses of Aloe preparation (�) or placebo(�) and gastric pepsin secretory rate at h2. +P < 0.05 vs. control group.
W. Suvitayavat et al. / Journal of Ethnopharmacology 90 (2004) 239–247 243
Table 2Effects of Aloe preparation, placebo and fresh Aloe gel on histamine-induced gastric pepsin secretory rate (U/g stomach/h) at h1, h2, h3 and h4
All values were expressed as mean ± S.E.M.∗P < 0.05, ∗∗P < 0.01 vs. basal secretion within group.+P < 0.05 vs. control group at the same time point.xP < 0.05 vs. 6.4 ml/kg fresh Aloe gel-treated group at the same time point.
than those of 8 ml/kg Aloe preparation- and placebo-treatedgroups at h2, h3 and h4. However, the secretory rate of2 ml/kg Aloe preparation- and placebo-treated rats wassignificant lower than those of 4 ml/kg Aloe preparationand placebo only at h3. The secretory rates at h2, h3 andh4 of Aloe preparation and placebo at doses of 4 and8 ml/kg treated rats were significantly higher than thoseof control group and 1 ml/kg of Aloe preparation- andplacebo-treated groups at the same time point. There wasno significant difference between the protein secretory ratesof Aloe preparation- and placebo-treated groups at the samedose.
Table 3Effects of Aloe preparation, placebo and fresh Aloe gel on histamine-induced gastric protein secretory rate (mg/g stomach/h) at h1, h2, h3 and h4
Treatment Gastric protein secretory rate (mg/g stomach/h)
All value were expressed as mean ± S.E.M.∗P < 0.05, ∗∗P < 0.01 vs. basal secretion within group.+P < 0.05 vs. control group at the same time point.Comparison between the doses of Aloe preparation- and placebo-treated groups at the same time point: aP < 0.05 vs. 8 ml/kg Aloe gel preparation-treatedgroup.bP < 0.05 vs. 8 ml/kg placebo-treated group.cP < 0.05 vs. 4 ml/kg Aloe gel preparation-treated group.dP < 0.05 vs. 4 ml/kg placebo-treated group.
3.2.4. Gastric soluble mucus secretionSimilar to the pepsin secretory rate profile pattern, the
histamine-stimulated soluble mucus secretory rate wassignificantly increased from the basal secretion only ath2 and then gradually decreased toward the basal levelat h4 (Table 4). The secretory rates of all doses of Aloepreparation- and placebo-treated groups were significantlyincreased at h2 and remained at the high level through h4period. These secretory rates at h2, h3 and h4, except thesecretory rate of 1 ml/kg Aloe preparation and placebo ath4, were significantly higher than that of control group atthe same time point. Both Aloe preparation and placebo
244 W. Suvitayavat et al. / Journal of Ethnopharmacology 90 (2004) 239–247
Table 4Effects of Aloe preparation, placebo and fresh Aloe gel on histamine-induced gastric soluble mucus secretory rate (�g/g stomach/h) at h1, h2, h3 and h4
All values were expressed as mean ± S.E.M.∗P < 0.05, ∗∗P < 0.01 vs. basal secretion within group.+P < 0.05 vs. control group at the same time point.Comparison between the doses of Aloe preparation- and placebo-treated groups at the same time point:aP < 0.05 vs. 8 ml/kg Aloe gel preparation-treated group.bP < 0.05 vs. 8 ml/kg placebo-treated group.cP < 0.05 vs. 4 ml/kg Aloe gel preparation-treated group.dP < 0.05 vs. 4 ml/kg placebo-treated group.eP < 0.05 vs. 2 ml/kg Aloe gel preparation-treated group.fP < 0.05 vs. 2 ml/kg placebo-treated group.
increased the mucus secretion in the dose-dependent man-ner (Table 4 and Fig. 4). There were also no significantdifference between the mucus secretory rates of Aloepreparation- and placebo-treated groups at the same dose.
3.2.5. Gastric visible mucus secretionThe visible mucus content on the gastric mucosal surface
of 1, 2 and 4 ml/kg Aloe preparation- and placebo-treatedgroups did not significantly differ from that of control group(Fig. 5). However, both Aloe preparation and placebo atdoses of 8 ml/kg significantly increased gastric visible mu-
0
1
2
3
4
5
0 1 2 3 4 5 6 7 8Dose (ml/Kg)
Pro
tein
(m
g/g
stom
ach/
hr)
abcd
abcdab
b
+
+
+
+
Fig. 3. Relationship between the doses of Aloe preparation (�) orplacebo (�) and gastric protein secretory rate at h2. +P < 0.05 vs.control group. Comparison between the doses of Aloe preparation- andplacebo-treated groups at the same time point: aP < 0.05 vs. 8 ml/kgAloe preparation-treated group. bP < 0.05 vs. 8 ml/kg placebo-treatedgroup. cP < 0.05 vs. 4 ml/kg Aloe preparation-treated group. dP < 0.05vs. 4 ml/kg placebo-treated group.
cus secretion from control group and other doses of Aloepreparation- and placebo-treated groups. There was no sig-nificant difference between Aloe preparation- and placebo-treated group at the same doses.
3.3. Effects on gastric secretion of Aloe preparationcompared to fresh Aloe gel
Effects of Aloe preparation were mostly parallel to thoseof placebo at all doses as shown in Figs. 1–5. Gastric se-cretory effect of Aloe preparation at a dose of 8 ml/kg was
0
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0 1 2 3 4 5 6 7 8
Dose (ml/kg)
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us (
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ach/
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abcd+*
abcd+*
abcd+*
abd+*
ab+*
ab+*
+*
+*
Fig. 4. Relationship between the doses of Aloe preparation (�) or placebo(�) and gastric soluble mucus secretory rate at h2. ∗P < 0.001 vs.basal secretion within group. +P < 0.05 vs. control group. Comparisonbetween the doses of Aloe preparation- and placebo-treated groups atthe same time point: aP < 0.05 vs. 8 ml/kg Aloe preparation-treatedgroup. bP < 0.05 vs. 8 ml/kg placebo-treated group. cP < 0.05 vs. 4 ml/kgAloe preparation-treated group. dP < 0.05 vs. 4 ml/kg placebo-treatedgroup.
W. Suvitayavat et al. / Journal of Ethnopharmacology 90 (2004) 239–247 245
Fig. 5. Effect of Aloe preparation (A), placebo (P), fresh Aloe gel(FA) and control (C) on the histamine-induced gastric visible mucussecretion. +P < 0.05 vs. control group. aP < 0.05 vs. 8 ml/kg Aloepreparation-treated group. bP < 0.05 vs. 8 ml/kg placebo-treated group.
the most prominent. Therefore, the additional group treatedwith fresh Aloe corresponding dose to 8 ml/kg of Aloepreparation were preformed to compare its effect to Aloepreparation.
Fresh Aloe gel at dose of 6.4 ml/kg which equivalentto Aloe preparation at dose of 8 ml/kg caused the acidsecretory rate profile pattern parallel to that of controlgroup. However, the secretory rate at h4 of 6.4 ml/kg freshAloe gel-treated group was still significantly higher thanits basal level (Table 1). This effect was contrast to thedecreased gastric acid secretory effect of Aloe preparationat the dose of 8 ml/kg.
The effect of fresh Aloe gel was also opposite from thestimulatory effect of Aloe preparation and placebo on pepsinsecretion. The pepsin secretory rate of fresh Aloe gel-treatedgroup had a tendency to decrease in h2, h3 and h4 (Table 2).Although the secretory rate of this group at h2 was not sig-nificantly different from control group, it was significantlylower than the other groups. In addition, at h3 and h4, thesecretory rate of fresh Aloe gel-treated group was signifi-cantly lower than most of those of Aloe preparation- andplacebo-treated groups at the same time point. Similar to theeffect on the pepsin secretion, fresh Aloe gel significantlydecreased the protein secretory rates at h3 and h4 (Table 3)whereas the Aloe preparation and placebo increased the pro-tein secretory rate at h2, h3 and h4.
Comparing the effect of Aloe preparation to fresh Aloegel on the mucus secretion found that Aloe preparation andplacebo clearly increased both soluble and visible mucussecretion whereas the effect of fresh Aloe was not signifi-cantly different from control group (Table 4). The rats treatedwith fresh Aloe gel had the similar gastric mucus secre-tory rate profile pattern as control group but the secretoryrates at h3 and h4 decreased less than the control group(Table 4). However, there was no significant difference be-tween these two groups. The gastric visible mucus contentof fresh Aloe gel-treated rats was slightly higher but not sig-nificantly different than those of control and 1, 2 and 4 ml/kg
Aloe preparation- and placebo-treated groups. It was signif-icantly lower than 8 ml/kg Aloe preparation-treated grouponly.
4. Discussion and conclusions
Aloe preparation has been formulated to stabilize theactive ingredients and make it convenient to use. The ef-fects of Aloe preparation on the secretion of gastric acid,pepsin and mucus were investigated and compared to thoseof placebo and freshly prepared Aloe (only high dose) inthe histamine-stimulated gastric fistula model in rats. Aloepreparation and placebo at doses lower than 4 ml/kg pro-duced no changes in gastric acid secretion. However, thedose of 8 ml/kg of both formulation inhibited gastric secre-tion at h2, h3 and h4. Since there was no significant differ-ence between Aloe preparation and placebo at the same dose,the inhibitory effect may be due to the acidic component inthe preparation and placebo. When pH in the autrum dropsto lower than 3, somatostatin is released to play a role in acidfeedback inhibition of acid secretion (Prinz et al., 1992).Somatostatin inhibits acid secretion by direct inhibition ofgastric parietal cell and indirect inhibition of histamine re-lease from enterochromafin like cell (Schubert et al., 1989;Chuang et al., 1993). In addition to somatostatin, the low lu-minal pH increases the synthesis of prostaglandin E whichexerts a strong inhibitory effect on parietal cell (Curtis et al.,1995). The acidity of gastric content expose to gastric mu-cosa was the sum of histamine-stimulated acid secretion andthe acidity of the test solution which could be lower than 3.Hence, the inhibitory effect of Aloe preparation and placeboat dose of 8 ml/kg may be resulting from the acid feedbackinhibition via somatostatin and prostaglandin. It is possiblethat other substances in the formula can also act directly onparietal cell to inhibit gastric acid secretion.
In contrast to the preparation, fresh Aloe gel showed thevery small stimulatory effect and the tendency to prolong thehistamine-stimulated secretion to h4-period. These findingswere correlated to the more inhibition of 8 ml/kg placebo-treated group than 8 ml/kg Aloe preparation-treated group.The stimulatory effect of Aloe gel was also found in gastricfistula dog when 15 ml of Aloe extract was orally adminis-tered daily for 20 days (Faitel’berg and Stambol’Skii, 1969).However, aloctin A isolated from the leaf gel inhibited acidsecretion when it was administered intravenously (Saitoet al., 1989). Since a very low amount of aloctin A would beabsorbed in oral administration of Aloe preparation or Aloeextract compared with intravenous route, it is possible thatthe inhibitory effect could not be observed in the presentexperiment. In addition, there may be other substances inAloe gel which has stimulatory effect on acid secretion.
Our former report showed that intraduodenal admin-istration of this Aloe preparation and placebo (7.5 and15 ml/kg) inhibited gastric acid secretion in Shay’s oper-ated rat (Suvitayavat et al., 1997). In contrast to this study,
246 W. Suvitayavat et al. / Journal of Ethnopharmacology 90 (2004) 239–247
Aloe preparation showed the significantly higher inhibi-tion of spontaneous gastric acid secretion than placebo.This significant inhibition might be due to the unidentifiedmechanism caused by intestinally absorbed Aloe and highdoses of the preparation. These unsimilar results may bedue to the different model of experiment and doses of Aloepreparation. Aloe gel may not be satisfactorily absorbedand has no direct effect on the parietal cell. The rats usedin the present study do not have spontaneous gastric se-cretion by Shay’s operation. The secretagogue-stimulatedgastric secretory model was then selected for this study.Furthermore, the dose of Aloe preparation could not applyhigher than 8 ml/kg because the high volume instilled intothe stomach stretched the wall causing the pressure to pushout the test solution. However, it is possible that the higherdose of the preparation may be needed to demonstrate theinhibitory effect of Aloe gel in this gastric fistula model.
Effect of Aloe preparation on the pepsin secretion wasopposite to its gastric acid inhibition. Both Aloe prepara-tion and placebo at doses of 2, 4 and 8 ml/kg caused an in-creased and prolonged effect of histamine stimulated pepsinsecretion. At the doses of 4 and 8 ml/kg, Aloe preparationand placebo significantly increased the gastric pepsin se-cretory rates at h2- to h4-period. This increased pepsin se-cretion was correlated with the increase in gastric proteinsecretion. Since gastric protein consisted mainly of prote-olytic enzyme, pepsin, the increase in pepsin secretion byAloe preparation and placebo might be due to the increasein pepsinogen secretion. In contrast to the preparation, freshAloe gel showed the inhibitory effect at h2- to h4-period.This result was correlated to the less stimulatory effects ofAloe preparation than those of the placebo at the same doses,even though, there was no significant difference between thesame doses of these two groups. The stimulatory effect ofthe Aloe preparation was mostly due to the component ofthe formula. The inhibitory effect of the gel agreed with theformer report that aloctin A inhibited pepsinogen secretion(Saito et al., 1989).
Similar to the effect on the gastric pepsin secretion, bothAloe preparation and placebo increased gastric soluble andvisible mucus. The gastric soluble mucus secretory ratesof 1, 2, 4 and 8 ml/kg Aloe preparation and placebo weresignificantly increased in the dose-dependent manner. Theeffect of Aloe preparation did not show any significantdifference from placebo groups at the same doses. Since,the fresh Aloe gel produced no effect on gastric solublemucus secretion, the increase in soluble mucus was prob-ably resulted from the instillated mucus content in the testsolution.
The gastric visible mucus content of fresh Aloe gel-treatedgroups showed the tendency to increase from controlgroup. The small effect of Aloe gel caused a slightly highervisible mucus content in 8 ml/kg Aloe preparation-treatedstomach than those of 8 ml/kg placebo-treated stomach.This slightly stimulatory effect of Aloe gel may be dueto endogenous prostaglandin (Robert et al., 1979) which
increases the cellular integrity of the gastric mucosa. Thetendency to increase visible mucus content in this experi-ment was similar to the previous reports (La-ongphanich,1987; Visuthipanich, 1988) which demonstrated the increasein mucus secretion in corpus and antrum of rat stomachafter oral administration of Aloe vera whole leaf extract(La-ongphanich, 1987) and Aloe vera gel (Visuthipanich,1988).
Data from this present study indicated that fresh Aloegel at the dose of 6.4 ml/kg slightly prolonged the effect ofhistamine-stimulated gastric acid secretion and had tendencyto stimulate mucus secretion but had tendency to inhibit thehistamine-induced pepsinogen secretion. Even though thisstudy showed that the Aloe preparation did not demonstratesignificant gastric acid inhibitory effect, the previous report(Suvitayavat et al., 1997) with higher dose of Aloe prepara-tion could significantly inhibit gastric acid secretion higherthan placebo. The higher dose and the longer period of treat-ment with this Aloe preparation may provide more effect. Itis possible that the antiulcer effect of fresh Aloe gel shouldbe mediated through the decrease in aggressive factors andincrease in protective factors. When formulated, the effect offresh Aloe gel was overwhelmed by the effect of the formula.Even though, this Aloe preparation was shown to increasepepsinogen secretion, it has been demonstrated to decreasein gastric acid secretion and increase in mucus secretion.Thus, this Aloe preparation decreases aggressive factor andincreases protective factor which provide the advantages forpatient with gastric ulcer. In addition, the Aloe vera gel hasbeen demonstrated to have wound healing (Heggers et al.,1993; Davis et al., 1989b; Chithra et al., 1998) and antiin-flammatory activities (Saito et al., 1982; Davis et al., 1989a)which promote its antiulcer effect. The effects of the Aloepreparation on chronic gastric ulcer models in rats shouldbe further investigated to support the antiulcer effect of thisAloe preparation.
Acknowledgements
This is partly supported by the M.S. research grant, Fac-ulty of Pharmacy, Mahidol University.
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Bovik, E.G., 1966. Aloe vera. Panacea or old wives’ tales? Texas DentalJournal 84, 13–16.
Chithra, P., Sajithlal, G.B., Chandrakasan, G., 1998. Influence of Aloevera on the glycoaminoglycans in the matrix of healing dermal woundsin rats. Journal of Ethnopharmacology 59, 179–186.
Chuang, C.N., Tanner, M., Lloyd, K.C.K., Wong, H., Soll, A.H., 1993.Endogenous somatostatin inhibits histamine release from canine gastricmucosal cells in primary culture. American Journal of Physiology 265,GT521–G525.
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Corne, S.J., Morrissey, S.M., Woods, R.J., 1974. The method for thequantitative estimation of gastric barrier mucus. Journal of Physiology(London) 242, 116P–117P.
Curtis, G.H., Mac Naughton, W.K., Gall, D.G., Wallana, J.L., 1995.Intraluminal pH modulates gastric prostaglandin synthesis. CanadianJournal of Physiology and Pharmacology 73, 130–134.
Davis, R.H., Leitner, M.G., Russo, J.M., Byrne, M.E., 1989a.Anti-inflammatory activity of Aloe vera against a spectrum of irri-tants. Journal of American Podiatric Medical Association 79, 263–276.
Davis, R.H., Leitner, M.G., Russo, J.M., Byrne, M.E., 1989b. Woundhealing: oral and topical activity of Aloe vera. Journal of AmericanPodiatric Medical Association 79, 559–562.
Faitel’berg, R.O., Stambol’Skii, M.M., 1969. Dynamics of the secretionof gastric juice in dogs during the long-term administration of vita-min, biogenic stimulant, and organic preparation. Vop Fiziol Pishch107–118.
Galal, E.E., Kandil, A., Hegazy, R., Ghoroury, M., Gobran, W., 1975. Aloevera and gastrogenic ulceration. Journal of Drug Research Egyptian7, 73–78.
Gjerstad, G., Riner, T.D., 1968. Current status of Aloe vera as a cure-all.American Journal of Pharmacy 140, 58–61.
Heggers, J.R., Pelley, R.P., Robson, M.C., 1993. Beneficial effect of Aloein wound healing. Phytotherapy Research 7, 548–552.
Hirata, T., Suga, T., 1977. Biologically active constituents of leaves androots of Aloe arborescens var. natalensis. Zeitschrift fur Naturforschung32c, 731–734.
Kandil, A., Gobran, W., 1982. Protection of gastric mucosa by Aloe vera.Bulletin of Islam Medicine 2, 503–511.
Koo, M.W.L., 1994. Aloe vera: antiulcer and antidiabetic effects. Phy-totherapy Research 8, 461–464.
La-ongphanich, S., 1987 Ulcer-healing effect of Aloe vera gel, Aloe verawhole leaf extract and cimetidine on rat gastric ulcer induced by fasting,refeeding and cortisol injection. M.Sc. Thesis in Anatomy, Bangkok,Faculty of Science, Mahidol University.
Limlomwongse, L., Nophakoon, J., Thithapanda, A., 1976. Mechanismof inhibition of gastric acid secretion by hypertonic solutions andvasopressin. Pfluegers Archives of European Journal of Physiology366, 159–165.
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Proteinmeasurement with the Folin phenol reagent. Journal of BiologicalChemistry 193, 265–275.
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Journal of Ethnopharmacology 90 (2004) 249–252
Hypolipidemic activity of seeds ofCassia tora Linn
Umesh K. Patila, S. Sarafb, V.K. Dixit a,∗a Department of Pharmaceutical Sciences, Dr. Hari Singh Gour University, Sagar 470003, MP, India
b B.R. Nahata College of Pharmacy, Mandsaur, MP, India
Received 22 March 2002; received in revised form 25 June 2003; accepted 7 October 2003
Cassia tora L. (Caesalpiniaceae) is a small shrub grow-ing as common weed in Asian countries. It constitutes anAyurvedic preparation “Dadhughnavati” which is one of thesuccessful antifungal formulations (Acharya and Chatterjee,1975; Hatano et al., 1999). The liver protective seed extractof Cassia tora yields two anthraquinone glycosides andtwo naphthopyran glycosides (chrysophanol-tetraglucoside,chrysophanol-triglucoside, rubrofusarin-6-gentiobioside,rubrofusarin-glycoside) (Shibata et al., 1969; Kaneda et al.,1969; Choi et al., 1994; Wong et al., 1989). The seed ex-tract is also reported for its hypotensive activity (Koo et al.,1976a, 1976b; Chan et al., 1976). Many medicinal prop-erties such as antimicrobial, antihepatotoxic and antimu-tagenic activities have been attributed to this plant (Wonget al., 1989; Choi et al., 1997; Yen and Chung, 1999).
The plant is claimed to be effective against a variety ofailments in indigenous medicine such as in treatment ofjaundice. In Chinese medicine, it is highly valued for thetreatment of hyperlipidemia. Several polyharbal formula-tions are available in Chinese market for preventing theformation of atherosclarosis plaque (Guan and Zhao, 1995;Kee, 1997). The aim of present work was to further evaluate
the hypolipidemic activity ofCassia tora which grows ascommon weed in India.
2. Material and methods
2.1. Plant material
Cassia tora seeds were collected in the month of Januaryfrom the area adjoining the University campus. The driedseeds were crushed to moderately coarse powder.
2.2. Extraction and fractionation
The drug powder was packed in a soxhlet apparatus andwas defatted with petroleum ether for 72 h. The defattedmaterial was completely freed of petroleum ether and themarc was extracted till exhausted completely. The ethano-lic extract so obtained was freed of solvent under vacuum(yield: 5.06% w/w). The solvent-free ethanolic extract wassuspended in distilled water and extracted with solvent ether.Ether soluble and water soluble fractions were thus obtained.
2.3. Chromatographic studies
TLC of ethanolic extract and its ether soluble and wa-ter soluble fractions was carried out using various solventsystems. A mixture of chloroform:ethyl acetate:formic acid
250 U.K. Patil et al. / Journal of Ethnopharmacology 90 (2004) 249–252
(40:40:30) gave the best resolution; five spots were de-tected in ethanolic extract and three spots in ether solublefraction. Water soluble fraction was best resoluted in amixture of chloroform:pyridine (1:1) and three spots wereobserved.
The most active fraction the water soluble fraction wassubjected to column chromatography with a view to isolateindividual component. The column was eluted with chloro-form : methanol mixture followed by increasing proportionof methanol. Three resulted components designated as I, IIand III. These components gave positive Borntrager test.λmax recorded for them was 258 nm for I, 275 nm for II and418 nm for III. Melting points of 165, 205 and 170◦C wasrecorded for I, II and III, respectively.
2.4. Screening for hypolipidemic activity
Screening for hypolipidemic activity was carried out inalbino rats (Wistar strain) of either sex weighing 90–120 gm.Triton WR 1339 (Tyloxapol) was purchased from SigmaChemicals Co., USA.
2.5. Preparation of test material
Ethanolic extract and its ether soluble were suspended indistilled water with PVP (4% w/v) whereas water solublefraction was dissolved in distilled water to give 50 mg/mlof respective suspension/solution. Triton (Sigma) was dis-solved in normal saline to give a 7% solution (Paoletti,1962).
2.6. Measurement of biochemical parameters
Albino rats were divided into five groups of six rats each.Group I served as normal. Groups III, IV, and V weregiven i.p. injection of Triton WR 1339 at dose of 400 mg/kgbody weight. After 24 h of Triton administration, animals ofGroups III, IV and V were treated with ethanolic extract,ether fraction and water fraction at dose 100 mg/kg bodyweight per day, respectively. Group II was kept as controland administered with PVP suspension only. The treatmentwas continued for 5 days with a view to see the effect ofdrug on lipid profile.
Table 1Effect of EtOH extract and fractions of EtOH extract on total cholesterol level in triton induced hyperlipidemic model
Group Initial Total cholesterol level afterthe administration of triton
Values are expressed as mean± S.D. for six animals in each group. Total cholesterol concentrations are estimated by standard method and the valuesare expressed as mg/dl serum.
∗ P < 0.05.
The blood samples were withdrawn from the eye veinand transferred directly into centrifuge tubes and allowedto clot at room temperature for 20–25 min and centrifugedfor 15–20 min at 2000 r.p.m. The supernatant clear serumthus obtained was transferred carefully with the help of mi-cropipette into small test tubes for estimation. The serumconcentration of total cholesterol, HDL-C, and triglyceridewere measured by standard procedure using auto-analyzer(RA-50 model, Miles India Ltd.), while the LDL-cholesterollevel was calculated with the help of the equation (Kurupand Krishnamurthy, 1993); LDL-C = TC− (HDL-C-TG/5).
2.7. Statistical analysis
Results of serum parameters are presented as Mean±S.D.
and percent inhibition against triton by the test extract andfractions. The percentage was calculated by considering thelevel parameters between rats treated with triton and controlrats as 100% degree of reversal. The difference among themeans were analyzed by the Student’st-test at 95% (P <
0.05) confidence level (Ostle, 1966; Armitage and Berry,1985).
3. Results
The results of the present study are given inTables 1–4.The yield of ethanolic extract was 5.06% w/w of crude drug.The ethanolic extract was fractioned into ether soluble frac-tion and water soluble fraction. The yield of these fractionswere found 5% w/w of ethanolic extract and 85% w/w ofethanolic extract, respectively. The rats treated with tritonshowed a two fold increase in serum cholesterol level ascompared to initial level. In ethanolic extract treated groupthe initial level of total cholesterol was found to be 59.35±5.24 which was increased upto 81.75±6.86 by triton. Afterthe treatment with test sample the total cholesterol level wasreduced upto 72.41± 4.22. In this group the initial HDL-Cwas 31.16± 4.50 which was increased upto 37.70 ± 3.60by triton and after the treatment with test drug it was in-creased upto 38.14± 1.20. The initial level of triglyceridewas 44.17±3.32 which was increased upto 64.62±2.74 bytriton and after the treatment with test drug it was decreased
U.K. Patil et al. / Journal of Ethnopharmacology 90 (2004) 249–252 251
Table 2Effect of EtOH extract and fractions of EtOH extract on HDL-C level in triton induced hyperlipidemic model
Group Initial HDL-C level after theadministration of triton
Values are expressed as mean±S.D. for six animals in each group. HDL-C concentrations are estimated by standard method and the values are expressedas mg/dl serum.
∗ P < 0.05.
Table 3Effect of EtOH extract and fractions of EtOH extract on triglyceride level in triton induced hyperlipidemic model
Group Initial Triglyceride level after theadministration of triton
Values are expressed as mean± S.D. for six animals in each group. Triglyceride concentrations are estimated by standard method and the values areexpressed as mg/dl serum.
∗ P < 0.05.
Table 4Effect of EtOH extract and fractions of EtOH extract on LDL-C level in triton induced hyperlipidemic model
Group Initial LDL-C level after theadministration of triton
Values are expressed as mean± S.D. for six animals in each group. LDL concentrations are estimated by standard method and the values are expressedas mg/dl serum.
∗ P < 0.05.
upto 59.11±2.19. In this group the initial LDL-C level was38.62±4.52 which was increased upto 56.97±3.10 and aftertreatment with test drug it was reduced upto 44.24± 5.11.
In ether soluble fraction treated group the initial levelof total cholesterol was found to be 58.75 ± 5.92 whichwas increased upto 92.66± 8.11 by triton. After the treat-ment with test sample the total cholesterol level was re-duced upto 78.60 ± 6.69. In this group the initial HDL-Cwas 29.10 ± 3.52 which was increased upto 34.33± 3.29by triton and after the treatment with test drug it was in-creased upto 35.23± 3.11. The initial level of triglyceridewas 45.76±4.12 which was increased upto 64.71±3.63 bytriton and after the treatment with test drug it was decreasedupto 58.93±2.61. In this group the initial LDL-C level was40.12±3.52 which was increased upto 71.27±2.62 and aftertreatment with test drug it was reduced upto 48.75± 5.21.
In water soluble fraction treated group the initial levelof total cholesterol was found to be 54.77 ± 2.73 which
was increased upto 80.57± 7.54 by triton. After the treat-ment with test sample the total cholesterol level was re-duced upto 57.20 ± 6.77. In this group the initial HDL-Cwas 34.13± 5.19 which was increased upto 41.35± 2.58by triton and after the treatment with test drug it was in-creased upto 42.71± 1.28. The initial level of triglyceridewas 42.25±4.30 which was increased upto 63.13±2.11 bytriton and after the treatment with test drug it was decreasedupto 55.12±3.42. In this group the initial LDL-C level was21.40±2.31 which was increased upto 51.87±4.12 and aftertreatment with test drug it was reduced upto 28.52± 3.28.
4. Discussion
The aim of present study was to asses the hypolipi-demic activity of seeds ofCassia tora. An increased riskof coronary heart disease is associated with a high serum
252 U.K. Patil et al. / Journal of Ethnopharmacology 90 (2004) 249–252
concentration of total cholesterol, low-density lipoprotein(LDL) and triglyceride. On the other hand, low serumconcentration of high-density lipoprotein (HDL) is also re-sponsible for coronary heart diseases. Atherosclarosis, con-gestive heart diseases and some other diseases are stronglyassociated with disorders of lipid metabolism and plasmalipoproteins.
Triton WR 1339 acts as a surfactant to block the uptakeof lipoprotein from the circulation by extra hepatic tissuesresulting in an increase in the level of circulatory lipopro-teins (Friedman and Bayer, 1957). In hyperlipidemic modelethanolic extract and its ether soluble fraction and water sol-uble fraction decreased serum levels of total cholesterol by42.07, 40.77 and 71.25%, respectively. The ethanolic ex-tract, ether and water fraction decreased the LDL-cholesterollevels by 69.25, 72.06 and 76.12%, respectively. The lipidlowering effect ofCassia tora seed extract in rats may bedue to inhibition of cholesterol biosynthesis and to increasedfaecal bile acid excretion.
The drug showed protective action as it slightly increasedthe HDL-cholesterol level. The ethanol extract, ether frac-tion and water fraction increased the serum HDL-cholesterollevels in triton induced hyperlipidemic model by 6.72, 17.20and 19.18%, respectively. The possible mechanism of ac-tivity may be due to enhancement of the activity of LCATand inhibition of the action of hepatic TG-lipase on HDL,which may contribute for a rapid catabolism of blood lipidsthrough extra hepatic tissues. The increased HDL facilitatesthe transport of triglyceride or cholesterol from serum toliver where it is catabolised and excreted out of the body.
The ethanolic extract, ether fraction and water fractiondecreased the serum triglyceride levels by 26.84, 35.74 and38.46%, respectively. The decrease in serum triglyceridelevel on administration of different fractions could be due toincreased catabolism of triglyceride and an inhibition of fattyacetyl-CoA activity and glycerophosphate acetyl transferase.
In C. tora some new naphtho-pyrone glycosides, 9-[{�-d-glucopyranosyl-(1 → 6)-O-�-d-glucopyranosyl}oxyl]-10-hydroxy-7-methoxy-3-methyl-1H-naphtho(2,3-c)pyron-1-one and 6-[{�-apiofuranosyl-(1→ 6)-O-�-d-glucopyra-nosyl}]-rubrofusarin, together with cassiaside and rubrofu-sarin-6-�-gentiobioside are reported from the seeds. Thesenaptho-�-pyrone glycosides are reported to have significanthepatoprotective activity (Tiwari and Behari, 1972; Wonget al., 1989; Maity et al., 2001). In our study it is found thatwater soluble fraction of ethanolic extract exhibited maxi-mum efficacy in lowering the elevated lipid levels. Keepingin view the protective activity of glycosides of the seeds,it appears that they contribute to observed hypolipidemicactivity of the drug.
Acknowledgements
One of the authors (UKP) is thankful to University GrantCommission, New Delhi, India for financial assistance.
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Journal of Ethnopharmacology 90 (2004) 253–259
Effect of Kuibitang on lipopolysaccharide-induced cytokine production inperipheral blood mononuclear cells of chronic fatigue syndrome patients
a Department of Pharmacology, College of Oriental Medicine, Kyung Hee University, 1 Hoegi-Dong, Dongdaemun-Gu, Seoul 130-701, South Koreab College of Pharmacy, VCRC of Wonkwang University, Iksan, Jeonbuk 570-749, South Korea
c SinnongC Oriental Clinic, Seongdonggu, Seoul, South Koread Department of Health Science, Graduate School of Public Health and Environment, Wonkwang University, Iksan, South Korea
e College of Phamacy, Woosuk University, Jeonju, Jeonbuk 565-701, South Koreaf Department of Herbal Resources, Professional Graduate School of Oriental Medicine, Wonkwang University, Iksan 570-749, South Korea
g Department of Biochemistry and Molecular Biology, College of Oriental Medicine, #707 Sukjang-Dong, Kyung-Ju, Kyungpook 780-714, South Koreah College of Oriental Medicine, Wonkwang University, Iksan, Jeonbuk 570-749, South Korea
Received 9 January 2003; received in revised form 18 June 2003; accepted 7 October 2003
The chronic fatigue syndrome (CFS) is characterized byunexplained chronic fatigue and pain. Although the patho-genesis of this disease is poorly understood, the Centers forDisease Control has estimated that 500,000 Americans areaffected by the disease. The etiology of CFS remains elu-sive, although some studies have suggested a role of im-mune dysfunction (Visser et al., 1998; Cannon et al., 1997;Bennett et al., 1997; Buchwald et al., 1997; Levy, 1994).Several reports have shown that CFS patients display an al-tered functioning of the immune response; i.e., altered cy-tokine production, low NK-cell function, and differences inthe expression of activation markers on lymphocytes (Bearnand Wessely, 1994; Strauss et al., 1993, 1994; Aoki et al.,1993; Caligiuri et al., 1987; Barker et al., 1994; Swanink
et al., 1993, 1996; Klimas et al., 1990; Chao et al., 1991;Mawle et al., 1997; Hassan et al., 1998; Tirelli et al., 1994).Others have suggested that an antiviral pathway in CFS pa-tients is upregulated as the consequence of an increased vi-ral reactivation in CFS patients (Visser et al., 1998; Landayet al., 1991); however, until now no infectious agent couldbe linked to the syndrome.
Cytokines have been suggested to play a role in the patho-genesis and clinical manifestations of CFS (Levy, 1994;Moutschen et al., 1994). CFS is an autoimmune disorderin which activated Th cells and different Th cell cytokinemight play an important role. Th cells are divided into threemain subset, Th1, Th2, Th3 cells. Th1 cells produce TNF-�,IFN-� whereas Th2 cells release IL-4 and IL-10 (Romagnaniet al., 1997). A characteristic of Th3 cells is their productionof the immune-modulating cytokine TGF-�1 (Chen et al.,1994; Fukaura et al., 1996). TGF-�1 inhibits T-cell pro-liferation and suppresses some Th1 and Th2 cell-mediatedautoimmune diseases (Kehrl et al., 1986; Holter et al., 1994;Mosmann and Sad, 1996; Bridoux et al., 1997). A large
254 H.-Y. Shin et al. / Journal of Ethnopharmacology 90 (2004) 253–259
number of immune abnormalities, including cytokine pro-duction in CFS, have been reported (Klimas et al., 1990;Buchwald and Komaroff, 1991; Gupta and Vayuvegula,1991; Gupta, 1992; Jone, 1991; Lloyed et al., 1989; Strauset al., 1989). Recent insights into autoimmune type dis-ease have suggested a pivotal role for the pro-inflammatorycytokine tumor necrosis factor (TNF)-�. TNF-� has beenshown to have a variety of in vivo effects including pain,inflammation and fatigue (Krakauer et al., 1999). Interferon(IFN) has been used therapeutically to treat a number ofdiseases, and the therapy is associated with a number ofadverse effects, including fatigue (Dusheiko, 1997). IL-10is a cytokine that is produced by a variety of cell types,including Th2 type T cells (Howard et al., 1992). IL-10inhibits cytokines produced by Th1 type T cells, includ-ing IL-2 and IFN-�. Furthermore, IL-10 inhibits monokineproduction by activated macrophages, including IL-6 andTNF-� (Fiorentino et al., 1991). Transforming growth fac-tor (TGF)-�1 is expressed as a large pro-protein (390–412amino acids), which includes TGF-�1 as its C-terminus, andTGF-�1 latency associated peptide (LAP) at its N-terminus,TGF-�1 remains in covalent association with LAP dueto limited intracellular proteolytic cleavage (Lopez et al.,1992). It appears that during both in vivo and in vitro my-cobacterial infections at least some TGF-�1 produced bymononuclear cells is detected in its active form (Aung et al.,2000; Hirsch et al., 1994). Most recently it was shownthat TGF-�1 is one of the key negative regulators of im-mune homeostasis and its absence leads to activation of aself-targeted immune response (Gorelik and Flavell, 2000).
Recently Kuibitang (KBT) has been successfully used forregulation of the immune response in South Korea (Limet al., 1999). KBT consists of 13 different herbs. This pre-scription was also composed on the basis of the theory ofOriental medicine to maximize its efficacy. Some main com-ponents they are known to have are as follows; saponins,glycosides, and polysaccharides etc (Matsuda et al., 1999;Ye et al., 2001).
Based upon the clinical presentation of CFS, we hypoth-esized that cytokines may play a role in the pathogenesisof the disease. In the present study, we investigated the ef-fect of KBT on lipopolysaccharide (LPS)-induced cytokinesproduction in PBMC of eight CFS patients and five healthycontrols. Our data demonstrated that KBT treatment inhib-ited production of TNF-�, IL-10, TGF-�1 and increasedproduction of IFN-� in PBMC of CFS patients.
2. Materials and methods
2.1. Reagents
Ficoll-Hypaque, LPS, bovine serum albumin (BSA),abidin-peroxidase and 2-azino-bis(3-ethylbenzithiazoline-6-sulfonic acid) tablets substrate (ABTS) were purchasedfrom Sigma (St. Louis, MO, USA). RPMI 1640, ampi-
cillin, streptomycin and fetal bovine serum (FBS) werepurchased from Gibco BRL (Grand Island, NY, USA).IL-6, IL-10, TGF-�1 and recombinant human IL-6, IL-10,TGF-�1 were purchased from Pharmingen (San Diego, CA,USA). Anti-human TNF-�, IFN-� and recombinant humanTNF-�, IFN-� were purchased from R & D Systems Inc.(Minneapolis, MN, USA).
2.2. Preparation of KBT
The plant materials were obtained from the WonkwangOriental Medicine Hospital (Chonju, Chonbuk) and au-thenticated by Professor Y.S. Lyu, College of OrientalMedicine, Wonkwang University. A voucher specimen(number 02-03-21) was deposited at the Herbarium of theCollege of Pharmacy, Wonkwang University. An extract ofKBT was prepared by decocting the dried prescription ofherbs with boiling distilled water. The duration of decoctionwas about 3 h. The decoction was filtered, lyophilized andkept at 4◦C. The yield of extraction was about 14% (w/w).The KBT water extract powder was dissolved in sterilesaline (50 mg/ml). The ingredients of KBT include 4 g ofAngelicae Gigantis Radix, 4 g of Longanae Arilus, 4 g ofZizyphi Spinosi Semen, 4 g of Polygalae Radix, 4 g of Gin-seng Radix Alba, 4 g of Astragali Radix, 4 g of AtractylodisRhizoma Alba, 4 g of Poria, 4 g of Prunellae Spica, 2 g ofSaussureae Radix, 1.2 g of Araliae Cordatae Radix, fivepiece of Zingiberis Rhizoma, two piece of Zizyphi Fruc-tus. These ingredients correspond to parts of the followingplants: Angelica gigasNakai (Umbelliferae), Euphorialongan (Lour.) Steud. (Sapindaceae),Ziziyphus spinosaHu. (Rhamnaceae),Polygala tenuifolia Willd. (Poly-galaceae),Panax ginsengC.A. Mey. (Araliaceae),Astra-galus membranaceusBunge (Leguminosae),AtractylodesmacrocephalaKoidz. (Compositae),Poria cocos(Schw.)Wolf (Polyporaceae),Prunella vulgarisvar. lilacina Nakai(Labiatae),Aucklandia lappaDecne. (Compositae),Gly-cyrrhiza uralensisFisch. (Leguminosae),Zingiber officinaleRosc. (Zingiberaceae),Zizyphus jujubaMill. var. inermisRehder(Rhamnaceae), respectively.
2.3. Patients
Patients between 20 and 50 years of age fulfilled the Cen-ters for Disease Control-criteria as defined byFukuda et al.(1994). Patients suffering from somatic and psychiatric dis-orders and patients using beta-blockers, psychotropic drugs,or diuretics were excluded from the study. Furthermore pa-tients with an alcohol intake of more than four units of alco-hol per day and a body mass index of greater than 45 wereexcluded from the study.
2.4. Recruitment of patients and controls
Patients suffered from severe fatigue for more than 6months resulting in a reduction of their daily activity by
H.-Y. Shin et al. / Journal of Ethnopharmacology 90 (2004) 253–259 255
more than 50%. Patients were included if more than fourof the following symptoms were present with a durationof at least 6 months: impaired memory or concentration,sore throat, tender cervical or axillary lymph nodes, musclepain, multi-joint pain, new headaches, unrefreshing sleep,and post-exertion malaise. Patients with CFS were recruitedthrough the Wonkwang Oriental Medicine Hospital (Jeonju,Jeonbuk). By the inspection of the available medical records,it was established whether the prospective patients met theabove-defined criteria. This study involved eight CFS pa-tients and five healthy controls. Informed consent was ob-tained from all the patients.
2.5. PBMC isolation and culture
PBMC (CFS patient with conscious disorder) from hep-arinized venous blood were isolated by Ficoll-gradientcentrifugation, washed three times in phosphated-bufferedsaline (PBS) solution and resuspended in RPMI 1640medium (GIBCO) supplemented with 2 mMl-glutamin,100 U/ml penicillin G, 100�g/ml streptomycin, and 10%FBS inactivated for 30 min at 56◦C. PBMC were culturedfor 24 h in 95% humidified air containing 5% CO2 (37◦C),in the presence or the absence of LPS, and the supernatantswere collected by centrifugation and stored at−20◦C. ForTGF-�1 assay, cell supernatants were adjusted to pH 3.0with 1N HCl and then the acidified samples were incubatedat 4◦C for 60 min. After incubation, the samples were neu-tralized by treating with 1N NaOH and stored at−70◦C.
2.6. MTT assay
Cell viability was determined by the MTT assay. Briefly,500�l of PBMC suspension (2.5 × 104 cells) was culturedin four-well plates for 24 h after treatment by each con-centration of purple bamboo salt. 20�l of MTT solution(5 mg/ml) was added and the cells were incubated at 37◦Cfor an additional 4 h. After washing the supernatant out, theinsoluble formazan product was dissolved in DMSO. Then,optical density of 96-well culture plates was measured us-ing an enzyme-linked immunosorbent assay (ELISA) readerat 540 nm. The optical density of formazan formed in un-treated control cells was taken as 100% of viability.
2.7. Cytokine assays
Cytokines were determined by modification of an ELISAas described previously (Scuderi et al., 1986). For the mea-surement of TNF-� and IFN-�, monoclonal anti-humanTNF-�, IFN-� (1�g/ml) were used as a capture antibody;biotinylated anti-human TNF-�, IFN-� (0.1�g/ml) wereused as a detecting antibody; whereas human recombinantTNF-�, IFN-� served as a standard. For the detection ofIL-10 and TGF-�1 the plates were coated with purified ratanti-human IL-10, TGF-�1 monoclonal antibody (1�g/ml)and biotinylated rat anti-human IL-10, TGF-�1 monoclonal
antibody were used in a concentration of 0.1�g/ml. Re-combinant human IL-10 and TGF-�1 that were diluted inPBS containing 0.05% tween 20 were used as a standard.The standard curves ranged from 1000 to 10 pg/ml.
Inhibition(%) = (a − b) × 100
a
where ‘a’ is cytokine secretion without KBT and ‘b is cy-tokine secretion with KBT.
2.8. HPLC analysis
The chromatographic system consisted of a pump (Shi-maduz LC-10AT HPLC pump), a UV detector (ShimaduzSPD-10A) and an autosampler (Shimaduz SIL-10AD au-tosampler). A Cosmosil 5C18-AR-IIcolumm (250 mm,Nacalai Tesgue Inc., Japan) was used. Acetonitrile-H2O-Acetic acid (100:900:10) was used as the mobile phase.Detection of the peaks at 254 nm and the sensitivity was setof 0.50 AUFS. The injection volume was 20�l and flowrate was 1.0 ml/min. Standard solution was prepared bydissolving in distilled water (10 mg/100 ml). The solutionwas filtered through 0.45�m membrane filter and appliedto HPLC.
2.9. Statistical analysis
The experiments shown are a summary of the datafrom at least three experiments and are presented as themean± S.E.M. Statistical analysis was performed using theMann–WhitneyU-nonparametric test for matched pairs. Re-sults withP < 0.05 were considered statistically significant.
3. Results
3.1. Inhibitory effect of KBT on LPS-induced TNF-α
production
We examined the effect of KBT on LPS-induced TNF-�production from PBMC of CFS patients and healthy con-trols. PBMC were stimulated with 10 ng/ml LPS in the ab-sence or presence of various concentrations of KBT, and thesupernatants were harvested after 24 h. Culture supernatantswere assayed for TNF-� levels by ELISA method. As shownin Table 1, addition of 1 mg/ml of KBT significantly in-hibited TNF-� production by 46.08 ± 0.59% (P < 0.05).PBMC cytotoxicity was not observed at the same conditionby KBT (Fig. 1).
3.2. Inhibitory effect of KBT on LPS-induced IL-10production
Next, we examined the effect of KBT on LPS-inducedIL-10 production from PBMC of CFS patients and healthycontrols. PBMC were stimulated with 10 ng/ml LPS in the
256 H.-Y. Shin et al. / Journal of Ethnopharmacology 90 (2004) 253–259
Table 1Inhibitory effect of KBT on LPS-induced TNF-� production from PBMCof healthy control and CFS patients
PBMC (2× 105) from healthy control and CFS patients were stimulatedwith 10 ng/ml LPS in the presence of KBT. The supernatants were har-vested after 24 h of culture. IL-10 secreted into the medium are presentedas the mean± S.E.M. of six independent experiments.
∗ P < 0.05: significantly different from LPS.
0 0.01 0.1 10
20
40
60
80
100
Cel
l via
bilit
y (%
)
KBT (mg/ml)
Fig. 1. Effect of KBT on the cell viability in PBMC. Cell viability wasevaluated by MTT assay 24 h after KBT treatment (0.01–1 mg/ml) inPBMC. The percentage of viable cells was over 95% in each condition.Data represent the mean± S.E.M. of six independent experiments.
absence or presence of various concentrations of KBT, andthe supernatants were harvested after 24 h. As shown inTable 2, KBT (1 mg/ml) significantly inhibited IL-10 pro-duction by 50.00± 0.27% (P < 0.05).
Table 2Inhibitory effect of KBT on LPS-induced IL-10 production from PBMCof healthy control and CFS patients
PBMC (2× 105) from healthy control and CFS patients were stimulatedwith 10 ng/ml LPS in the presence of KBT. The supernatants were har-vested after 24 h of culture. IL-10 secreted into the medium are presentedas the mean± S.E.M. of six independent experiments.
∗ P < 0.05: significantly different from LPS.
Table 3Inhibitory effect of KBT on LPS-induced TGF-�1 production from PBMCof control and CFS patients
Treatment TGF-�1 production (ng/ml) Inhibition (%)
PBMC (2× 105) from healthy control and CFS patients were stimu-lated with 10 ng/ml LPS in the presence of KBT. The supernatants wereharvested after 24 h of culture. TGF-�1 secreted into the medium arepresented as the mean± S.E.M. of six independent experiments.
∗ P < 0.05: significantly different from LPS.
3.3. Inhibitory effect of KBT on LPS-induced TGF-β1production
LPS-induced TGF-�1 production was measured fromPBMC of CFS patients and healthy controls after KBTtreatment. PBMC were stimulated with 10 ng/ml LPS inthe absence or presence of various concentrations KBT,and the supernatants were harvested after 24 h. All super-natants were measured as described inSection 2. As shownin Table 3, KBT (1 mg/ml) significantly inhibited TGF-�1production by 50.71± 0.59% (P < 0.05).
3.4. Inhibitory effect of KBT on LPS-induced IFN-γ
production
We finally examined the inhibitory effect of KBT onLPS-induced IFN-� production from PBMC of CFS patientsand healthy controls. PBMC were stimulated with 10 ng/mlLPS in the absence or presence of various concentrationsKBT, and the supernatants were harvested after 24 h. Asshown inTable 4, KBT (0.01 mg/ml) significantly increasedIFN-� production (P < 0.05).
Table 4Effect of KBT on LPS-induced IFN-� production from PBMC of controland CFS patients
PBMC (2× 105) from healthy control and CFS patients were stimulatedwith 10 ng/ml LPS in the presence of KBT. The supernatants were har-vested after 24 h of culture. IFN-� secreted into the medium are presentedas the mean± S.E.M. of six independent experiments.
∗ P < 0.05: significantly different from LPS.
H.-Y. Shin et al. / Journal of Ethnopharmacology 90 (2004) 253–259 257
Fig. 2. HPLC chromatogram of the KBT. Standard solution of KBT wasprepared by dissolving in distilled water (10 mg/100 ml). The injectionvolume was 20�l and the detection was made at 254 nm.
3.5. Characterization of the principal components of KBT
The KBT was analyzed by HPLC. Chromatogram of theKBT is shown inFig. 2. Peaks of the principal componentshave not yet been identified in this study (Fig. 2).
4. Discussion
It has been suggested that abnormal production of cy-tokines may play a role in the pathogenesis and clinicalmanifestations of CFS (Buchwald and Komaroff, 1991). Anumber of investigators have reported levels of certain cy-tokines in serum and in culture supernatants. TNF-� is apro-inflammatory molecule that appears to play a role in thepathogenesis of AIDS and multiple sclerosis, both associ-ated with chronic fatigue (Matsuyama et al., 1991; Brosnanet al., 1988). TNF provokes slow-wave sleep when placedin the lateral ventricles of experimental animals (Shohamet al., 1987). Chao et al. reported increased TNF-� pro-duction by PBMC of CFS in response to LPS (1991). Inthe present study, we observed a significant inhibition ofLPS-induced TNF-� production in PBMC of CFS patientsby KBT. In CFS patients, spontaneously produced IL-10 byboth adherent monocytes and non-adherent lymphocytes and
by phytoagglutinin-activated non-adherent monocytes weredecreased (Gupta et al., 1997). Fatigue is a condition whichis also associated with multiple sclerosis. Multiple sclerosispatients show decreased IL-10 levels, in particular, duringactive disease (van Boxel-Dezaire et al., 1999). LPS-inducedcytokine secretion in whole blood cultures showed a signif-icant increase in IL-10 as compared with controls (Visseret al., 2001). TGF-�1 has been implicated in the pathogen-esis of a number of diseases including infection with intra-cellular pathogens (Border and Ruoslahti, 1992; Koyanagiet al., 2000; Barral et al., 1993; Reed, 1999). Serum bioactiveTGF-�1 levels were higher in patients with CFS and Chronicidiopathic thrombocytopenic purpura (ITP) than in controlsubjects (Chao et al., 1991). Chronic ITP patients with ac-tive disease had a reduced PBMC production of the TGF-�1(Andersson et al., 2002). In the present study, LPS-inducedTGF-�1 production was increased in PBMC cultures de-rived from patients with CFS and controls. However, KBTinhibited production of TGF-�1 in LPS induced PBMC ofCFS patients. These results indicate that KBT can regulateTGF-�1 production in PBMC treated with LPS.
Our results showed that LPS-induced IFN-� productionwas slightly increased comparing to unstimulated PBMCand that LPS-induced IFN-� production was increased byKBT. IFN-� is an immunoregulatory molecule, enhancingboth NK cell cytotoxicity and causing inhibition of suppres-sor T lymphocyte activity (Targan and Stebbing, 1982; Knopet al., 1982). Two groups have found impaired IFN-� pro-duction on mitogenic stimulation of PBMC from CFS pa-tients (Visser et al., 1998; Klimas et al., 1990). In contrast,Morte et al. (1988), observed normal interferon production,andAltmann et al. (1988), andRasmussen et al. (1991), ob-served increased production in CFS (1988, 1991). Furtherresearch in this area will shed more light on the usefulnessof IFN-� in the diagnosis and treatment of CFS patients.
We can reasonably conclude that KBT treatment pro-foundly affect the LPS-induced TNF-�, IL-10, TGF-�1,and IFN-� production in PBMC of CFS patients. However,the effect was not directly proportional to concentration.We assume that may be because KBT is a crude extract orwas slightly absorbed by the cells, but further detailed studyis needed. In addition, the immune-profiling of CFS patientswill provide valuable aid for the individuals to guaranteehomogeneity of the PBMC.
Acknowledgements
This work was supported by Korea Research FoundationGrant (KRF-2001-042-F00118).
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Journal of Ethnopharmacology 90 (2004) 261–265
Cytotoxic oleanane triterpenoids from the rhizomes ofAstilbe chinensis (Maxim.) Franch. et Savat.
Hong-Xiang Suna,b,∗, Yi-Ping Yec, Yuan-Jiang Pana,∗a Department of Chemistry, Zhejiang University, Hangzhou 310027, PR China
b College of Animal Sciences, Zhejiang University, Hangzhou 310029, PR Chinac Institute of Materia Medica, Zhejiang Academy of Medical Sciences, Hangzhou 310013, PR China
Received 4 April 2003; received in revised form 25 September 2003; accepted 7 October 2003
Astilbe chinensis (Maxim.) Franch. et Savat. (Saxifra-gaceae) is a perennial herbaceous plant growing at an alti-tude of 390–3600 m in China, Russia, Japan, and Korea. Itsrhizome, known as “Luo Xinfu” (Chinese name), has beenused for headache, arthralgia, chronic bronchitis, and stom-achalgia in traditional Chinese medicine (Pan, 1985; Pan,1995). Pharmacological experiments indicated the extractsfrom Astilbe chinensis had antineoplastic and immunopoten-tiating activities (Chen et al., 1996). However, until reportedhere, no constituents have been elucidated as principles forthe antineoplastic action of the rhizomesAstilbe chinensis.Previously we isolated�-sitosterol palmitate, daucosterol,�-sitosterol, and bergenin from the rhizomes ofAstilbe chi-nensis (Sun et al., 2002). In the course of this investigation,the petroleum ether-soluble extract from the rhizomes ofAstilbe chinensis was found to exhibit distinctive cytotoxicactivity. Bioassay-directed purification of the extract usingcolumn chromatography afforded four cytotoxic oleanane
triterpenoids. To our knowledge, oleanane-type triterpenoidshave seldom shown the presence of a carboxyl group at C-14position; moreover, their cytotoxicity has not been reportedpreviously. This paper describes the details of isolation andin vitro cytotoxicity toward HO-8910, Hela, and HL60 ofthese compounds, as well as an apoptotic effect on the HO-8910 cells by compound1.
2. Materials and methods
2.1. General
Melting points were determined on a Shimadzu LIBRORAEC-200 instrument. IR spectra were obtained for a KBrpellet on Perkin-Elmer 577 spectrometer. HREIMS, EIMSdata were obtained by VG AUTOSPEC 800 MS spectrom-eter with glycerol as matrix.1HNMR, 13CNMR, DEPT,1H−1H COSY, HMQC, and HMBC spectra were recordedin CDCl3 or C5ND5 at 500 MHz for1H, and 125 MHz for13C on a Bruker DRX-500 NMR spectrometer using tetram-ethylsilane (TMS) as an internal standard. Column chro-matography procedures (CC) were performed with Silicagel (200–300 mesh, Qingdao Haiyang Chemical Co. Ltd.).
262 H.-X. Sun et al. / Journal of Ethnopharmacology 90 (2004) 261–265
Analytical thin layer chromatography was carried out withKiesel gel 60 F254 (Merck, Germany), and the spots werevisualized by spraying with 10% H2SO4 in C2H5OH fol-lowed heating (100◦C).
2.2. Plant material
The rhizomes ofAstilbe chinensis were collected in Anjicounty, Zhejiang province, China in August 2001. Theplants were identified asAstilbe chinensis (Maxim.) Franch.et Savat. by prof. Xiang-Ji Xue, College of Pharmaceu-tical Science, Zhejiang University. A voucher specimen(No. 200120) was deposited in Laboratory of Nature andBiochemistry, College of Science, Zhejiang University.
2.3. Extraction and isolation
The rhizomes ofAstilbe chinensis were dried at 40◦Cin the dark, in a ventilated hood, and ground. The mate-rial (5.0 kg) was extracted with MeOH three times (each25 l) at room temperature. The MeOH extract was con-centrated, suspended in H2O, and sequentially partitionedwith petroleum ether, EtoAc, andn-BuOH. The bioactivepetroleum ether extract (40.0 g) was absorbed onto silica gel(60 g) and chromatographed on a silica gel (600 g) columneluted with petroleum ether–EtoAc (50:1, 30:1, 15:1, 5:1,3:1, 2:1) gradients. The eluted fractions were evaluated byTLC and combined to give Frs. 1–19, respectively. Frs. 11,12, and 14 were recrystallized with EtOAC–CH3OH (1:1) toafford compounds1 (1.1 g),2 (1.8 g), and3 (0.6 g), respec-tively. Fr. 10 (1.2 g) was separated on silica gel (24 g) col-umn with CH3Cl (0.9 l) to afford pure compound4 (0.2 g).
2.4. Human tumor cell lines
The human ovarian carcinoma cells (HO-8910), humancervical squamous carcinoma cells (Hela), and humanleukemic cells (HL60) were obtained from ATCC (Ameri-can Type Culture Collection). All cell lines are propagatedas monolayers in RPMI-1640 containing 10% fetal bovineserum (Gibco Co Ltd.), 20 mM HEPES, 2 mMl-glutamine(Sigma), and gentamycin (100�g/ml) (Shanghai 4th PharmLtd.) at 37◦C in a 5% CO2 humidified atmosphere.
2.5. MTT assay
Assessment of in vitro antineoplastic activity was deter-mined according to the methods (Fatope et al., 1996). Briefly,cells were seeded in 96-well plates with 5×104 cells/ml andincubated at 37◦C for 48 h prior to drug addition to allowattachment of cells. The extract or compounds were solu-bilized in 100% DMSO and further diluted in RPMI-1640(Sigma) containing 10% fetal bovine serum (Gibco Co Ltd.),20 mM HEPES, 2 mMl-glutamine (Sigma), and gentamycin(100�g/ml) (Shanghai 4th Pharm. Ltd.) to a maximal fi-nal DMSO concentration of≤0.25%, which is not growth
inhibitory. Each cell line was treated with five concentra-tions of the extract or compounds (two long ranges). After48 h incubation, 50�l of MTT solution (5 mg/ml) (Sigma)was added to each well. After further 4 h incubation, 200�lof dimethyl sulfoxide (DMSO) working solution was addedto each well and formazan crystals in each well dissolvedby vibration. Optical density was determined at two wave-lengths (590 and 700 nm) by an ELISA reader (Shanghai,China). Each test was performed in triplicate.
2.6. Determination of cell growth curve
The effect of compounds1–4 on the growth curve ofHO-8910 cells was determined according to the methods(Fei et al., 2002). Briefly, cells (3× 104 cells per well)were seeded into flat-bottom microtiter plate (Nunc), andthen various drug concentrations (final drug concentration1.0�g/ml, 4.0�g/ml) or medium were added to each well.The plates were incubated at 37◦C in a 5% CO2 humidifiedatmosphere. The numbers of the cultured cells were countedwith a haemocytometer following staining with 0.4% trypanblue every other 24 h, and then cell saturation density, killingpercent and doubling time were calculated.
2.7. Apoptotic assay
The apoptotic effects of compound1 on HO-8910 cellswas determined according to the methods (Chen and Chen,2002). Briefly, HO-8910 cells (4×104 cells/ml) were seededinto flat-bottom microtiter plate (Nunc). The plates were thenincubated at 37◦C in a humid atmosphere with 5% CO2.Various drug concentrations (final drug concentration 40,20, 10, 5, 2.5�g/ml) or DMSO were added to the cultures48 h after subculture. After 48 h, the acridine orange solution(100�g/ml) (Sigma) was added to each well. After a further10 min incubation, cultured cells were harvested, fixed andobserved using phase contrast and fluorescence microscope.
3. Results and discussion
In the present study aiming at the identification of sec-ondary metabolites with cytotoxic activity from the rhizomesof Astilbe chinensis, its methanol extract was investigated.Among the partitioned fractions of the methanol extract, theEtoAc and petroleum ether-soluble extract showed someinhibitory effect on HO-8910 cells. Their 50% inhibitoryconcentration (IC50) values were 52.90 ± 2.65�g/ml and118.75 ± 7.18�g/ml against HO-8910 cells, respectively,while those ofn-BuOH, and water-soluble fractions weremuch higher than 100�g/ml. This result implied that thewater andn-BuOH extracts are essentially inactive againstHO-8910 and the active principles were distributed to theEtoAc and petroleum ether-soluble extract. The petroleumether extract was subjected to silica gel column chromatog-raphy affording four active compounds, which belong to
H.-X. Sun et al. / Journal of Ethnopharmacology 90 (2004) 261–265 263
Fig. 1. Inhibitory effect of compounds1–4 on human ovarian carcinoma cells (HO-8910,�), human cervical squamous carcinoma cells (Hela,�) andhuman leukemic cells (HL60,�). After 48 h exposure to these compounds, cell viability was determined by a MTT assay as described in the text. Thevalues are expressed as means± S.D. (n = 3).
oleanane-type triterpenoids. The structures of compounds1–4 were identified as 3�-hydroxy-olean-12-en-27-oic acid,3�,6�-dihydroxy-olean-12-en-27-oic acid, 3�-acetoxy-olean-12-en-27-oic acid, and 3�-hydroxy-urs-12-en-27-oicacid, respectively (Nagai et al., 1969; Takahashi et al., 1972).
The structures of compounds1–4 were elucidated on thebasis of chemical and spectral evidence. 2D NMR tech-niques including1H−1H COSY, HMQC, HMBC spectra,and HREIMS were extensively applied to establish the struc-tures. The structure of compound1 was confirmed by a sin-gle crystal X-ray diffraction analysis. Copies of the originalspectra are obtainable from the authors.
As shown inFig. 1, compounds1–4 exhibited strong cy-totoxic activity against HO-8910, Hela, and HL60 in vitro ina dose-dependent manner. The 50% inhibitory concentration(IC50) values of compound1 were 8.01±0.89�g/ml, 3.94±0.13�g/ml, and 3.67± 0.19�g/ml against HO-8910, Hela,and HL60, respectively, those of compound2 were 22.24±0.57�g/ml, 11.91 ± 1.02�g/ml, and 24.24 ± 3.82�g/ml,respectively, those of compound3 were 12.90±1.92�g/ml,6.11 ± 0.38�g/ml, and 12.55 ± 1.27�g/ml, respectively,and those of compound4 were 8.71± 1.83�g/ml, 4.05±0.33�g/ml, and 7.22 ± 0.96�g/ml, respectively. With theIC50 values against three tumor cells of less than 10�g/ml,compounds1 and4 should be an effective tumor inhibitor.
To investigate the effect of compounds1–4 on the growthcurve of HO-8910 cells, the cell killing percent, doublingtime (TD), and saturation density were measured by trypan
blue dye staining and are shown inTable 1. Among fourcompounds, compound1 has the highest growth-inhibitoryeffect on HO-8910 cells, the most prolonged rate ofTD andthe highest cell killing percentage.
When HO-8910 cells were treated with compound1,marked morphological changes were observed as compared
Table 1Effect of four compounds on growth curve of HO-8910 cells
264 H.-X. Sun et al. / Journal of Ethnopharmacology 90 (2004) 261–265
Fig. 2. Apoptotic effects of compound1 on human ovarian carcinoma cells (HO-8910). After 48 h treatments with indicated concentration of compound1,the morphological changes of the cells were analyzed by acridine orange fluorescence stain. Densely Kelly fluorescence stain indicated apoptotic bodies.
with DMSO control. As shown inFig. 2, the HO-8910cells treated with compound1 showed nuclear conden-sation, nuclear fragmentation, membrane blebbing, andapoptotic bodies. Compound1 induced apoptotic cell deathin a dose-dependent manner, ranging from 2.5 to 40�g/ml.However, the untreated cells did not show these apoptoticcharacteristics.
In conclusion, the petroleum ether-soluble extract fromthe rhizomes ofAstilbe chinensis exhibited a significant in-hibitory effect on HO-8910, Hela, and HL60 cells. From theextract, four active oleanane-type triterpenoids were isolatedwith cytotoxic activity against HO-8910, Hela, and HL60in vitro in a dose-dependent manner; moreover, compound1 induced apoptotic cell death of the HO-8910. These re-sults suggest that the presence of these active compounds inrhizomes ofAstilbe chinensis might be responsible, at leastin part, for the antineoplastic action of the traditional crudedrug.
Acknowledgements
This work was supported by Grant-in-Aid from theZhejiang Provincial Natural Science Foundation of China(No. 300456), Zhejiang Provincial Science and TechnologyAdministration of Traditional Chinese Medicine Council(021103008) (2002KF02) and Zhejiang Province.
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Pan, J.-T., 1995. A study on the tribe Astilbeae Miq. (Saxifragaceae).Acta Phytotaxonomica Sinica 33, 390–402.
Sun, H.-X., Ye, Y.-P., Yang, K., 2002. Studies on the chemical constituentsin Rhizoma Astilbe Chinensis. China Journal of Chinese Materia Med-ica 27, 751–754.
Takahashi, K., Kanayama, K., Tanabe, Y., Takani, M., 1972. Studies onthe constituents of medicinal plants. XI. Constituents of the roots ofAstilbe thunbergii Miquel var. congests H. Boissieu. Chemical andPharmaceutical Bulletin 20, 2106–2111.
Journal of Ethnopharmacology 90 (2004) 267–272
Protection against murine endotoxemia by treatment withRutaChalepensis L., a plant with anti-inflammatory properties
Liliana Iauka,∗, Katia Manganoa, Antonio Rapisardab, Salvatore Ragusab, Luigi Maiolinoc,Rosario Musumecia, Rosaria Costanzoa, Agostino Serrac, Annamaria Specialea
a Department of Microbiological and Gynaecological Sciences, Section of Microbiology, University of Catania, Via Androne 81, Catania 95124, Italyb Pharmaco-Biological Department, University of Messina, Villaggio Annunziata, Messina 98168, Italy
c ENT Department, University of Catania, Catania, Italy
Received 23 April 2003; received in revised form 29 September 2003; accepted 7 October 2003
Abstract
The anti-inflammatory effect of the extract ofRuta chalepensis L. (Rutaceae) on the course of lethal endotoxemia in BALB/c mice wasstudied. When administered by gavage as 1 g/kg per day starting 14 or 7 days prior to injection of 0.75 mg endotoxin (LPS: lypopolisaccharide),the extract markedly reduced lethality (32.5% in both experiments versus approximately 85% of the control mice). A delay in lethality, butnot cumulative lethality, was observed when prophylaxis was given 24 and 1 h prior to LPS challenge. The effect was associated with reducedLPS-induced blood levels of nitrite, an indicator of nitric oxide production.
Ruta chalepensis L. (Rutaceae) is a perennial herb widelydiffused in the Mediterranean area, usually growing on rockyslopes.Ruta Chalepensis is characterised by glabrous, alter-nate bi-pennatisect leaves with narrow oblong-lanceolate orobovate segments and cymose inflorescence.
Ruta Chalepensis L. has pleiotropic pharmacologicalproperties, attributed to the high content of alkaloids,flavonoids, phenols, amino acids, furocoumarins andsaponins found in the leaves and young stems of the plant(Hnatyszyn et al., 1974). Ruta Chalepensis L. is used in thetraditional medicine of many countries for the treatment ofa variety of diseases.
In Saudi Arabia, a decoction of the aerial parts of theplant is used as an analgesic and antipyretic and for thetreatment of rheumatism and mental disorders. The plant isprescribed in the Indian system of medicine for the treatmentof dropsy, neuralgia, rheumatism and menstrual and other
bleeding disorders. In China, a decoction of the roots of theplant is used as an anti-venom.
The leaves of this plant infused with vinegar are given tochildren for the treatment of convulsion and other nervousdisorders. An aqueous decoction of the leaves is used forthe treatment of fever in Africa (Mansour et al., 1990).
In addition to its described emmenagogue, abortifi-cient, antihelmintic and spasmolytic effects (Font Quer,1962; Di Stasi et al., 1994), Ruta Chalepensis L. also hasanti-inflammatory properties (Atta and Alkofahi, 1998).
To gain insights into the anti-inflammatory prop-erties of this medicinal plant, we studied the effectsof prophylactic treatment withRuta Chalepensis L.endotoxin-induced lethality in mice. This is a prototyp-ical type 1 cytokine-dependent inflammatory conditionwhich serves as a model for human endotoxemia (Glauseret al., 1991). It is provoked by the release into the bloodstream of vasoactive and proinflammatory mediators fromLPS-activated monocytes/macrophages, including nitric ox-ide (NO), oxygen radicals, tumour necrosis factor (TNF),Interleukin (IL)-1, and Interferon (IFN)-� (Bendtzen, 1988;Alexander et al., 1991; Ashkenazi et al., 1991; Glauseret al., 1991; Moncada et al., 1991; Abraham, 1999). On
268 L. Iauk et al. / Journal of Ethnopharmacology 90 (2004) 267–272
the other hand, type 2 anti-inflammatory mediators si-multaneously secreted during the course of the syndromeconstitutes a homeostatic anti-inflammatory attempt. Thesemediators include IL-4, IL-6, IL-10 and IL-13, and thesemay counteract and limit the lethal effect of lypopolisac-charide (LPS) (Gèrard et al., 1993; Muchamuel et al., 1997;Nicoletti et al., 1997a,b, 2001).
The results show that prolonged prophylactic treat-ment withRuta Chalepensis L. markedly reduced the highLPS-induced lethality. The effect was associated with re-duced blood levels of nitrite which is an indicator of NOproduction (Rabinovitch, 1998) but not with LPS-inducedTNF, IL-6 or IL-10.
2. Materials and methods
2.1. Animals
Female, 6-week-old BALB/c mice were purchased fromCharles River (Calco, Italy).
The mice, kept under standard laboratory conditions withfree access to food and water, were allowed to adapt for1 week to the environment before commencing the experi-ments.
2.2. Plant material
Leaves ofRuta Chalepensis L. were collected in the areaaround Messina (Italy) in April 1999. The identification ofthe plant was confirmed according to previously describedmethods by a plant taxonomist (Professor G. Gramuglio,Department of Botany, University of Messina, Italy) usingpreviously described methods (Pignatti, 1982). A voucherspecimen of the plants was deposited in the herbarium ofthe Pharmaco-Biological Department of the University ofMessina.
The fresh material was lyophilised and powdered.
2.3. Preparation of plant extract
For the preparation of the ethanol extract of leaves, anexhaustive extraction of 100 g of the drug was carried out inSoxhlet with 800 ml of ethanol 95%.
The extract was then dried under vacuum. The residue ofthe ethanol extract of the leaves was 26 g.
One gram of ethanol extract ofRuta Chalepensis L. wassolubilised in sterile 2 ml polyethylene glycol and 3 ml oliveoil.
2.4. Induction of shock and treatment regimen
Each mouse was treated with one single i.p. dose of0.75 mg of Escherichia coli-derived lypopolisaccharide,serotype, 055:B5 (Sigma Chimica, Milan, Italy). Previousexperiments performed with this batch of LPS showed thatthis dosage results in 75–100% lethality within 72 h of in-
jection to the same strain of mice. At different time pointsprior to LPS injection, theRuta Chalepensis L. extract wasadministered by gavage at a dosage of 1 g/kg. Control micewere either treated with the vehicle alone or left untreated.
2.5. Measurements of cytokines
Heparinised blood was obtained by cardiac puncture un-der ether anaesthesia. Blood samples were obtained prior to,and 2 and 8 h after i.p. injection of 200�g LPS. TNF, IL-10and IL-6 are not normally measurable in plasma of healthyanimals. This dose of LPS was chosen on the basis of pre-liminary studies showing its ability to induce measurableplasma levels of cytokines (respectively, in control mice:TNF 242± 144, IL-10 228± 82, IL-6 323± 162, in treatedmice TNF 247±124, IL-10 221±86.5, IL-6 318±138), butnot sufficiently high to kill the mice. TNF, IL-6 and IL-10were measured in duplicate by specific solid-phase ELISA.Kits were purchased from Endogen (Cambridge, MA, USA)and used according to the manufacturers’ instructions. Thelower limit of sensitivity of the assays was 10 pg/ml. Theintra- and inter-assay coefficients of variations were within10 and 20%, respectively.
2.6. Measurement of nitrite
In vivo generation of NO was quantified by determinationof nitrite production in serum samples obtained from cardiacblood of mice 2 or 8 h after LPS challenge (Rasmussen et al.,1994). Briefly, serum samples were incubated for 10 min atroom temperature with an equal volume of Griess reagent(0.5% naphthalene diamine dihydrochloride, 1% sulfanil-amide and 2.5% concentrated H3PO4). The absorbance at550 nm was measured in a spectrophotometer against a stan-dard curve.
2.7. Statistics
Mortality were compared using the Log rank (MantelCox) test. The cytokine levels were evaluated by the Mann–Whitney rank sum test.P-values<0.05 were considered sig-nificant.
3. Results
3.1. Effect of prophylactic treatment with Ruta chalepensisL. extract on LPS-induced lethality
As expected, most of the control mice died within 3 daysof LPS injection (Fig. 1a–c). The group lethalities werecomparable regardless of whether they were left untreated,or were treated with the vehicle used to administerRutaChalepensis L. extract (Fig. 1).
In contrast, prophylactic treatment withRuta Chalepen-sis L. extract, administered 7 or 14 days prior to LPS
L. Iauk et al. / Journal of Ethnopharmacology 90 (2004) 267–272 269
Fig. 1. (a–c) Prolonged prophylactic treatment withRuta chalepensis L. diminishes LPS-induced lethality in mice.Ruta Chalepensis L. was administeredby gavage to mice (closed circles) as1 g/kg per day starting 14 days (a), 7 days (b) or 24 and 1 h prior (c) to i.p. injection of 0.75 mg endotoxin(LPS). Control mice were treated with vehicle alone (open circles) or were left untreated (open squares). Each group consists of 40 mice. Data arerepresentative of three independent experiments. Since data were always reproducible (inter-experiment variation less than 5%) data are merged andshown as a single experiment. Cumulative lethalities for (a) were 32.5% (13/40) for theRuta Chalepensis L.-treated mice vs. 87.5% (35/40) and 85%(34/40) vs. vehicle-treated and untreated mice (P < 0.0001 vs. both groups by the Mann–Whitney rank sum test). For (b), cumulative lethalities were32.5% (13/40) for theRuta Chalepensis L.-treated mice vs. 82.5% (33/40) and 85% (34/40) vs. vehicle-treated and untreated mice (P < 0.0001 vs. bothgroups by the Mann–Whitney rank sum test). For (c), cumulative lethalities were 72.5% (29/40) in the group of mice treated withRuta Chalepensis L.vs. 85% (34/40) and 87.5% (35/40) of the vehicle-treated and untreated mice (P = 0.043 vs. both groups by the Mann–Whitney rank sum test).
challenge, greatly improved the survival of the treated mice(P < 0.01 (Fig. 1a and b)). “Prolonged” prophylaxis withRuta Chalepensis L. extract did not merely delay the lethalaction of LPS, as none of the remaining mice from theRutaChalepensis L.-treated died during a follow-up period of 1week. “Short” prophylaxis treatment withRuta ChalepensisL. extract 24 or 1 h prior to LPS challenge was less effec-tive than the prolonged treatment regime, even if the effectwas still statistically significant as the drug delayed thekinetic of mortality induced by LPS (Fig. 1c). The effectof Ruta Chalepensis L. extract was dose-dependent sincethe mice receiving the plant on “prolonged” prophylactictreatment at the dose of either 0.1 or 0.001 g/kg exhibitedcumulative lethality and kinetic of mortality induced byLPS that were very similar to those found in control mice(Fig. 2).
3.2. Effect of Ruta chalepensis L. on the in vivo cytokineand NO response to LPS
The effects ofRuta Chalepensis L. on the release of TNF,IL-6 and IL-10 induced by LPS were studied using three
Fig. 2. Dose-dependent effect ofRuta chalepensis L. in counteracting thelethal effects of LPS.Ruta Chalepensis L. was administered by gavageto mice (closed circles) as either 0.1 g/kg (closed circles) or 0.001 g/kg(open circles) per day starting 14 days prior to i.p. injection of 0.75 mgendotoxin (LPS). Control mice were left untreated (open squares). Eachgroup consists of 20 mice. Data are representative of two independentexperiments. Since data were reproducible (inter experiment variation lessthan 5%) data are merged and shown as a single experiment. Comulativelethalities were 85% (17/20) and 90% (18/20) in the mice treated withRuta Chalepensis L. vs. 85% (17/20) of those left untreated (P = NS).
270 L. Iauk et al. / Journal of Ethnopharmacology 90 (2004) 267–272
Fig. 3. Effects ofRuta chalepensis L. prophylaxis on LPS-induced TNF,IL-6 and IL-10 in plasma of BALB/c mice. Mice, treated for sevenconsecutive days with either 1 g/kg per dayRuta Chalepensis L. (closedcircles) or with the vehicle (open circles), were killed prior to and 2 and 8 hafter i.p. injection of 200�g LPS. Plasma samples from individual micewere obtained from cardiac blood to measure TNF, IL-6 and IL-10. Eachgroup consisted of 15 mice. Data are representative of three independentexperiments. Since data were highly reproducible and inter-experimentvariability was always less than 20% the data are merged and shown hereas the result of a single experiment.
groups of mice treated with the plant extract, its vehicle,or left untreated during the 7 days prior to LPS adminis-tration. For cytokine and nitrite measurements, animals inboth groups were killed before LPS injection (time 0), and2 and 8 h thereafter. As shown inFig. 3, the plasma levelsof TNF, IL-6 and IL-10, all below the limit of sensitivityof the assays at time 0, increased considerably 2 and/or 8 hafter challenge with LPS.
There were no significant differences between theRutaChalepensis L.-treated mice and the vehicle-treated controlmice (Fig. 2). In contrast, the levels of nitrite found in seraof Ruta Chalepensis L.-treated mice 2 and 8 h after LPSinjection were significantly lower than those found both invehicle-treated and in untreated mice (Fig. 4).
Fig. 4. Effects ofRuta chalepensis L. prophylaxis on LPS-induced nitrite inserum of BALB/c mice. Serum samples from cardiac blood were obtainedfrom mice using the same experimental conditions described in the legendto Fig. 2. Closed circles are mice treated withRuta Chalepensis L. andopen circles the mice treated with the vehicle alone. Each group consistedof 15 mice. Data are representative of three independent experiments.Since data were highly reproducible and inter experiment variability wasalways less than 20% the data are merged and shown here as the resultof a single experiment.∗P < 0.001 vs. vehicle-treated mice by ANOVA.
4. Discussion
The results of the present study demonstrate for the firsttime that prophylactic treatment withRuta chalepensis L.extract counteracts the lethal effects of LPS in BALB/c mice.
Phytochemical screening showed that the aerial parts ofRuta chalepensis L. yielded coumarins (Ulubelen et al.,1986), as well as alkaloids (Ulubelen et al., 1986) andflavonoids (Mansour et al., 1990).
Pharmacological investigations clearly indicated thatanti-inflammatory activity in many plants has been attributedto their flavonoid and sterol contents (Mansour et al., 1990).
We suppose in this study that the anti-inflammatory ac-tivity of ethanol extract ofRuta chalepensis L. is due toflavonoids, although their mechanisms of action are far fromclear.
The strongest effect was achieved when the plant extractwas administered daily for as long as 7 or even 14 days priorto LPS challenge. When treatment was commenced 24 and1 h prior to LPS injection, there was a significant delay inlethality, but not a reduction of mortality.
In spite of the clinical efficacy ofRuta ChalepensisL. in reducing LPS-induced lethality, the underlying im-munopharmacological mode of action remains poorly un-derstood.
In fact, neither the LPS-induced blood levels of TNF,nor those of IL-6 and IL-10 (Gèrard et al., 1993) whichexert pathogenic (Ashkenazi et al., 1991) and protective(Gèrard et al., 1993; Rasmussen et al., 1994; Dalrympleet al., 1996; Nicoletti et al., 1997a) actions, respectively, inthis model differed significantly between the mice treatedwith Ruta Chalepensis L. and the controls. This finding wasunexpected as drugs capable of diminishing LPS-induced
L. Iauk et al. / Journal of Ethnopharmacology 90 (2004) 267–272 271
lethality in mice almost always reduce circulating TNFlevels or increase those of IL-6 and IL-10 (Nicoletti et al.,1995; Tarazona et al., 1995; Durez et al., 1999). However,reduction in LPS-induced TNF is not a necessary require-ment for drug-induced reduction in endotoxin lethality(Rasmussen et al., 1994).
Nonetheless, because it is known that endogenous TNF,along with other proinflammatory mediators, plays a pivotalrole in lethal murine endotoxemia (Ashkenazi et al., 1991)interfering with TNF synthesis and/or production it may cer-tainly be an important part of any protective effect exertedby compounds exerting beneficial effects in murine endo-toxemia.
It is possible, for example, thatRuta Chalepensis L. pro-phylaxis inhibited the function rather than the synthesis ofTNF, for example, by down-regulating the expression ofTNF receptors on the surface membrane of the target cellsor by augmenting the blood levels of soluble TNF receptor,which serves as a naturally occurring inhibitor of circulatingTNF. It is also possible thatRuta Chalepensis L. prophylaxisinhibited the synthesis/action of other type 1 proinflamma-tory cytokines involved in the pathogenesis of lethal endo-toxemia such as IL-1 and IFN-� (Heinzel, 1990; Ohlssonet al., 1990; Alexander et al., 1991; Nicoletti et al., 2001).
Finally, an effect ofRuta Chalepensis L. on type 2 cy-tokines other than IL-6 and IL-10 such as, for example, IL-4and IL-13 can not be ruled out (Muchamuel et al., 1997;Nicoletti et al., 1997b; Baumhofer et al., 1998). Studies arein progress to test these hypotheses.
Increased production of NO has repeatedly been observedduring murine and human endotoxemia. It occurs after in-duction of nitric oxide synthase (iNOS) in endothelial cellsby LPS and proinflammatory cytokines (IL-1, TNF) (seeMoncada et al., 1991; De Angelo, 1999for a review). Al-though NO plays a role in some of the clinical featuresof endotoxemia, such as hypotension, collapse, myocardialand kidney dysfunctions and pulmunary oedema (Hnatyszynet al., 1974; Ullrich et al., 2000) conflicting results havebeen obtained regarding its role on LPS-induced lethalityin mice, with some studies supporting and other denying apathogenic role for NO in this system (Moncada et al., 1991;MacMicking et al., 1995; De Angelo, 1999; Hollenberget al., 2000; Iskit and Guc, 2001).
We therefore do not know whether the observed reductionin NO production observed inRuta Chalepensis L.-treatedmice injected with LPS is either an epiphenomenon or thecause of increased survival. To clarify this issue, we arepresently studying whether the effect of the plant extractdisappears in a system in which NO is pharmacologicallyor genetically (iNOS−/− mice) suppressed.
Ruta Chalepensis L. prophylaxis needed to be started atleast 7 days before LPS challenge for its preventive effectsto became fully evident. The reason for this is unknown, butit suggests thatRuta Chalepensis L. needs time to triggeranti-inflammatory pathways that prime the animals for in-creased LPS resistance. If the results can be transferred to
the clinical setting, the necessity to start prophylaxis withRuta Chalepensis L. long before LPS challenge limits thepotential use of this plant in humans, where anti-LPS strate-gies almost always have to be effective in patients with al-ready established symptoms of endotoxemia (seeAbraham,1999for a review).
This study clearly shows the properties ofRuta Chalepen-sis L. in a well-known model of acute immunoinflamma-tion, so it would be interesting to continue our studiesin other experimental models where NO has pathogenicrelevance. These models include type 1 diabetes mellitusin NOD mice, systemic lupus erythematosus-like syn-drome in MRL-lpr mice, murine type II collagen-inducedarthritis and adotpively-transferred experimental allergicencephalomyelitis (Weinberg et al., 1994; Gold et al., 1997;Rabinovitch, 1998; Vermeire et al., 2000).
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Journal of Ethnopharmacology 90 (2004) 273–277
Modulation of rat and human cytochromes P450 involved in PhIP and4-ABP activation by an aqueous extract ofPhyllanthus orbicularis
a Unitat de Microbiologia (Ciències), Dpt. de Genètica i de Microbiologia, Universitat Autònoma de Barcelona,Edifici Cn 08193, Bellaterra, Barcelona, Spain
b Departament de Farmacologia, Terapéutica i Toxicolog´ıa, Facultat de Veterinaria, Edifici V 08193, Bellaterra, Barcelona, Spainc Laboratorio de Genética Toxicológica, Facultad de Biolog´ıa, Universidad de La Habana, Calle 25 No. 455, Ciudad de la Habana, Cuba
d Centro de Aplicaciones Tecnológicas y desarrollo Nuclear (CEADEN), Calle 30 No. 502 e/5ta y 7ma,P.O. Box 6122, Miramar, Playa, Ciudad de la Habana, Cuba
Received 22 May 2003; received in revised form 1 October 2003; accepted 7 October 2003
Over the last few decades, major efforts have been madeto identify natural products and dietary constituents whichcan protect against DNA damage caused by environmentaland endogen mutagens. The mechanisms whereby antimu-tagenic compounds exert their effects are not clear. Specialattention has been paid to intracellular mechanisms basedeither on blocking or competition reactions with xenobioticmetabolising enzymes, trapping electrophiles and free rad-icals and protection of nucleophilic sites in DNA either onmodulation of metabolising enzymes, such as the enhance-ment of enzymes involved in detoxification of mutagens orthe inhibition of metabolic activation of promutagens (De
Flora and Ramel, 1988; De Flora, 1998; De Flora et al.,2001; Dashwood, 2002).
Phyllanthus orbicularisHBK (Euphorbiaceae) is an en-demic plant of Cuba, its aqueous extract having differentantiviral activities. Besides these properties, we have re-ported that this plant extract also has antimutagenic proper-ties. Thus, it protects against damage induced by hydrogenperoxide because reduction of both the number of chromo-some aberrations and revertants in Chinese hamster ovary(CHO) cells and in theSalmonellaassay, respectively, wasfound (Sánchez-Lamar et al., 1999; Ferrer et al., 2002).
In theSalmonellaassay, this plant extract also has a clearantimutagenic effect against different promutagenic aro-matic amines (AAs), the main mechanism involved in the de-crease of the mutagenesis mediated bym-phenylenediamine,2-aminoanthracene and 1-aminopyrene was interactions ofsome components of the extract with microsomal enzymes(Ferrer et al., 2001). Given these results, we decided to
274 M. Ferrer et al. / Journal of Ethnopharmacology 90 (2004) 273–277
investigate if the plant extract can modulate the activityof the CYP1A subfamily of cytochromes P450 (CYPs),which are the main enzymes involved in the metabolism of2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP)and 4-aminobiphenyl (4-ABP) to mutagenic compounds(Weisburguer, 1988; Zhao et al., 1994; Windmill et al.,1997). Both AAs are recognized animal (PhIP and 4-ABP)and human (4-ABP) carcinogens. PhIP is the most abundantheterocyclic amine detected in burned meats of consump-tion (Knize et al., 1997). In humans, PhIP can be found inthe urine, indicating preliminary evidence that PhIP–DNAadducts occur in human colon tissues and suggesting apossible contribution of this compound to human colorectalcancer risk (Gooderham et al., 2002). In rats, PhIP inducestumours in mammary glands and in the colon (IARC, 1993).4-ABP appears to be an environmental pollutant arisingfrom combustion sources such as cigarette smoke and syn-thetic fuels, and it is a demonstrated bladder carcinogen inhumans and animals (IARC, 1987).
To conduct the proposed study, experiments were per-formed usingSalmonellastrains YG1024 and YG1029(Watanabe et al., 1990), expressing high levels of acetyl-CoA:N-hydroxylarylamineO-acetyltransferase, and espe-cially sensitive to PhIP and 4-ABP, respectively. Liver mi-crosomal fractions from induced rats and humans were usedas metabolic activation systems of both AAs to study the an-timutagenesis of the plant extract against both compounds.Inhibition of alkoxyresorufinO-dealkylation activities ofboth microsomal fractions by thePhyllanthus orbicularisextract was determined.
2. Materials and methods
2.1. Chemicals
Glucose-6-phosphate (CAS No. 54010-71-8), resorufin(CAS No. 34994-50-8), methoxyresorufin (CAS No. 5725-89-3), ethoxyresorufin (CAS No. 5725-91-7) and 4-ABP(CAS No. 92-67-1) were from Sigma (Sigma-Aldrich Co.,USA). PhIP (CAS No. 105650-23-5) was from Wako (Japan)and NADP (CAS No. 24292-60-2) and NADPH (CAS No.2646-71-1) were supplied by Roche (Germany).
2.2. Aqueous extract of Phyllanthus orbicularis
Phyllanthus orbicularis HBK (Euphorbiaceae) plantswere collected from Cajálbana, Pinar del Rıo, Cuba. Thespecimens were authenticated and stored at the CubanBotany Garden (No. 7-220-HAJB) by Professor Dr. Ros-alina Berazaın.
The plant stem and leaves were dried at 50◦C for 3 days,before grinding. The ground material was suspended 1:10(w/v) in distilled water and shaken for 4 h in a water bath at70◦C. The resulting suspension was filtered and the filtratecentrifuged at 10,000 rpm. using a JA-10 rotor in a Beckman
JA-21 centrifuge. The supernatant was lyophilised for furtheruse (del Barrio and Parra, 2000).
2.3. Salmonella mutagenicity assay
The Salmonella assay was performed following themethod of plate incorporation as previously described, us-ing three plates per dose (Maron and Ames, 1983). Ratliver S9 was supplied by MOLTOXTM, USA, obtained fromphenobarbital/�-naphthoflavone-induced rats. Likewise, hu-man liver microsomal fraction (MOLTOXTM, USA) wasprepared from a pool of healthy livers of human donorsdeath by car accident and rejected for transplantation. Bothmetabolic activation system were used at 10% (v/v) in theactivation mix, according to that previously reported for thestudied promutagens (Dang and McQueen, 1999; Hakuraet al., 1999). Salmonellastrains YG1024 and YG1029 werekindly provided by Dr. T. Nohmi.
2.4. Determination of the antimutagenic effect
Antimutagenesis was expressed as the percentage of Re-maining Mutagenesis (%RM). This value was calculatedfrom a minimal of three independent experiments as follows:
%RM = 100×
number of induced revertants per plate in thetreatment with both AA and plant extract
number of induced revertants per plate withAA and without plant extract
.
The number of induced revertants was calculated by sub-tracting the number of spontaneous revertants obtained inthe negative control of each experiment.
2.5. Alkoxyresorufin O-dealkylation assays
Demethylation (MROD) and deethylation (EROD) ofalkoxyresorufins by induced rat and human fractions weredetermined in the presence of increasing concentrationsof plant extract, following the fluorimetric method asdescribed (Burke et al., 1985). A final concentration of5.0�M for both substrates (ethoxy and methoxyresorufin)was employed. Because it is known that using S9 fractions,resorufin may be metabolised by DT-diaphorase (quinonereductase, EC 1.6.99.2) which reduce resorufin producedin the dealkylation reaction (Nims et al., 1984), the inclu-sion of controls without the addition of plant extract in ourassays solved this possible interference. The assays wereconducted in a total volume of 2 ml, containing 2 mg ofmicrosomal proteins per ml, and reaction was started by theaddition of 0.5 mM NADPH. The formation of resorufinwas continuously measured for 3 min in a fluorescencespectrophotometer RF-551S (Shimadzu, Japan) with theexcitation and emission wavelength set at 530 and 585 nm,respectively.
M. Ferrer et al. / Journal of Ethnopharmacology 90 (2004) 273–277 275
3. Results
Although hepatic activation of AAs is mainly carried outby CYP activities, it is known that the level of mutagenic re-sponse is different when these compounds are metabolisedby liver enzymes from induced rats and humans (Burkeet al., 1985; Johnson et al., 1996; Dang and McQueen, 1999;Hakura et al., 1999). According to this and due to the dif-ferent CYP1A levels in rat induced and human microso-mal fractions used, it was necessary to select the suitableconcentration of both AAs for further antimutagenesis stud-ies. Preliminary experiments were performed, following theSalmonellaplate incorporation method with both fractions,to know the mutagenic response of PhIP and 4-ABP instrains YG1024 and YG1029, respectively. In these assays,several concentrations of PhIP and 4-ABP were tested, be-ing chosen 5 and 3�g/plate of PhIP and 4-ABP, respectively,for further experiments with induced rat S9. Likewise, andaccording to the number of induced revertants per plate inpresence of the promutagens, 50 and 30�g/plate of PhIPand 4-ABP, respectively, were the selected doses for assayswith human fraction. At these doses, and using the same ex-perimental conditions, the antimutagenesis of the plant ex-tract in a range of concentrations from 100 to 1000�g/platewas studied. Results found indicated that the plant extractproduced a significant decrease on the mutagenesis inducedby these promutagens, the optimal antimutagenesis beingreached at 1000�g/plate of the extract (Table 1). The bestantimutagenic effect was found when human enzymes acti-vated both AAs, although it was more marked for the 4-ABP.
Following the same approaches that we used in studies ofantimutagenesis of other AAs (Ferrer et al., 2001), we de-termined that the main mechanism involved in the decreaseof the mutagenesis mediated by 4-ABP and PhIP was inter-actions of some components of the extract with microsomalenzymes. Likewise, the most relevant antimutagenic effectwas observed in presence of human S9 (data not shown).
Assays of dealkylation of alkoxyresorufins allow for theaccurate identification of induction and inhibition of CYP
Table 1Optimal antimutagenic effect of thePhyllanthus orbicularisextract against 4-ABP and PhIP, using rat and human S9
4-ABP 3 0 R 391.0± 17.0 1003 1000 R 220.0± 12.7 52.2
30 0 H 311.3± 12.5 10030 1000 H 56.0± 3.1 7.56
PhIP 5 0 R 1970.0± 17.7 1005 1000 R 1025.7± 8.3 51.2
50 0 H 880.0± 1.5 10050 1000 H 259.0± 2.4 26.4
Standard deviations from three or more independent experiments are shown.R, rat S9; H, human S9.YG1024 and YG1029 spontaneous revertants per plate with rat S9 were 33.8±1.5 and 38.5±2.5 and with human S9 35.2±0.3 and 44.7±1.2, respectively.YG1024 and YG1029 revertants per plate with S9 and 1000�g/plate of plant extract were 35.5 ± 0.9 and 40.0 ± 1.2 using rat S9, and with human S938.8 ± 0.5 and 46.0 ± 1.8, respectively.
0
20
40
60
80
100
0 20 40 50 60 100 120 140 160
Plant extract (µg/ml)
AR
OD
(%
from
the
cont
rol) EROD
MROD
Fig. 1. Inhibition of alkoxyresorufinO-dealkylation activities of rat liver S9by thePhyllanthus orbicularisextract. Standard deviation from a minimalof three independent experiments are shown. The basal EROD and MRODactivity (100%) corresponds to 63.0 and 34.5 nmols of resorufin/min/mgprotein of S9, respectively.
isoforms (Burke et al., 1985, 1994). In order to determinewhether the plant extract has some compound able to inhibitor modulate the CYP1A activities of both fractions, MRODand EROD activities were measured in the presence of dif-ferent concentrations of plant extract. Results found indi-cated that the plant extract was able to reduce both activi-ties of rat S9, MROD having a higher reduction than EROD(Fig. 1). Assays using human fraction showed a less markeddecrease of alkoxyresorufinO-dealkylation activities as a re-sult of the plant extract addition, the levels of inhibition ofboth EROD and MROD being similar (Fig. 2).
4. Discussion
Recently, we have demonstrated the antimutagenic poten-tial of the Phyllanthus orbicularisaqueous extract againstdifferent promutagenic AAs, showing that the plant extractcontains compounds capable of interfering with microsomal
276 M. Ferrer et al. / Journal of Ethnopharmacology 90 (2004) 273–277
0
20
40
60
80
100
0 20 40 50 60 100 120 140 160
Plant extract (µg/ml)
AR
OD
(%
from
the
cont
rol) EROD
MROD
Fig. 2. Inhibition of alkoxyresorufinO-dealkylation activities of humanliver S9 by thePhyllanthus orbicularisextract. Standard deviation froma minimal of three independent experiments are shown. The basal ERODand MROD activity (100%) corresponds to 2.05 and 1.95 nmols of re-sorufin/min/mg protein of S9, respectively.
enzymes, involved in the activation of these AAs (Ferreret al., 2001). Likewise, this mechanism was also the mostimportant in reducing the mutagenesis of the carcinogenicaromatic amines PhIP and 4-ABP byPhyllanthus orbicu-laris extract (data not shown), which had proved to be moreeffective when both promutagens were activated by liver en-zymes from human fraction (Table 1).
Against both AAs, the most effective concentration toreach the greatest antimutagenic effect of plant extract was1000�g/plate. The optimal antimutagenic effect of plant ex-tract was more marked when human enzymes activated bothAAs, decreasing the mutagenicity induced by PhIP by about75%, and those due to 4-ABP almost completely. In contrast,the decrease of the mutagenicity of both AAs, activated byinduced rat fraction, was only of a 50%.
N-Hydroxylation is thought to be the first step in theAAs activation, the CYP1A subfamily being responsible forthe activation of promutagens here studied (Weisburguer,1988; Kadlubar et al., 1992; Zhao et al., 1994; Windmillet al., 1997). In human livers, CYP1A2 is the most abundantisoform from this subfamily, CYP1A1 being mainly extra-hepatic (Venkatakrishnan et al., 2002). Rat liver also con-tains CYP1A2, but CYP1A1 is strongly induced in livers ofrat treated with phenobarbital/�-naphthoflavone (Callanderet al., 1995). Therefore, in our study, it can be assumed thatCYP1A2 is the main isoform involved in PhIP and 4-ABPactivation by human fraction, meanwhile both CYP1A2 andCYP1A1 are responsible for this activation by induced ratfraction. The finding that the antimutagenesis mediated bythePhyllanthus orbicularisextract was highest when 4-ABPand PhIP were activated by human fractions, seems to sug-gest that one of the antimutagenic mechanisms of the ex-tract against PhIP and 4-ABP could be a selective inhibi-
tion of CYP1A isoforms, and particularly of CYP1A2. Thissuggestion was confirmed by data obtained regarding theeffects of the plant extract in decreasing alkoxyresorufinO-dealkylation activities of both induced rat and human frac-tions (Figs. 1 and 2). It has been described that both CYP1A1and CYP1A2 contribute to EROD activity, whereas onlyCYP1A2 catalyses MROD (Burke et al., 1985, 1994). Ac-cording to this, no significant differences were seen betweenthe potency of MROD and EROD inhibition by plant extractin assays with human enzymes, which is consistent with thefact that human microsomes only contain CYP1A2. How-ever, the fact that the plant extract inhibits more MROD thanEROD activity of enzymes of induced rat fraction stronglysuggests a selective inhibition or modulation of CYP1A2 bysome component of the plant extract, although interactionswith CYP1A1 must also occur. In this sense, it must be in-dicated that different natural compounds, such as polyphe-nols and glucosinolates, have been identified as inhibitorsor inducers of CYP1A (Canivenc-Lavier et al., 1996; Zhaiet al., 1998; Hammons et al., 1999; Bear and Teel, 2000;Rose et al., 2000; Steinkelner et al., 2001; Takahashi et al.,2002).
It is also remarkable that the plant extract reduced the4-ABP mutagenesis by approximately 90% whereas the de-crease of PhIP mutagenesis only was by about 75%, whenboth AAs were activated by human fractions. This differ-ential effect could be explained by different affinities ofCYP1A2 for its substrates, 4-ABP, PhIP and antimutageniccompounds of the extract.
In conclusion, the results presented in this study showa significant antimutagenic effect of thePhyllanthus or-bicularis aqueous extract against the carcinogenic aromaticamines PhIP and 4-ABP, activated by enzymes of both in-duced rat and human fractions. Plant extract has shown tobe more effective in reducing PhIP and 4-ABP mutagene-sis when both AAs are activated by human enzymes. Thisreduction is related to inhibition or modulation of CYP1Asubfamily activities, and possibly of CYP1A2. The antimu-tagenic activity ofPhyllanthus orbicularisextract here stud-ied, in addition to before reported properties, highlight therelevance of this plant extract as a valuable source of an-timutagens.
Acknowledgements
The authors wish to thank Dr. T. Nohmi for the sup-ply of the bacterial strains, M.Sc. Gladys Fonseca for herhelp in the preparation of the aqueous extract ofPhyllan-thus orbicularisHBK and Mr. Chuck Simmons, our Englishteaching-university colleague, for his help in the revisionand correction of the English version of this paper. This re-search has been supported by a grant (2001SGR-206) of theComissionat per a Universitats i Recerca de la Generalitatde Catalunya (Spain) and with the help of the UniversitatAutònoma de Barcelona (Spain).
M. Ferrer et al. / Journal of Ethnopharmacology 90 (2004) 273–277 277
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Journal of Ethnopharmacology 90 (2004) 279–284
Evaluation of ethnobotanically selected Benin medicinalplants for their in vitro antiplasmodial activity
Bernard Wenigera, Latifou Lagnikab, Catherine Vonthron-Sénécheauc,Tomabu Adjobimeyb, Joachim Gbenoud, Mansourou Moudachiroud,
Reto Brune, Robert Antona, Ambaliou Sannib,∗a Unité de Pharmacognosie, UMR ULP/CNRS 7081, Faculté de Pharmacie, Université L. Pasteur, Strasbourg, France
b Laboratoire de Biochimie et de Biologie Moléculaire, Département de Biochimie et de Biologie Cellulaire,Faculté des Sciences et Techniques, Université d’Abomey-Calavi, Cotonou 04 BP 0320, Benin
c UFR des Sciences et Technologie des Aliments, Université d’Abobo-Adjamé, 02 BP 801 Abidjan 02, Ivory Coastd Laboratoire de Pharmacognosie et Huiles Essentielles, Faculté des Sciences et Techniques,
Université d’Abomey-Calavi, Cotonou 01 BP 2126, Benine Swiss Tropical Institute, Medical Parasitology and Infection Biology, Socinstrasse 57, Basel CH-4002, Switzerland
Received 26 May 2003; received in revised form 1 October 2003; accepted 7 October 2003
The increasing prevalence of malaria exhibiting resis-tance ofPlasmodium falciparumto cheap standard treat-ments has led to searches for new antimalarial compounds(Wellems and Plowe, 2001). Traditional treatments havebeen investigated and a significant success was achievedwith Quinghaosu, isolated from a Chinese herbal medicine,Artemisia annua(Quinghaosu Antimalarial CoordinatingResearch Group, 1979).
Thus, in our search for natural products with antiprotozoalactivity, we investigated traditional antimalarial treatmentsused in Benin, West Africa. A registry of 88 traditional reme-dies used to cure malaria by traditional healers in Benin,compiled by the “Direction de la Protection Sanitaire” of
the Benin Ministry of Health, was our starting point. Dif-ferent databases (NAPRALERT and PRELUDE databases,MEDLINE/PubMed, ISI Web of Science, Chemical Ab-stracts) were consulted to examine the literature concern-ing the plants which appear in this registry. Consequently, 9species, belonging to 9 genera and 8 families, were selectedand collected in the field. We chose species widely used bythe traditional healers but which had never been evaluatedfor antiplasmodial activity before or, for two of them, onlypartially studied.
Apolar and polar tannin free extracts were preparedfrom each selected drug and then evaluated for antiplas-modial activity in vitro towards two strains ofPlasmod-ium falciparum. First, by assessing their ability to inhibitthe uptake of [3H]hypoxanthine into thePlasmodiumfalciparum K1 chloroquine resistant strain, secondly,by measuring their ability to inhibit parasites matura-tion of the 3D7 chloroquine sensitive strain to schizontstage.
280 B. Weniger et al. / Journal of Ethnopharmacology 90 (2004) 279–284
2. Methodology
2.1. Plant material
The selected species (seeTable 1) were collected betweenDecember 2001 and January 2002 in their natural habitatsin Benin:
• in the Ouémé region (Southeastern Benin) forCanthiumsetosumHiern. (Rubiaceae),Pavetta corymbosa(DC.)F.N. Williams (Rubiaceae),Schrankia leptocarpaDC.(Mimosaceae),Hybanthus enneaspermus(L.) F. Muell.(Violaceae) andDichapetalum guineense(DC.) Keay(Dichapetalaceae);
• in the Zou region (Centre Benin) forSecamone afzelii(Schutt.) K. Schum. (Asclepidaceae).
• and in the Atlantic region (Southern Benin) forThaliageniculata L. (Maranthaceae),Gomphrena celosioidesMart. andCroton lobatusL. (Euphorbiaceae).
Botanical determination was performed by taxonomistsfrom the Herbier National of Abomey-Calavi University inBenin and voucher specimens were deposited at the sameHerbarium (BENIN).
2.2. Preparation of crude extracts
Air dried plant material of each species (10 g) wasground (0.2 mm sieve) and defatted overnight with hex-ane (200 ml) at room temperature. The defatted plantmaterial was extracted twice for 30 min with methylenechloride (60 ml) at 40◦C in a semi-automatized Soxhletextractor (Soxtec Avanti 2055 apparatus, Foss Tecator AB,Box 70, Se-263 21, Höganäs, Sweden). The same plantmaterial was then extracted twice with methanol (60 ml)for 30 min at 64◦C. The filtrates were taken to drynessunder vacuum and the residues were stored at room tem-perature until testing. Tannins were removed from thecrude methanolic extracts using Sephadex LH-20 exclu-sion chromatography, according to the method describedby Houghton and Raman (1998). Both methylene chlo-
Table 1Selected plants for in vitro antiplasmodial activity
Botanical name/Family Local names UP CA Voucher No
Canthium setosum/Rubiaceae Avovoun (G, F), Igielera (Y.N.) AP O Houngnon 3435Croton lobatus/Euphorbiaceae Gbodupogo (G), Aloviaton (F), Erugale (Y.N.) R, AP A Hp 565aDichapetalum guineense/Dichapetalaceae Agbagloma (F. Mi), Alo (Y.N.) L A Av 1551Gomphrena celosio¨ıdes/Amaranthaceae Nyohohwawe (G), Hounvama (F), Eseleri arugbo elewu (Y.N.) AP A Souza 46cHybanthus enneaspermus/Violaceae Abiwelema (G), Semegblele (F), Abiwere (Y.N.) AP O Houngnon 3708Pavetta corymbosa/Rubiaceae Dakplasuma (F), Lohuma (G), Idofrin igbo (Y.N.) AP O Houngnon 4870Schrankia leptocarpa/Mimosaceae Ahosibwasa (G), Danhunkan (F), Kpatanwo olokun (Y.N.) AP O Houngnon 954bSecamone afzelii/Asclepiadaceae Zunjikuzwi (F), Zunkuju (G), Ewe ayibu (Y.N.), Anosika (Mi) AP Z Souza 1202aThalia geniculata/Marantaceae Aflema (G), Aflima (F), Tetegu (N) R R Av 2468
G: Goun; F: Fon; N: Nagot; Y.N.: Yoruba and Nagot; Mi: Mina; UP: used part; L: leaf; AP: aerial part; R: root; CA: collection area; O: Oueme area,Southeastern Benin; A: Atlantic area near Calavi, Southern Benin; Z: Zou area, Central Benin.
ride and tannin free methanolic extracts yields are listed inTable 2.
2.3. Antimalarial assay
2.3.1. K1 chloroquine resistant strainQuantitative assessment of in vitro antimalarial activity
against the K1 resistant strain was determined by meansof the microculture radioisotope technique based upon themethod previously described byDesjardins et al. (1979)andmodified byRidley et al. (1996). The assay uses the uptakeof [3H]hypoxanthine by parasites as an indicator of viability.Continuous in vitro cultures of asexual erythrocytic stagesof Plasmodium falciparumwere maintained followingthe methods ofTrager and Jensen (1976). Plant extracts weretested on K1 strain (multi-drug pyrimethamine/chloroquineresistant strain;Thaithong and Beale, 1981). Initial con-centration of each plant extract was 30�g/ml diluted withtwo-fold dilutions to make 7 concentrations, the lowestbeing 0.47�g/ml. After 48 h incubation of the parasiteswith the extract at 37◦C, [3H]hypoxanthine (AmershamInt. Buckinghamshire, UK) was added to each well andthe incubation was continued for another 24 h at the sametemperature. IC50 was calculated by linear interpolationbetween the two drug concentrations above and below 50%(Huber and Koella, 1993). Chloroquine and artemisininwere used as positive references. The values given inTable 2are means of two independent assays; each assay was run induplicate.
2.3.2. 3D7 chloroquine sensitive strainThe test procedure for in vitro antimalarial activity against
the 3D7 sensitive strain was based upon a method previ-ously described byRieckmann et al. (1978)and adapted byWHO to be used as an epidemiological monitoring emer-gency of antimalarial drug resistance. In this technique, onlyring stage parasites were used. Thus, the end point mea-sured is the ability of each extract to inhibit parasites mat-uration to schizont stage. Continuous in vitro culture of thePlasmodium falciparum3D7 strain was maintained follow-ing a slightly modified version of the method ofTrager and
B. Weniger et al. / Journal of Ethnopharmacology 90 (2004) 279–284 281
Table 2In vitro antiplasmodial activity of the selected plant extracts against K1 resistant strain and 3D7 sensitive strain ofPlasmodium falciparum
Family Plant species Part used Extract Yield (w/w) Antiplasmodial activityPlasmodiumfalciparum IC50 (�g/ml)
3D7 K1
Rubiaceae Canthium setosum AP Da 0.6 2.77± 2.29 4.80± 0.05Mb 8.0 6.21± 1.10 >20
Euphorbiaceae Croton lobatus R D 0.3 4.42± 1.43 2.80± 0.13M 0.5 6.56± 3.71 4.91± 0.70
Croton lobatus AP D 0.6 3.74± 0.36 3.64± 0.47M 9.5 0.38± 1.85 >20
Dichapetalaceae Dichapetalum guineense L D 0.4 >20 7.35± 1.62M 4.1 >20 15.70± 2.07
Amaranthaceae Gomphrena celosio¨ıdes AP D 0.3 14.41± 4.76 6.83± 1.24M 11.6 4.26± 0.40 14.97± 1.25
Violaceae Hybanthus enneaspermus AP D 0.7 >20 2.57± 0.36M 9.9 >20 >20
Rubiaceae Pavetta corymbosa AP D 0.7 >20 5.54± 0.79M 8.2 >20 17.50± 2.25
Mimosaceae Schrankia leptocarpa AP D 0.6 16.58± 2.34 3.38± 0.57M 12.4 8.00± 5.49 >20
Ascleapidaceae Secamone afzelii AP D 0.9 >20 6.48± 1.25M 17.8 >20 >20
Marantaceae Thalia geniculata R D 0.2 >20 14.33± 1.74M 6.3 2.83± 0.69 6.38± 2.32
Chloroquine 0.0039 0.069± 0.025
Artemisinin – 0.0015± 0.0005
IC50 values are means± S.D. (n = 2), P < 0.05 (Snedecor and Cochran, 1980).a Methylene chloride extract.b Methanolic extract after tannin removal.
Jensen (1976). Parasite cultures were synchronized with 5%d-sorbitol. Initial concentration of each plant extract was128�g/ml diluted with two-fold dilutions to make 8 concen-trations, the lowest being 1�g/ml. After 24–36 h incubationof the parasites with each dilution at 37◦C under candlejar conditions, thin blood films were made and stained withGiemsa or Diff Quick Stain. The number of schizonts wascounted in each well and IC50 was determined graphically.In our in vitro testing conditions, IC50 of chloroquine was3.9 ng/ml.
3. Results
The parts traditionally used of each of the nine selectedplants were extracted to give 20 extracts. In vitro antiplas-modial activities of the 20 extracts are summarized inTable 2.
3.1. Antiplasmodial activity against the sensitive strain(3D7 strain)
The highest antiplasmodial activity was found for themethanolic extract ofCroton lobatusaerial part (IC50 =
0.38�g/ml). Eight other extracts showed good antiplas-modial activities (IC50 < 10�g/ml), i.e. methanolic andmethylene chloride extracts ofCanthium setosumandCro-ton lobatus(root), methanolic extracts ofSchrankia lepto-carpa andThalia geniculataas well as methylene chlorideextracts ofCroton lobatusaerial part andGomphrena celo-sioıdes.
3.2. Antiplasmodial activity against the resistant strain(K1 strain)
The highest antiplasmodial activities were found for themethylene chloride extracts ofHybanthus enneaspermusand Croton lobatus(root) (IC50 = 2.57 and 2.80�g/ml,respectively). The methanolic extract ofCroton lobatus(root) and methylene chloride extracts ofCanthium se-tosum, Croton lobatus(aerial part) andSchrankia lepto-carpa all showed IC50 values<5�g/ml. The methanolicextract of Thalia geniculataand methylene chloride ex-tracts ofPavetta corymbosa, Secamone afzelii, Gomphrenacelosio¨ıdesand Dichapetalum guineensewere less active,with IC50 values between 5 and 10�g/ml. Four extractsshowed mild antiplasmodial activities (10�g/ml < IC50 <
20�g/ml), i.e. the methylene chloride extract ofThalia
282 B. Weniger et al. / Journal of Ethnopharmacology 90 (2004) 279–284
geniculata and methanolic extracts ofGomphrena celo-sioıdes, Dichapetalum guineenseandPavetta corymbosa.
4. Discussion
4.1. Gomphrena celosioides
This species is traditionally used in Tanzania as an anti-malarial (Gessler et al., 1994). Our results are comparableto those found previously for an aqueous leaf extract of theplant (Gbeassor et al., 1989).
4.2. Dichapetalum guineense
This species is traditionally used as an antimalarial inTogo, a border country of Benin. Antiplasmodial activity hasbeen previously described for this species, with IC50 valuesbetween 15 and 22�g/ml (Gbeassor et al., 1989). These dataare comparable with our results.
4.3. Pavetta corymbosa
Pavetta spp. are traditionally used in the treatment ofmalaria in East Africa (Vlietinck et al., 1995; Gessler et al.,1994; Chhabra et al., 1991; Chagnon M., 1984). Alka-loids and polyphenols have been described in the genus(Arbain et al., 1989; Balde et al., 1990, 1991). Little isknown about the biological activities ofPavetta corymbosa.Nevertheless, the close related speciesPavetta crassipesfrom Togo showed antiplasmodial activity with an IC50value below 7.5�g/ml (Gbeassor et al., 1989). Two ex-tracts of Pavetta crassipesfrom Tanzania, tested for invitro antiplasmodial activity, showed mild antiplasmodialactivities (Gessler et al., 1994). These activities are con-sistent with those found here (IC50 ranging from 5 to90�g/ml).
4.4. Croton lobatus
Although severalCroton species are known to be toxic(Lewis and Elvin-Lewis, 1977), many of them are tradition-ally used as antimalarials throughout the endemic malariaareas (Prozesky et al., 2001; Milliken W., 1997; Johns et al.,1994; Jain et al., 1994; Kasa M., 1991; Mazzanti et al.,1987; Nyazema N.Z., 1984; Kumar et al., 1980; Kokwaro,1976). The genus is known to contain alkaloids (Veraet al., 1990; Sanchez et al., 1982; Khier and Salih, 1979;Levy-Appert-Collin and Levy, 1976; Bouquet and Debray,1974) and antiplasmodial activity was demonstrated in vitrofor the related speciesCroton pseudopulchellusand Cro-ton tonkinensis(Prozesky et al., 2001; Be Thi and Truong,1991). However, to our knowledge, no antiplasmodial activ-ity has been described before forCroton lobatus, the mostactive plant in our assays.
4.5. Canthium setosum
Canthium species are largely used as traditional anti-malarials: the leaf ofCanthium corneliain Guinea, the stembark of Canthium phyllanthoideumand the root ofCan-thium zanzibaricumin Tanzania (Vasileva B., 1969;Gakunjuet al., 1995; Chhabra et al., 1991). However, no biologicalstudy supported the traditional use ofCanthiumspecies asantimalarial agents. The quite good antiplasmodial activityfound here forCanthium setosumsupports its traditional useas an antimalarial in Benin.
4.6. Hybanthus enneaspermus
The flowers of this species are used against jaundice inIndia (Gopal and Shah, 1985). Other reports mention thetraditional use of the plant to treat male sterility in IvoryCoast (Kheraro and Bouquet, 1950), urinary tract infec-tions and water retention in India (Pushpangadan and Atal,1984; Selvanayahgam et al., 1994), and venomous snakebites (Majumder et al., 1979). Although chemical studiesshowed the presence of alkaloids, flavonoids, triterpenes andsteroids in this species, no data are available concerning itsbiological activities (Saxena H.O., 1975; Majumder et al.,1979). As far as we know, the results obtained here withHybanthus enneaspermusconstitute the first description ofantiplasmodial activity in the genus.
4.7. Thalia geniculata
Neither biological nor chemical data on the genusThaliais available in the literature, but the antiplasmodial activityof the methanolic extract ofThalia geniculatafound here to-wards both chloroquine-sensitive and chloroquine-resistantstrains is consistent with its traditional use as antimalarialin Benin.
4.8. Schrankia leptocarpa
This species is traditionally used against eruptive feversand hypertension (Adjanohoun et al., 1989). Alkaloids weredescribed in the fruit ofSchrankia uncinata(Smolenskiet al., 1973). As it was the case for the genusHybanthus,this work provides, to our knowledge, the first evidence ofantiplasmodial activity in the genusSchrankia.
4.9. Secamone afzelii
Despite a wide range of traditional uses (Adjanohounet al., 1989), little information is available for the genusSecamone. Antimicrobial effect on some pathogenic bac-teria has been described forSecamone afzelii(Ebana andMadunagu, 1993). As far as we know, this is the first reportof antiplasmodial activity in this genus.
One extract, at least, of each of the nine selected speciesshowed in vitro antiplasmodial activity below 10�g/ml.
B. Weniger et al. / Journal of Ethnopharmacology 90 (2004) 279–284 283
These results are consistent with the traditional use of theseplants as antimalarial agents in Benin. The ethnopharma-cological approach used in our study may explain the highrate of positive hits. Only two species out of nine studied,Gomphrena celosio¨ıdesand Dichapetalum guineense, hadbeen previously investigated for antiplasmodial activity. Forthe seven other species, the first evidence of antiplasmodialactivity is provided here.
Regarding the methylene chloride extracts, 3 out of 10showed in vitro antiplasmodial activity below 10�g/mlagainst the 3D7 chloroquine sensible strain, whereas 9 outof 10 were active in the same range against the K1 chloro-quine resistant strain. Among the methanolic extracts, 6out of 10 showed in vitro antiplasmodial activity below10�g/ml against the 3D7 strain, whereas only 2 out of 10were active in the same range against the K1 strain.
Some of the extracts were markedly more active againstone strain: this was the case of the methanolic extract ofCroton lobatus(aerial part), which was about a factor of50 more active against the 3D7 sensitive strain than againstthe resistant one. On the other hand, the methylene chlorideextract ofHybanthus enneaspermuswas surprisingly 10-foldmore active against the K1 resistant strain than the 3D7sensitive strain. The 11 most active extracts towards the K1resistant strain exhibited IC50 values<8�g/ml, comparableto those reported elsewhere for ethanolic extracts of bothArtemisia annua(IC50 = 3.9�g/ml; K1 resistant strain) andAzadirachta indica(IC50 = 4.1–7.2�g/ml; FcB1 resistantstrain) in the in vitro microdilution test (O’Neill et al., 1985;Benoit et al., 1996; Udeinya, 1993).
At least five species,Hybanthus enneaspermus, Crotonlobatus, Canthium setosum, Schrankia leptocarpa, Thaliageniculata, andGomphrena celosioidesseem to be of partic-ular interest for further investigations, as they showed highin vitro activities towards one or both of the two strains(IC50 < 5�g/ml). Cytotoxicity evaluation as well as biogu-ided fractionation of the corresponding extracts are underprocess.
Acknowledgements
The authors wish to thank the traditional healers fromBenin for their willingness to share with us their knowledgeabout plants. The “Centre Béninois de la Recherche Scien-tifique et Technique” (CBRST) and his Director Professor F.Toukourou are thanked for financial support to the work per-formed in Benin. One of the authors (L. Lagnika) benefitedfrom a fellowship of the French Mission of Cooperation inBenin.
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Antiinflammatory evaluation of alcoholic extractof galls ofQuercus infectoria
Gurpreet Kaura, Hinna Hamidb, Asif Ali b, M Sarwar Alamb,∗, Mohammad Atharaa Department of Medical Elementology and Toxicology, Jamia Hamdard, Hamdard Nagar, New Delhi 110062, India
b Department of Chemistry, Faculty of Science, Jamia Hamdard, Hamdard Nagar, New Delhi 110062, India
Received 25 October 2002; received in revised form 7 May 2003; accepted 9 October 2003
Abstract
Galls ofQuercus infectoriaOlivier (Fagaceae) possess pleiotropic therapeutic activities, with particular efficacy against inflammatory dis-eases. The present study was undertaken to evaluate the effect of alcoholic extract ofQ. infectoriagalls on various in vivo and in vitro experi-mental models of inflammation. Oral administration of gall extract significantly inhibited carrageenan, histamine, serotonin and prostaglandinE2 (PGE2) induced paw oedemas, while topical application of gall extract inhibited phorbol-12-myristate-13-acetate (PMA) induced ear inflam-mation. The extract also inhibited various functions of macrophages and neutrophils relevant to the inflammatory response. In vitro exposure ofrat peritoneal macrophages to gall extract ameliorated lipopolysaccharide (LPS) stimulated PGE2 and nitric oxide (NO) production and PMAstimulated superoxide (O2•−) production in a dose dependent manner. Gall extract also scavenged NO and O2
Quercus infectoriaOlivier (Fagaceae), is a small tree ora shrub mainly present in Greece, Asia Minor, Syria andIran. The tree capitulates galls that emerge on its shoots asa consequence of assault of gall wasp, Cypnis gallae tinco-toriae (Samuelson, 1992). The galls ofQ. infectoriahave agreat medicinal value and have pharmacologically been de-ciphered to be astringent, antidiabetic, antitremorine, localanaesthetic, antipyretic and antiparkinsonian (Hwang et al.,2000; Dar et al., 1976; Dar and Ikram, 1979). The con-stituents of galls comprise a large amount of tannins, gallicacid, syringic acid, ellagic acid,�-sitosterol, amentoflavone,hexamethyl ether, isocryptomerin, methyl betulate, methyloleanate, hexagalloyl glucose, (Hwang et al., 2000; Daret al., 1976; Ikram and Nowshad, 1977). In Asian countries,the galls ofQ. infectoria have been used for centuries inoriental traditional medicines for treating inflammatory dis-eases (Anonymous, 1995; Galla, 1911). Gargle of hot wa-
ter extract of galls is very effective against inflamed ton-sils, while direct application of boiled and bruised galls onskin effectively cures any swelling or inflammation (Chopraet al., 1956). The application of powdered galls in the formof ointment also cures hemorrhoids caused by inflammationof the skin (Anonymous, 1995). However, the antiinflamma-tory activity of galls has never been evaluated.
An inflammatory response implicates macrophages andneutrophils, which secrete a number of mediators (eicosi-noids, oxidants, cytokine and lytic enzymes) responsiblefor initiation, progression and persistence of acute orchronic state of inflammation (Lefkowitz et al., 1999).Prostaglandin E2 (PGE2) and nitric oxide (NO) are mostimportant amongst these mediators and are produced inmacrophages by cyclooxgenase-2 (COX-2) and induciblenitric oxide synthase (iNOS), respectively (Harris et al.,2002; MacMicking et al., 1997). PGE2 is implicated ininducing the production of various chemoattractants andproinflammatory cytokines (Harris et al., 2002), while NOis responsible for vasodilatation, increase in vascular per-meability and oedema formation at the site of inflammation(Moncada et al., 1991). NO along with superoxide (O2•−)
286 G. Kaur et al. / Journal of Ethnopharmacology 90 (2004) 285–292
and the products of their interaction, also initiates a widerange of toxic oxidative reactions causing tissue injury(Hogg, 1998). Likewise, the neutrophils too produce oxi-dants and release granular constituents comprising of lyticenzymes performing important role in inflammatory injury(Yoshikawa and Naito, 2000). Inhibition in the release ofthese mediators is a potential strategy to control inflamma-tion and is implicated in mechanism of action of a numberof antiinflammatory drugs including the representative oneslike dexamethasone (Bourke and Moynagh, 1999).
In the present investigation we evaluated the antiinflam-matory activity of alcoholic extract of galls ofQ. infectoriain various in vivo models of inflammation and also inves-tigated the mechanism underlying the antiinflammatory ac-tivity by determining the effect of gall extract on functionsof macrophages and neutrophils relevant to the inflamma-tory process. The results demonstrated the in vitro inhibitoryeffects on cell function exerted by the extract, which alsoexhibited antiinflammatory activity in vivo.
2. Materials and methods
2.1. Chemicals
Thioglycollate (TG) broth (Brewer) was purchased fromDIFCO (Detroit, MI). Carrageenan, histamine, serotonin,PGE2, RPMI-1640, phorbol 12-myristate 13-acetate (PMA),lipopolysaccharide (LPS) (fromEscherichia coli, serotype:0127:B8), xanthine oxidase, napthylethylenediamide di-hydrochloride, sodium nitroprusside andN-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP) were purchasedfrom Sigma Chemicals, St. Louis, MO, USA.l-[3H]-Arginine was purchased from Amersham Corporation (UK).
2.2. Plant material
The air-dried galls ofQuercus infectoriawere purchasedfrom Khari Baoli, New Delhi, India and authenticated byDr. M.P. Sharma, Reader in the Department of Botany in ourUniversity. A voucher specimen is kept in our laboratory.
2.3. Preparation of gall extract
To prepare the extract, 125 g of powdered galls/l of al-cohol were extracted in soxhlet. The extract was filtered,concentrated and then defatted with petrol. The defattedalcoholic extract was 18.3% of the starting material. The ex-tract was suspended in 0.15 M NaCl for in vivo assays anddissolved in dimethylsulfoxide (DMSO) for in vitro assays.The final concentration of DMSO never exceeded 0.1%.
2.4. Experimental animals
Male Wistar rats (200–220 g) and male Swiss albino mice(25–30 g) were used in this study. The animals were pro-cured from Central Animal house, Jamia Hamdard and were
maintained at 25◦C in polypropylene cages. Animals had afree access to water and pellet diet (Hindustan Lever Ltd.,Bombay, India).
2.5. Study of gall extract on in vivo models ofinflammation
2.5.1. Carrageenan induced paw oedemaOedema was induced by subcutaneous injection of
0.1 ml of 1% freshly prepared suspension of carrageenan(commercial grade from Irish Moss) into the hind pawsof the rats. Following the administration of carrageenan,the volume of the injected and counter-lateral paws wasmeasured hourly for 10 h using a plethysmometer (Winteret al., 1962). The test groups were administered withgall extract (300 and 600 mg/kg, p.o.) or a standarddrug, indomethacin (25 mg/kg) 30 min before administra-tion of carrageenan. The control group received only thesaline.
2.5.2. Histamine, serotonin and prostaglandin E2 (PGE2)induced paw oedema
Oedema was induced by subcutaneous injection of 0.05 ml1% freshly prepared solutions of histamine, serotonin orPGE2 (20�g in 0.05 ml) into the hind paws of the rats after30 min of oral administration of gall extract. The volume ofthe injected and counter-lateral paws was measured 3 h afteradministration of the phlogistic agents.
2.5.3. PMA induced mouse ear oedemaOedema was induced by topical application of 2.5�g
PMA in 20�l acetone to the right ear of mice (Carlsonet al., 1989). The left ear served as control and received onlyacetone. Gall extract (0.5, 1 and 2.5 mg per ear) was ap-plied topically in acetone before PMA application. After 4 h,mice were subjected to light anesthesia by ether and thenkilled by cervical dislocation. The earlobes were punchedand weighed. The increase in weight of the right ear punchover that of left indicated the oedema.
2.6. Study of gall extract on macrophage functions
2.6.1. Isolation and culture of macrophagesAll assays were performed on thioglycollate-elicited rat
peritoneal macrophages prepared according toLi et al.(1997). Following isolation macrophages were washed twicewith HBSS and finally suspended at a density of 1×106/mlin ice-cold phenol red free RPMI-1640 medium supple-mented with 10% fetal bovine serum, 100 U/ml penicillinand 100�g/ml streptomycin. Cell viability of≥95% wasconfirmed by trypan blue exclusion assay. Cells were incu-bated at 37◦C and 5% CO2 for 2 h to allow macrophagesto adhere to the surface of culture plates. The non adherentcells were removed by vigorously washing with HBSS andadherent monolayers of macrophages were maintained inabove-mentioned medium at 37◦C and 5% CO2.
G. Kaur et al. / Journal of Ethnopharmacology 90 (2004) 285–292 287
2.6.2. Nitrite analysis
2.6.2.1. NO assay.Macrophage (1× 106 per well) wereincubated with 10�g/ml LPS in the presence of gall extract,0.1�M dexamethasone (positive control) or vehicle alonefor 24 h at 37◦C. The amount of nitrite (taken as index ofNO) released in the culture supernatant was determined byGriess reagent (Green et al., 1982). Following aspiration ofmedia for nitrite determination, viability of macrophageswas determined by incubating the macrophages with5 mg/ml MTT for 1 h.
Scavenging of NO was determined by incubating 5 mMsodium nitroprusside (SNP) with gall extract at 25◦C. At dif-ferent time intervals, 0.5 ml of incubation solution was with-drawn and nitrite content determined with Griess reagent.
2.6.2.2. iNOS (inducible nitric oxide synthase) assay.iNOS activity was assayed in macrophage cytosol by themethod ofMoeslinger et al. (2000). Macrophages cultured inthe presence of 10�g/ml LPS for 24 h were disrupted by 3–4freeze-thaw cycles in 50 mM Tris–HCl containing 0.1 mMEDTA, 0.1 mM EGTA, 1 mM phenylmethylsulfonylfluo-ride, 1�M pepstatin A and 0.1% 2-mercaptoethanol. Thecell lysate was centrifuged at 15,000× g for 30 min toobtain the cytosol. iNOS was determined in cytosol byconversion ofl-[3H]-arginine tol-[3H]-citrulline. The as-say mixture consisting of 1 mM NADPH, 10�M FAD,1 mM dithiothreitol, 100�M tetrahydrobiopterin, 10�Ml-arginine, 0.3�Ci [3H]-l-arginine and 100�l macrophagecytosol was incubated at 37◦C for 60 min. The reaction wasstopped by addition of 2 ml ice-cold stop buffer (30 mMHEPES, 5 mM EDTA, pH 5.5).l-[3H]-Citrulline was sepa-rated froml-[3H]-arginine on Dowex 50 WX 4 cation ex-change column (sodium form) and quantified by scintillationcounter
2.6.2.3. Arginine uptake.To study arginine uptake,macrophage monolayers were incubated with 2�Ci l-[3H]-arginine for 60 min after the treatment with gall extractor 0.1�M dexamethasone for 24 h. Following incubation,media was aspirated and the cells were lysed with 0.3 MNaOH containing 1% sodium dodecyl sulphate after a thor-ough washing. The amount of radioactivity in cell lysatewas quantified by scintillation counter.
2.6.3. Prostaglandin E2 assayMacrophages (1× 106 per well) were incubated with
10�g/ml LPS in the presence of gall extract, 0.1�M dex-amethasone or vehicle alone for 24 h at 37◦C. PGE2 levelwas assayed in culture supernatant by radioimmunoassay(Moroney et al., 1988).
2.6.4. O2•− production (respiratory burst)
The macrophages (2× 106 per well), pretreated with gallextract for 2, 4 or 6 h at 37◦C were incubated with 0.1�MPMA and 80�M ferricytochrome c for 90 min. Following
incubation, amount of superoxide released was determinedaccording toKeisari and Pick (1981).
O2•− scavenging ability was determined by incubat-
ing 100�M hypoxanthine, 60�M nitroblue tetrazolium,0.07 U/ml xanthine oxidase in the presence or absence(control) of gall extract at 25◦C for 15 min. Following incu-bation, the absorbance was immediately recorded at 560 nmagainst a blank which did not contain the enzyme.
2.7. Neutrophil degranulation
The method ofEscrig et al. (1997)was followed to iso-late the neutrophils. Viability was greater than 95% by thetrypan blue exclusion test. The cell suspension was preincu-bated with gall extract at 37◦C for 30 min, then challengedwith 1�M fMLP for 1 h. Lysozyme and�-glucuronidaseactivities were determined in the supernatant. The total con-tent was measured after treatment of the cell suspensionwith triton-X-100 and the percent of enzyme released wascalculated. Lysozyme was assayed by measuring the rate oflysis of Micrococcus lysodeikticussuspension (Smith andIden, 1979). �-Glucuronidase was assayed by fluorimetericmethod (Fuhrman et al., 1994).
2.8. Statistics
All values are expressed as mean± S.E. Statistical sig-nificance of the difference was assessed by Student’st-test.P values lower than 0.05 were considered significant.
3. Results
3.1. Effect of gall extract on in vivo models of inflammation
Oral administration of gall extract significantly and dosedependently inhibited carrageenan induced paw oedema(Fig. 1). Gall extract also showed a dose dependent in-hibition in histamine, serotonin and PGE2 induced pawoedemas (Table 1). Topical application of gall extract alsosignificantly and dose dependently inhibited PMA inducedear oedema (Fig. 2).
3.2. Effect of gall extract on macrophage function
3.2.1. Effect of gall extract on no production in LPSstimulated macrophages
The normal, unactivated macrophages produced 3.42�Mnitrite (measured as index of NO) after 24 h of incubationat 37◦C. Exposure to LPS induced a huge amount (about11.47 folds) of nitrite in macrophages (Table 2). Gall extractdose dependently ameliorated LPS stimulated nitrite produc-tion (Table 2), with an IC50 value of 34�g/ml. The viabil-ity of macrophages remained intact during 24 h incubationperiod with gall extract indicating the inhibition obtainedin nitrite release not to be the consequences of cytotoxic-
288 G. Kaur et al. / Journal of Ethnopharmacology 90 (2004) 285–292
Table 1Effect of gall extract and indomethacin on paw oedema induced by histamine, serotonin and prostaglandin E2
Each point represents the mean± S.E. (n = 6). aP < 0.05, bP < 0.01, cP < 0.001 vs. control group.
Fig. 1. Inhibition of carrageenan induced paw oedema (�) by gall ex-tract at the doses of 300 (�), 600 (�) mg/kg, p.o. and by 25 mg/kg in-domethacin ( ) when given 30 min prior to carrageenan administration.Each point represents the mean± S.E. (n = 6). aP < 0.05, bP < 0.01,cP < 0.001 vs. control group.
Table 2Effect of gall extract and dexamethasone on nitrite production, iNOS levels, arginine uptake and viability of macrophages after 24 h of lipopolysaccharidestimulation
Each point represents the mean± S.E. (n = 5). aP < 0.05, bP < 0.01, cP < 0.001 vs. normal, non treated group,dP < 0.05, eP < 0.01, f P < 0.001 vs.LPS-treated control.
Fig. 2. Inhibition of PMA induced ear oedema by gall extract and in-domethacin applied topically before PMA application. Each point repre-sents the mean± S.E. (n = 4). aP < 0.05, bP < 0.01, cP < 0.001 vs.control group.
ity of gall extract. We investigated the mechanism respon-sible for NO inhibition in macrophages. Gall extract sig-nificantly and dose dependently scavenged nitrite releasedby SNP (Fig. 3). The IC50 was 125�g/ml. We next deter-
G. Kaur et al. / Journal of Ethnopharmacology 90 (2004) 285–292 289
Fig. 3. Scavenging of nitrite produced from 5 mM SNP (�) by 100�g/ml(�) or 250�g/ml (�) gall extract at indicated time interval duringincubation at 20◦C. Each point represents the mean± S.E. (n = 4).aP < 0.05, bP < 0.01, cP < 0.001 vs. control group.
mined the effect of gall extract on iNOS activity. The cy-tosolic preparations from macrophages stimulated with LPSfor 24 h were used as source of iNOS. Addition of increas-ing concentrations of gall extract (0–200�g/ml) to iNOSassay mixture did not show any statistically significant in-hibition (data not shown). In contrast 0.1 mMl-NMMA re-duced iNOS activity by 72.54%. However, iNOS activityin cytosolic preparations from macrophages cultured in thepresence of LPS as well as gall extract (10–100�g/ml) re-vealed a dose dependent amelioration (Table 2) indicatingthe gall extract to suppress NO generation by ameliorat-ing the induction of iNOS rather than its enzymatic activ-ity. To obtain further insight into NO inhibitory mechanismof gall extract, we investigated whether it inhibited the up-take of arginine, a precursor of NO. The gall extract didnot show any inhibition in arginine uptake by macrophages(Table 2).
3.2.2. Effect of gall extract on PGE2 production in LPSstimulated macrophages
The normal, unstimulated macrophages produced 0.31 ngPGE2. Exposure to LPS induced PGE2 production by 9.03folds. Co-incubation of LPS stimulated macrophages withgall extract ameliorated PGE2 production dose dependently(Fig. 4).
3.2.3. Effect of gall extract on O2•− production in PMAstimulated macrophages
Rat peritoneal macrophages exposed to 0.1�M PMAproduced around 63.1 nmol/mg protein O2
•− indicating
Fig. 4. Effect of gall extract on LPS stimulated PGE2 production in ratperitoneal macrophages. Each point represents the mean± S.E. (n = 5).aP < 0.05, bP < 0.01, cP < 0.001 vs. normal, non treated group,dP < 0.05, eP < 0.01, f P < 0.001 vs. LPS-treated control.
significant amount of respiratory burst. Pretreatment ofmacrophages with gall extract time and dose dependentlyameliorated O2•− production (Fig. 5). The IC50 value was38�M. The pretreatment of macrophages with 1�M dex-amethasone for 6 h produced 28% inhibition in PMA stim-ulated O2
•− production. The gall extract also scavengedO2
•− with an IC50 value of 260�g/ml (Fig. 6).
Fig. 5. Effect of gall extract on PMA stimulated O2•− production in rat
peritoneal macrophages. Each point represents the mean± S.E. (n = 5).aP < 0.05, bP < 0.01, cP < 0.001 vs. control group.
290 G. Kaur et al. / Journal of Ethnopharmacology 90 (2004) 285–292
Fig. 6. Scavenging of O2•− by gall extract. O2•− was generated byxanthine/xanthine oxidase system and measured by NBT reduction. Eachpoint represents the mean± S.E. (n = 4). aP < 0.05, bP < 0.01,cP < 0.001 vs. control group.
3.3. Effect of gall extract on neutrophil degranulation
Stimulation of neutrophils with fMLP induced degranu-lation process releasing enzymes like�-glucuronidase andlysozyme. Pretreatment of fMLP stimulated neutrophils withgall extract dose dependently inhibited the release of boththese enzymes (Fig. 7). The IC50 values for�-glucuronidase
Fig. 7. Effect of gall extract onN-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP) stimulated lysozyme and�-glucuronidase releasefrom neutrophils. Each point represents the mean± S.E. (n = 5).aP < 0.05, bP < 0.01, cP < 0.001 vs. control group.
and lysozyme were 45 and 60�g/ml, respectively. Neitherof these enzymes was directly inhibited by gall extract.
4. Discussion
Galls of Q. infectoria have widely been used for treat-ment of inflammatory diseases in Asian countries. Thepresent study established the antiinflammatory activity ofthe alcoholic extract of galls ofQ. infectoria in various invivo models of inflammation and also revealed some of themechanisms implicated in the antiinflammatory activity:suppression in the release of certain key inflammatory me-diators, viz. NO, PGE2 and O2
•− from macrophages andlytic enzymes from neutrophils.
Our study showed that gall extract inhibits experimen-tal inflammation after oral or topical administration. Car-rageenan induced paw oedema in rats is one of most com-monly used models of inflammation and has been acceptedas a useful phlogistic tool for investigating new antiin-flammatory agents. Gall extract significantly inhibited thisoedematous response over all time periods and at all dosesassayed. The initial phase of carrageenan paw oedema ismediated by histamine and serotonin, while later phaseby prostaglandins, producing oedema after mobilizationof leukocytes (Castro et al., 1968). Gall extract effectivelyinhibited oedema produced by serotonin, histamine andPGE2, which suggests that the antiinflammatory activityof gall extract is possibly mediated by either inhibiting thesynthesis, release or action of these mediators. In additionto these mediators, NO also plays an important role incarrageenan induced paw oedema (Salvemini et al., 1996).iNOS is expressed in this model within 4 h after injection ofcarrageenan. The subsequent production of NO maintainsthe oedema. Gall extract significantly inhibited carrageenaninduced paw oedema up to 10 h. Scavenging of NO and in-hibition in its generation in macrophages by gall extract maybe implicated in suppression of paw oedema at later stages.
Topical application of PMA offers a skin inflamma-tion model appropriate for evaluating antiinflammatoryagents. PMA induces inflammation by activating PLA2,which subsequently activates the release and metabolismof arachidonic acid. The COX-2 inhibitors are very effec-tive in suppressing PMA induced ear oedema, indicatingthe role of prostaglandins. Topical application of gall ex-tract significantly inhibited PMA induced ear inflammationsuggesting that the extract can act as a topical antiinflam-matory agent and that prostaglandins are implicated in itsantiinflammatory activity.
It is appealing in perspective of antiinflammatory agents toexplore the probable mechanism underlying their potentiallybeneficial activity to ascertain their mode of action. To thisend, effect of gall extract was tested on some of the functionsof macrophages and neutrophils relevant to inflammation.
Inflammatory macrophages constantly express iNOS pro-ducing a large amount of NO, which contributes to inflam-
G. Kaur et al. / Journal of Ethnopharmacology 90 (2004) 285–292 291
mation by increasing vascular permeability, causing oedemaformation, inducing synthesis of prostaglandins and due itsdirect cytotoxicity (Grisham et al., 1999; Moncada et al.,1991). Gall extract not only potently inhibited the gen-eration of NO in macrophages, but also effectively scav-enged it. The mechanism responsible for NO inhibition inmacrophages seems to involve direct scavenging of NOalong with attenuation in induction of iNOS. The catalyticactivity of iNOS was not affected by gall extract. The ex-tract also does not hamper the uptake of arginine (precursorof NO) by the macrophages. The decrease in iNOS levelsby gall extract may be the result of decrease in expressionof iNOS mRNA or decrease its stability. Since the expres-sion of iNOS is mediated by the transcription factor, nuclearfactor-�B (NF�B), which is induced by ROS (D’Acquistoet al., 1998), the decrease (if any) in the expression of iNOSmRNA by gall extract may be attributed to O2
•− scavengingand inhibition of O2
•− generation in macrophages by gallextract.
Inflammatory macrophages also express COX-2 leadingto production of a heap of PGE2, which contributes to patho-physiology of inflammation by chemoattraction of leuko-cytes, their activation and by inducing the release of proin-flammatory cytokines like interleukin-1 (IL-1) (Harris et al.,2002). Production of PGE2 by COX-2 expression has beenreported in various experimental models of acute and chronicinflammation (Kang et al., 1996; Vane et al., 1994). Gallextract exerted a dose dependent inhibition in release ofPGE2 from macrophages. NO also induces the productionof proinflammatory prostaglandins including PGE2 (Vaneet al., 1998). Inhibition of NO by gall extract could be im-plicated in suppression of the related PGE2 generation.
The exposure of macrophages to appropriate stimuli acti-vates metabolic pathway known as respiratory burst, whichgives rise to O2•−, which is itself toxic and may furtherproduce potentially reactive radicals like hydroxyl and per-oxynitrite, which are implicated in a number of oxidativereactions causing tissue injury. The gall extract inhibitedPMA activated respiratory burst in macrophages and alsosignificantly scavenged O2•− in the cell-free system. Theinhibition observed in O2•− production in macrophages bygall extract is apparently because of its ability to scavengeO2
•− and/or gall extract may directly inhibit the componentsof respiratory burst machinery of NADPH oxidase system.
The gall extract also inhibited the release of lytic en-zymes:�-glucuronidase, lysozyme from fMLP stimulatedneutrophils suggesting the gall extract also has an inhibitoryinfluence on the functions of neutrophil, which may also bean important component of mechanisms of its antiinflam-matory activity.
In summary, the present study establishes the antiinflam-matory activity of galls ofQ. infectoria. The antiinflam-matory activity of the galls may be related to inhibitionin functions of macrophages and neutrophils wherein theextract inhibits the release of inflammatory mediators, viz.PGE2, NO, O2
•− and lytic enzymes from these cells. The
profile and potency of gall extract may have relevance forcuring different inflammatory pathologies.
Acknowledgements
Council of Scientific and Industrial Research (CSIR) isacknowledged for providing the financial assistance to oneof us (GK).
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Journal of Ethnopharmacology 90 (2004) 293–316
Herbal mixtures in the traditional medicine of Eastern CubaJuan Hernández Canoa, Gabriele Volpatob,∗
a BIOECO, Centro Oriental de Ecosistemas y Biodiversidad, José A. Saco 601 esq. Barnada, 90100, Santiago de Cuba, Cubab Laboratorio di Agroecologia ed Etnobiologia, Dipartimento di Biologia, Università di Padova, Via U. Bassi, 58/b 35121 Padova, Italy
Received 20 December 2002; received in revised form 3 January 2003; accepted 9 October 2003
The study of ethnomedical systems and of plants as ther-apeutic agents is of paramount importance to addressinghealth problems of traditional communities and third worldcountries as well as of industrialized societies. As well, alarge number of studies have been conducted in the past fewdecades on the traditional pharmacopoeia of indigenous peo-ples and rural communities throughout the tropics. Many ofthem have been carried out in the Americas.
Nevertheless, these ethnobotanical studies are rarely fo-cused on herbal mixtures. Both the botanical and ethnobi-ological aspects of such complex preparations have oftenbeen disregarded, and very little attention has been paid tothem in the Caribbean (but see, for example,Longuefosseand Nossin, 1996; Garcıa et al., 2000; Ososki et al., 2002).
In Cuba, medicinal plants are traditionally arranged in asurprising number of herbal mixtures using at times elabo-rate procedures. Some recipes have already been reported(Seoane, 1984; Fuentes, 1988; Moreno et al., 1994), al-though to date no ethnobotanical research on multi-speciesformulas has ever been conducted in Eastern Cuba. Cubanpeople rely for food and medicine on a mixed culturethat draws upon wisdom originating mainly from Indian,African, Spanish, French-Haitian, and Antillean ethnic
∗ Corresponding author.E-mail address:[email protected] (G. Volpato).
groups (Guanche, 1983; Fuentes, 1984b; Rivero de la Calle,1992; Núñez and González, 1999). This multi-ethnic legacyhas resulted in a rich pharmacopoeia, particularly in moun-tainous areas of the eastern provinces of Cuba (Hernández,1985, 2000). A peculiarity of this unique herbalism is itsrichness in multi-species formulas that have been evolvingacross the centuries, some of which are labeled with spe-cific denominations. These mixtures thus represent a socialheritage, and their ethnobotanical investigation can addmuch to the understanding of local folk medical systems.
In Cuba, medicinal plants are not only of anthropologicalinterest: they have an essential role for people, who cannotrely on pharmaceuticals for their health needs, due to eco-nomic difficulties following the breakup of the Soviet Unionin 1989 and to the US blockade against the country (Kuntz,1994; Kirkpatrick, 1996; AAWH, 1997; Garfield andSantana, 1997). Among the healthcare strategies adoptedto confront medicine shortages of the so-calledperiodo es-pecial, a national complementary medical system based onlocal folk medicine has developed (Soler and Porto, 1997;Suárez, 1997; Acosta de la Luz, 2001), as has the use ofacupuncture and homeopathy (Fuentes, 1996; Abreu andMateo, 1997). Ethnobotanical, phytochemical and pharma-cological studies for the development of local cheap ther-apeuticals are thus emphasized (i.e.Carbajal et al., 1983;Martınez et al., 1996; Payo et al., 1997; Guerra et al., 2001).
This paper focuses on the medicinal plants and other prod-ucts used in the preparation of traditional herbal mixtures
294 J.H. Cano, G. Volpato / Journal of Ethnopharmacology 90 (2004) 293–316
in Eastern Cuba. We will discuss: (1) the ethnobotanicalaspects of herbal and non-herbal components and of theirjoint use within mixtures, (2) the role within local culture ofcomplex formulas with specific denominations and, (3) theecological distribution and phytogeographical aspects of thespecies used.
2. Material and methods
The data presented in this paper are part of a wider studyon folk medicine in Eastern Cuba. Some 130 knowledge-able people (most of them traditional healers,yerberosandcuranderos) have been interviewed in cities and villages ofthe provinces of Santiago de Cuba and Guantánamo by oneof the authors (J.H.C.) since 1983. Eighty per cent of theinterviews were conducted in the city of Santiago de Cuba,thirteen per cent in villages of the homonymous province(Simpatıa, Ramón de las Yaguas, La Talı, La Chotera, ElCaney, Perseverancia, Loma del Gato, Bella Pluma, El Dián,Palma Mocha, Las Cuevas, Ocujal); the remaining five percent have been conducted in the province of Guantánamo(Baracoa, Rancho de Yaguas, Santo Domingo, San Mateo)(Fig. 1).
For each mixture, plant components, part(s) used, vernac-ular names, products other than botanicals used, means ofpreparation and application, number of reports, and genericand specific illnesses treated have been recorded. Illnessesare reported according to local ethno-medical terminologyand classification reported by interviewees. Open-endedconversations were carried out with people interviewed.Voucher specimens of the plants cited were collected, identi-fied, and deposited at BIOECO Herbarium (BSC), Santiagode Cuba. Taxonomical nomenclature followsLeón (1946),
Fig. 1. The location of the study area and major cities.
León and Alain (1951, 1953, 1957), andAlain (1964, 1974),and the identification of the specimens was performed withthe help of taxonomists of BIOECO. Finally, the uses of thespecies in mixtures have been compared with those referredto in the most important sources for Cuban medicinal plants(Roig, 1965, 1974; Seoane, 1984; Fuentes, 1984a, 1988).
3. Results and discussion
3.1. Herbal and non-herbal components of mixtures
Table 1 lists plant species used in the preparation ofmedicinal mixtures in alphabetical order of scientific name,along with their botanical families, vernacular names asthey have been recorded during the fieldwork, and voucherspecimens. For each species, the number of different mix-tures in which it is present and the labels of these mixturesare reported as well. Ingredients others than plant species,such as products derived from plants (i.e. oils), animals(i.e. fats, excrements), or of industrial origin (i.e. aspirin,salts) are listed inTable 2. Mixtures (components, partsused, preparation, means of use and number of reports) aregiven in Table 3according to groups of illnesses. Withineach generic illness category, mixtures cited without furthermedical specifications are first reported, followed by mix-tures treating specific afflictions; each group of mixtures isfurther arranged by increasing number of components. Thecategory “other mixtures” includes all those mixtures thatare used to treat ailments that do not fall within any previouscategory, and for which it makes no sense to build a sep-arate category. We created a three-letter (corresponding tothe generic illness category to which each mixture belongs)increasing number labelling system which identifies each
J.H. Cano, G. Volpato / Journal of Ethnopharmacology 90 (2004) 293–316 295
Table 1Medicinal species used in herbal mixtures in Eastern Cuba
Botanical taxon Cuban phytonym Voucher number Labels of mixtures Number of mixtures
different recipe, in order to cite mixtures throughout thetext. There are 170 different species used in the preparationof 199 herbal mixtures that are made to treat both minorailments and life-threatening diseases; 22 of them are com-plex mixtures with specific denominations (galones, miel degüira, pru) and therapeutical aims.Galonesare mainly usedto treat pneumonia and venereal diseases,miel de güiraisused as an anticatarrhal and to treat gynaecological prob-
Table 2Other products used in mixtures
Aspirin®
BeerBee’s honeyBovine lung (bofe)Castor oilCondensed milkCow’s bileCow’s milkCricket’s legsDried cod liver oilDry wine (not necessarily of grapes)Duck’s eggsEpsom saltsFat of maja (Epicrates anguliferBibron, Boidae)Ram’s fatGoat’s excrementsGoat’s hoofGoat’s milkMagnesium powderMule’s hoofNest of comejen (American termite;Nasutitermessp., Termitidae)OilRumSalt (sodium chloride)ScorpionSoapSoap ofCastilla (washing-soap)SugarSweet wine (not necessarily of grapes)Tincture of iodineTobacco’s topsUrine of the mother of the person being sickVinegarWhale’s spermWheat mealYolk of hen’s eggs
lems, andpru is used as a hypotensive, depurative and di-gestive. The species belong to 71 families, with a prevalenceof Fabaceae (9.4%), Asteraceae (7.6%), Lamiaceae (4.7%),Euphorbiaceae and Poaceae (4.1%), Rutaceae, Solanaceaeand Verbenaceae (2.9%). Asteraceae (96), Poaceae (96),and Fabaceae (93) are also families represented by moregenera within the Cuban flora (Acevedo, 1991). Thirty-fivefamilies are represented in mixtures by just one species,accounting for the 20.6% of the total species.
Cocos nucifera(27), Bidens pilosa(22), Cissus sicy-oides (16), Erythroxylum havanense(15), Stachytarphetajamaicensis(15), Chiococca albaandRoystonea regia(13)are the species most frequently reported.Cocos nucifera,Chiococca alba, Erythroxylum havanense, and Roystonearegia are major components of complex mixtures (galones)for pneumonia and venereal diseases, whileBidens pilosaandCissus sicyoidesare principal in mixtures for respiratoryproblems, which form the major ethno-medical category interms of number of preparations. Although most speciespresent one or a few main therapeutical use(s) throughoutdifferent mixtures and are concentrated in specific illnesscategories, others are used without any apparent specificity.Interestingly, congeneric species are often used in mixtures,each with a specific medicinal purpose. WhileSolanumamericanumhas a wide-ranging pattern of use,Solanumtorvum is a species commonly used in anticatarrhal for-mulas;Ocimum basilicumis preferred in sedative mixtures(nervios; Fuentes and Granda, 1982), Ocimum tenuiflorumis used as an anti-diabetic, whereasOcimum campechi-anumis the only species of the genus to be used for renalafflictions. The latter, as well asAcanthospermum humile,Craniolaria annuaandVitex agnus-castusare reported herefor the first time as used in Cuban popular medicine. As wewould have expected, species reported as “new” to Cubanmedicine are collected from the wild rather than cultivated,and their use could either belong to specific informants’knowledge or pertain to specific isolated areas of EasternCuba. This is not the case forVitex agnus-castus, whichcan be found for sale in herbal markets of Santiago deCuba for religious purposes (Hernández, 2000), and that iswell known and used worldwide for menstrual problems(Roberts et al., 2001; Barnes et al., 2002).
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Table 3Herbal mixtures in Eastern Cuba
Label Components and plant parts Preparation Way of use Cit. Specific illness (ifreported)
Gastro-intestinal afflictions (GAS)GAS01 Lippia alba (ap), Mentha spicata(ap), Mentha×
piperita var. citrata (ap)de or 1
GAS02 Cassia grandis(ap), Lippia alba (ap), Origanummajorana(ap)
de or 1
GAS03 Mentha spicata(ap), Psidium guajava(ap) ma or 1 Stomach painsGAS04 Origanum majorana(wp), Mentha spicata(le) de or 1 Stomach painsGAS05 Origanum majorana(ap), Mentha spicata(ap),
GAS14 Jatropha curcas(le), Mentha spicata(ap) de or 1 Anti-emeticGAS15 Arundo donax(ro), Cecropia peltata(fr) de or 1 LaxativeGAS16 Chromolaena odorata(ap), Jatropha curcas(se),
Trichostigma octandrum(ap), oilde or 1 Laxative
GAS17 Psidium guajava(le), Punica granatum(ep) de or 1 DiarrhoeaGAS18 Annona squamosa(ap), Ceiba pentandra(ep),
Psidium guajava(le)de or 1 Diarrhoea
GAS19 Koanophyllon villosum(ap), rum, salt de or 1 Disentery
Liver and vesicular afflictions (LIV)LIV01 Lonchocarpus domingensis(le), Thouinia elliptica(le) de or 1LIV02 Cassia fistula(fr), Cassiasp. (le),Senna occidentalis
LIV12 Chiococca alba(ro), Erythroxylum havanense(ro) de or 1 Vesicular gall-stonesLIV13 Citrus aurantifolia(fr), Xanthium strumarium(wp), oil de or 1 Vesicular gall-stonesLIV14 Opuntia cochenillifera(st), Pimpinella anisum(fr), oil de or 1 Vesicular gall-stones
Renal afflictions (REN)REN01 Acanthospermum humile(ap), Guazuma ulmifolia(ba) de or 1REN02 Guazuma ulmifolia(ba),Salpianthus purpurascens(le) de or 1REN03 Petiveria alliacea(ro), Plantago major(le) de or 1REN04 Bambusa vulgaris(ro), Cocos nucifera(ro) de or 1
J.H. Cano, G. Volpato / Journal of Ethnopharmacology 90 (2004) 293–316 303
Table 3 (Continued)
Label Components and plant parts Preparation Way ofuse
REN10 Commelina erecta(ap), Salpianthus purpurascens(ap) de or 1 (Painful mintion) disuriaREN11 Peperomia pellucida(wp), Salpianthus purpurascens
(le)de or 2 Diuretic
REN12 Cyperus rotundus(ro), Stachytarpheta jamaicensis(ap) de or 1 DiureticREN13 Cyperus rotundus(ro), Peperomia pellucida(ap) de or 1 CalculusREN14 Chiococca alba(ro), Erythroxylum havanense(ro) de or 1 CalculusREN15 Guazuma ulmifolia(ba), Peperomia pellucida(wp),
Salpianthus purpurascens(ap)de or 1 Calculus
REN16 Cocos nucifera(fr,ro), Lepidium virginicum(ap) de or 1 Bactericide
RES11 Hibiscus rosa-sinensis(wp), whale’s sperm de or 1 CatarrhRES12 Bidens pilosa(ap), Citrus aurantifolia (fr) de or 2 CatarrhRES13 Bidens pilosa(ap), Ocimum tenuiflorum(ap) de or 1 CatarrhRES14 Bidens pilosa(ap), Solanum torvum(le) je or 1 CatarrhRES15 Chromolaena odorata(ap), Gossypium arboreum(ap) de or 1 CatarrhRES16 Origanum majorana(ap),Plectranthus amboinicus(le) je or 1 Catarrh
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Table 3 (Continued)
Label Components and plant parts Preparation Way ofuse
Cit. Specific illness(if reported)
RES17 Bidens pilosa(ap), Solanum torvum(le), fat of maja(Epicrates angulifer)
je or 1 Catarrh
RES18 Coriandrum sativum(ap), Oxandra lanceolata(ba), oil de or 1 CatarrhRES19 Annona muricata(le), Bidens pilosa(ap), Cissus
SKI10 Koanophyllon villosum(ap), cow’s bile, fat of ram je,fr to 1 Dandruff
Nervousness, insomnia (NER)NER01 Justicia pectoralis(ap), Lactuca sativa(ro) de or 1 Sedative, somniferousNER02 Justicia pectoralis(ap), Lawsonia inermis(fl) de or 1 Sedative, somniferousNER03 Justicia pectoralis(ap), Ocimum tenuiflorum(ap) de or 2 Sedative, somniferousNER04 Ocimum basilicum(ap), Tilia europea(fl) de or 1 Sedative, somniferousNER05 Justicia pectoralis(ap), Lippia alba (ap), Ocimum
NER09 Cymbopogon citratus(le), Justicia pectoralis(ap) de or 1 Sedative, hypotensive
Parasites (PAR)PAR01 Allium sativum(bu), Chenopodium ambrosioides(ap) de or 1PAR02 Brachiaria mutica(ro), Cocos nucifera(fr) de,je or 1PAR03 Cajanus cajan(le), Carica papaya(la) je or 1PAR04 Chenopodium ambrosioides(le,ap),Cocos nucifera(fr) je or 4PAR05 Cocos nucifera(fr), Momordica charantia(ap) je or 1PAR06 Jatropha aethiopica(le), Solanum americanum(ap) in or 1PAR07 Luffa cylindrica (fr), washing-soap, salt de or 1PAR08 Chenopodium ambrosioides(ap), cow’s milk,
FEV05 Alpinia zerumbet(le), Citrus aurantifolia (le) de ba,or 1 Malaria
Other afflictions (OTH)OTH01 Senna occidentalis(ro), Senna uniflora(ro) de or 1 Blood depurativeOTH02 Annona muricata(le), Annona reticulata(le) de or 1 HypotensiveOTH03 Annona muricata(le), Citrus aurantifolia (le) de or 1 HypotensiveOTH04 Annona muricata(le), Bidens pilosa(ap), Ocimum
tenuiflorum(ap)de or 1 Hypotensive
OTH05 Tobacco’s tops, urine de or 1 SpasmsOTH06 Fevillea cordifolia (se),Swietenia mahagoni(ba), rum de or 1 SpasmsOTH07 Bursera simaruba(le,ba),Swietenia mahagoni(ba),
castor-oil, nest of comejen (Nasutitermessp.)de or 1 Spasms
Part(s) used: ap, aerial part; ba, bark; bu: bulb; ep, fruit epicarp; fl, flowers; fr, fruits; ft, flowering tops; la, latex; le, leaves; ls, leaf stalks; re, resin; rh,rhizome; ro, root/ tuber; se, seeds; sg, stigma; sh, shoots; st, stems; uf, unripe fruits; wo, wood; wp, whole plant. Preparation: de, decoction (cocimiento);fr, frying; in, infusion; je, juice extraction; ma, maceration; tr, trituration. Way of use: ba, bath; en, enema; fr, frictionating; gu, gurgles; or,oral ingestion;to, topical application. Cit., citations (number of individual reports for each mixture).
Eight species (Canavalia nitida, Diospyros grisebachii,Erythroxylum havanense, Garcinia aristata, Guibourtia hy-menifolia, Protium cubense, Ravenia leonis, Thouinia ellip-tica), corresponding to 4.8% of the total species, are Cubanendemisms (cf.León and Alain, 1951, 1953, 1957; Lópezet al., 1994a, 1994b, 1995). A decoction of the rootE. ha-vanenseis a main component ofgalonesand it is used inCuba for liver and renal afflictions and as a powerful diuretic(Roig, 1974), and when combined with the root ofChio-cocca albais used to treat vesicular gallstones and renal cal-culus (LIV12; REN14). The other species are sparsely cited,and often belong to themateria medicaof specific healers,particularly of producers ofgalones. Endemic species of thegenusPinus(Pinus caribaeaMorelet ssp.caribaeaMorelet,Pinus cubensisGriseb., andPinus maestrensisBisse) are ap-parently used without distinction to makepru (PRU01,02),and are commonly cultivated in Eastern Cuba as part of re-forestation efforts and as shade trees for coffee plantations(Esquivel et al., 1992; Del Risco, 1999).
Four species not belonging to Cuban flora but on sale inchemist’s shop (farmacias) have been assimilated into theCuban pharmacopoeia through a form of syncretism be-tween traditional folk remedies and cosmopolitan medicine(Laguerre, 1987). Cassiasp. andRheumsp. are compo-nents of mixtures for hepatitis (LIV09,10,11),Illicium
sp. for intestinal and stomach gases (GAS07,08,09), andTilia europeais used in sedative (NER04) and anti-diabetic(DIA09) formulas. These species are reported by heal-ers in combination with Cuban folk medicinal species(i.e. Corchorus siliquosus, Lepidium virginicum, Solanumamericanum), and sometimes have been assimilated withmedicinal purposes other than those that they are sold for(i.e. Cassia sp. in formulas for jaundice). Cosmopolitanmedicinal species are testimony to cultural and commercialexchanges, on the one hand, with the Far East and China(Cassiasp.,Rheumsp.), and on the other hand with Europe(Tilia europea). Since World War II,Justicia pectoralis, themajor component of mixtures for insomnia and nervousness,is widely used in Cuba as sedative (Roig, 1974; Morenoet al., 1994). At the time, imports ofTilia europea’s driedflowers from Europe could not keep up with local demand(Fuentes, 1984b). People thus started using the aerial partof Justicia pectoraliswhich, in some Cuban regions, hasthe same vernacular name asTilia europea(tilo). The latteris still used when available at the drugstore instead of theformer species, as also occurs with NER05.
A total of thirty-six products other than plant species areused in the mixtures, representing forms of syncretisms withcosmopolitan medicine (aspirin, beer, wine) and with othermedical traditions (i.e. African). For example, beer and
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aspirin are added to GYN03 and GYN04, respectively. Bothare used as abortifacients. These combinations of alternative(i.e. non-official) uses of industrial products with traditionalherbal medicine have also been recorded bySchultes andRaffauf (1990)in the Amazonia and byMoreno et al. (1994)in Cuba. Meanwhile, the concoction of the roots ofCleomegynandraandRoystonea regiawith cricket’s (genusAcheta)legs (REN05) is likely to have an African origin. The use ofinsects or parts of insects (and arthropoda) in treatments, i.e.scorpions which are macerated alive in alcohol for rheuma-tisms (RHE09), can act at a psychological level thanks tothe symbolic value of some species, but their therapeuti-cal efficacy should not be excluded (seeMotte-Florac andRamos-Elorduy, 2002). Some of the products used representtraditional therapeutical resources to local people, and theircultural and material importance can be appreciated in mix-tures. In RES17, the juice of the leaves ofSolanum torvumandBidens pilosais ingested along with the fat ofEpicratesangulifer Bibron (Boidae), locally known asmajá or majáde Santa Mar´ıa (Alvarez and Melián, 1997). Like oil, the fatof majá may serve as a fluidifying agent to allow the veg-etal products to be more easily ingested and, like ram’s fat(RES37,38), it is also used topically as an anti-inflammatory,i.e. in mumps, leprosy, and eczemas (Seoane, 1984).
Although to this point single-component cases have beendiscussed in this paper, patterns of association of plants andother products will be best understood by focusing on therecipes themselves rather than on single components per se.
3.2. Herbal mixtures
In this paper, we define herbal mixtures as concotions ofplants or parts of plants and other products to treat specifichealth afflictions. These formulas can be more or less com-plex, commonly known home remedies used to treat minorillnesses or complex preparations employed by traditionalhealers for life-threatening diseases. About 65% of mix-tures are composed of two or three plants, 22% of four orfive plants, and 13% of from six to nineteen different plantspecies. Decoction of fresh herbal components is by far thepreferred means to prepare the mixtures, as was pointedout by Fuentes (1984b)in relation to Cuban traditionalmedicine. Dosis and quantification are variable among thepeople interviewed and they heavily rely on personal ex-perience and family customs. Herbal components are alsosometimes macerated in water (GAS03,12; LIV07) or inalcohol (i.e. in mixtures for rheumatisms), triturated (i.e. forsinusitis, RES38,39, when applied topically for boils andscalds, SKI02,04,05), fried in oil, or the juice is extracted(i.e. in mixtures against catarrh and intestinal parasites).Ingestion is the preferred means to take the remedies; top-ical application as a pomate is used mainly to treat skinproblems, while frictioning is preferred with preparationsfor rheumatisms and artrosis. Plant parts used include:aerial parts (29.4%), roots (24.7%), leaves (13.5%), fruits(12.7%), stem barks (5.5%), flowers (3.2%), stems (2.7%),
whole plants (2.3%), rhizomes (2.1%), resins (1.3%), seeds(1.3%), bulbs (0.8%), and latex (0.5%). The use of rootsand rhizomes (26.8% as a whole) in the preparation ofCuban medicinal mixtures will be discussed later.
In their simplest form, mixtures are concoctions of two orthree species with the same popular medicinal use that areprepared jointly to enhance the therapeutical effect of thetea. These low-component remedies are often well knownin local communities and are prepared at home for minorailments (i.e. insomnia, digestive disorders), or for theiracute manifestation (i.e. diarrhoea, fever). The principleaim of mixtures seems to be potentiating the therapeutic-ity of single-plant preparations, and species with commonuses are often used jointly. Thus, plants used in stom-achic and sedative mixtures are mainly aromatic species(i.e. Mentha spp.), rich in therapeutically-active essentialoils (Etkin, 1981; Pérez et al., 1996; Pino et al., 1997;Sánchez et al., 1998; Pascual et al., 2001; Barnes et al.,2002), and singly used for their antispasmodic, antibacte-rial and stomach-soothing properties (Roig, 1974; Morton,1981; Seoane, 1984; Fuentes, 1988). Instead, mixtures forstomach ulcer (GAS11,12) and diarrhoea (GAS17,18) arecharacterized by species with high tannin content, popu-larly used as astringents and antimicrobials (Etkin, 1981;Morton, 1981; Seoane, 1984; Mota et al., 1985; Alvarezet al., 1999; Akinpelu, 2001).
Three mixtures are reported to be used againstpadrejónandmadrejón(OTH13,14,15). These illnesses are describedby informants as “a sensation of pain at the pit of the stom-ach, which the person feels like a cramp, or like it werejumping or palpitating”. When it happens to a man it iscalledpadrejón(from padre, father), to a womanmadrejón(from madre, mother). This taxonomical differentiation bysex of a stomach pain is unique in the ethno-medical termscollected. Herbal and non-herbal components of these mix-tures are of European origin and probably of Spanish culturalinfluence, and their provision by people who diagnose theillness is accompanied by the recitation of specific popularprayers (Seoane, 1984). This illness, also locally calledelhistérico(the hysterical one), is likely to be of nervous ori-gin. Ruta graveolensis used as an anti-hysteric in the Amer-icas (Morton, 1981; Duke and Vásquez, 1994) andAlliumsativumhas antispasmodic properties (Barnes et al., 2002).
The number of components in complex remedies, andthe level of knowledge and time needed to collect theplants and prepare the mixtures, mean that it is unlikelythat they will be prepared as home remedies. Instead,yer-beros sometimes sell them in bottles (i.e.pru). As couldbe expected, more complex mixtures are usually used totreat less common and more serious diseases (i.e. vene-real diseases, pneumonia). While green parts of plants arepredominant in few component preparations such as stom-achic (green parts: 93.75%; GAS01-06), sedative (95.65%;NER01-09), and anti-asthmatic and anti-catarrhal remedies(82.1%; RES01-28), ligneous parts such as roots, rhizomes,stems, and barks are increasingly present in mixtures for
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pneumonia (ligneous parts: 53.85%; RES30-32), to treatgynaecological problems and to induce abortion (50%;GYN01-10), and in complex mixtures likegalones(70.6%;GAL01-15). Thus, pneumonia (RES30,31,32) and spasms(which according to informants are caused by suddenchanges in temperature, i.e. “roasting coffee and then takinga bath or staying in the open air”; OTH06,07) are treatedwith combinations of roots and barks of the same species(Swietenia mahagoni, Bursera simaruba, Protium cubense),and the bark ofCinnamomum verumis the main compo-nent of abortifacient mixtures (GYN03,04,05). Abortiveherbal remedies have been widely used in many traditionalsocieties, and also among indigenous Cubans before the ar-rival of the Europeans (Bachiller, 1879). Although rare, thispractice is still used in rural Cuba, and midwives (parteras,comadronas) are repositaries of knowledge about plantsused in gynaecological and reproductive afflictions and forwomen’s health (Moreno et al., 1994).
If most mixtures are combinations of plants with the samepopular use, some include species claimed to have differenttherapeutical actions and are used in combination to achievespecific goals. In anti-hepatitis formulas (LIV09,10,11),Cassiasp. andRheumsp., which are well-known laxativeand anti-constipation agents (Roberts et al., 2001; Barneset al., 2002), are found in combination withCorchorussiliquosus, used in Cuba as an anti-venereal agent and forkidney problems (Roig, 1974; Barreto et al., 1992), and withthe fruit of Cassia fistula, which is well-known for treatingliver and vesicular illnesses and for its hepatoprotectiveactivity (Fuentes and Granda, 1982; Bhakta et al., 1999).Carminative mixtures (GAS07,08,09,10) includeIlliciumsp., which possesses antimicrobial, stomachic, and carmina-tive properties (Roberts et al., 2001; De et al., 2002), alongwith Lepidium virginicum, traditionally used as diuretic,and with fruits and leaves ofPimenta dioica, regarded inCuba as a stomachic and digestive (Roig, 1974; Fuentes andGranda, 1982). Whatever the complexity of a mixture, theseremedies are likely to be means to enhance the therapeuticvalue or other properties of traditional single-componentpreparations (see alsoElvin-Lewis, 2001). Plants and otherproducts may also be used in combination possibly in or-der to detoxify plant allelochemicals. Termite (comején)mounds are found in association with the bark ofSwieteniamahagoniand other barks and roots to treat pneumonia(RES30,31), spasms (OTH07,08), and venereal diseases(GAL05). Cubans believe that the triterpenes-rich stem barkof Swietenia mahagonihas powerful astringent, bitter, andfebrifuge properties (Roig, 1974; Murthy et al., 1991). Theconsumption of clays from termite mounds as a means ofdetoxication has been observed in many nonhuman primates(Oates, 1978; Davies and Baillie, 1988) and seems to playan important role in humans as well (Johns, 1990). Claysact by binding and neutralizing the toxicity of alkaloids andtannins (Johns, 1990and references within); adding termitemounds to herbal decoctions could thus represent an em-pirical attempt to deal with toxic allelochemicals present in
barks and roots of medicinal species while getting thera-peutical benefit from them.
Some mixtures are medicinal-food formulas (Etkin andRoss, 1982) rather than herbal preparations, and fruits (i.e.Cocos nucifera) and vegetables (i.e.Solanum americanum)traditionally consumed as food (Esquivel et al., 1992) arerecognized as having specific pharmacological properties,i.e. as an antihelmintic. Particularly, fruits ofCapsicumfrutescensand seeds ofMyristica fragrans, along with eggs,milk, and wine, are macereted together and eaten as a foodto treat impotence and sterility (GYN07). Seeds ofMyris-tica fragransand duck’s or hen’s eggs are also used in bothmixtures cited as reconstituents (OTH09,10), confirmingtraditional Cuban perceptions of impotence and sterility asa “lack of strength in the body” (Seoane, 1984).
3.3. Galones, miel de güira and pru
Among complex mixtures, some are well known recipeswith definite cultural and ethnomedical features. On the ba-sis of the variety of different components, the most impor-tant is a kind ofchicha (fermented beer-like drink) knownas galón, frucanga, or nowadays asbotella (GAL01-15).It is obtained by preparing a decoction of plant parts, andsometimes by first macerating the components in order toachieve a light fermentation. No exhaustive study has yetbeen conducted ongalones, and the only references are inRoig (1965, 1974). The termgalón comes from the recipi-ent containing the mixture, traditionally potted in one galloncontainers; they are presently decanted in wine-like bottles,hence the use of the termbotella (bottle). Information hasbeen collected on 66 plant species used in the preparationof galones, as well as onCaesalpinia bonducandSchaeffe-ria frutescens, which have been reported as components ofgalones, although they have not been cited in any specificrecipe. Fifteen different formulas are claimed to treat asthma(1), catarrh (1), pneumonia (3), venereal diseases (7), impo-tence (1), and as a depurative of the blood (1); one more hasbeen cited asfrucangaand is the only one to be applied top-ically against boils (granos). Frucangaseems to be a wordof African origin, with the same suffix offricanga (frittersprepared from cassava andCapsicum frutescens), from theCongo Kanga (to fry) (Ortiz, 1956). Although the recipecollected is a decoction, indeed it seems reasonable that itcould be also prepared by frying, like other mixtures, to beapplied locally.Galón recipes are variable, and plant num-bers range from five (VEN03) to nineteen (RES41), accord-ing to family traditions and local availability of specific plantspecies. When this kind of mixture is prepared with five orfewer components, it is sometimes called apreparado.
Few plant species appear to be characteristic ofgalones,while most of the components possibly are a “summary”of socially-acquired herbal knowledge and anti-microbialplants known by the informant. This is likely to explainthe complexity ofgaloneswith their possible cultural roleas a means for transmitting medicinal plant knowledge
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through healers’ generations. Moreover, the compositionin plant species seems to be culturally determined at threelevels. The first level includes those species that “make”the galón: the roots ofCocos nucifera(9), Erythroxylumhavanense(9), andRoystonea regia(9) are the main com-ponents. These species are widely used throughout Cuba fortheir anti-bacterial, tonic and stimulant properties, and areconsidered as “magic plants” within Afro-Cuban religions(Seoane, 1984; Aguilar and Herrera, 1995; Moreno et al.,1995). Species in the second level give to each mixture atherapeutical specificity (i.e. venereal disease versus pneu-monia). Thus,Chiococca alba, Bambusa vulgaris, Smilaxdomingensis, and Tournefortia hirsutissimaare more fre-quently used ingalonesthat are claimed to treat venerealdiseases, whereasSolanum torvumand Swietenia mahag-oni characterize those for pneumonia and other respiratoryafflictions. Smilax species (i.e.Smilax officinalisGriseb)have traditionally been used worldwide and observed tobe effective for the treatment of both acute and chroniccases of syphilis (Vermani and Garg, 2002). Cassia fistula,Cleome gynandra, Polypodium aureum, Senna occidentalis,andWaltheria indicaare used with some level of agreementthroughoutgaloneswith no ailment-dependence, and arelocally regarded as depuratives and diuretics (Roig, 1974;Fuentes and Granda, 1982). Species belonging to the thirdlevel are often plants cited in one or few recipes; they aremainly collected from the wild (Campyloneurum phyllitidis,Craniolaria annua, Critonia aromatisans, Cyrtopodiumpunctatum, Desmodium canun, Gerascanthus collococ-cus, Philodendron lacerum), some are Cuban endemisms(Canavalia nitida, Ravenia leonis), and they include medic-inal species known and used by single healers.
The important role of roots (48.6% of plant parts) ingaloneshas to be stressed. In Caribbean traditional knowl-edge, roots are considered as the “strongest” part of plants;“root” medicine is likely to have an African origin andis widespread throughout the Caribbean, where Afro-American healers are often called “root doctors” (Cabrera,1954; Laguerre, 1987). Indeed, the effect of root harvestingcan be very damaging to the plant (Cunningham, 2001).People interviewed stated that they are aware of and harvestthe roots through what they call “capar”, i.e. harvesting onlyfew tap-roots for each plant individual (Hernández, 2000).
Cleome spinosaJacq.,Pisonia aculeataL., andMorindaroyoc L., cited byRoig (1974)as components ofgalones,have not been reported for this purpose in our interviews.Instead, we report the use ofCleome gynandra; this con-generic has the same folk name asCleome spinosa(uña degato), and they are very likely to be used indistinctly. Theleaves ofPisonia aculeataare part of a mixture (SKI08)applied topically to extract the spines of the same plant,which are reported to be very painful (hinconosas) and tocause infection.Morinda royocis reported byRoig (1965,1974) as a component ofgalones, of pru, and to be usedas an aphrodisiac. However, this species has not been re-ported to be used in this nor in other recent works on
Another complex mixture,miel de güira(güira’s honey;MIE01-05), has as a main component the mesocarp of thefruit of the güira (Crescentia cujete), specifically of thewild variety calledgüira cimarrona. The fruit is put intodecoction for a long time, and then the decoction is stirredand honey, sugar and/or rum are added, as in MIE01. Thefruit contains iridoids, iridoid glucosides and other com-pounds (Binutu, 1997; Kaneko et al., 1997, 1998), and isused throughout Cuba as a pectoral and depurative (Roig,1974; Fuentes and Granda, 1982; Seoane, 1984). Four mix-tures have been reported as anti-catarrhal, the fifth is claimedto treat coolness (frialdad) of the uterus (matriz) and men-strual irregularity (dismenorrhea), by cleaning women’s ab-domen (vientre) promoting either abortion or pregnancy (seealso Moreno et al., 1994). Women who became pregnantafter a “depurative process” withmiel de guira“should thennot smell the preparation in order not to loose the baby”.Plant components other thanCrescentia cujetevary muchwithin miel de güira’s formulas. OnlyBidens pilosaandCissus sicyoidesare used with much agreement to enhanceanti-inflammatory and pectoral properties of the formulas(MIE03,04,05).Miel de güirais likely to have a Caribbean,or at least American origin, asgüira is taken internally forgonorrhea in Haiti and the Dominican Republic (Weniger,1991), and the green fruits’ juice is used for bronchialasthma and the decoction of the ripe fruit in the preparationof an abortive tea in Guayana (Grenand et al., 1987). TheSpanish could have disseminated Indian medicinal knowl-edge about the plant from tropical America and throughoutthe Caribbean during their trips throughout the continent(Wilson, 1997), functioning as a material and culturalbridge.
Pru (PRU01,02) is a medicinal-food beverage of proba-ble Afro-Haitian origin (Ortiz, 1956; Volpato and Godınez,submitted for publication) produced from the decoction ofvarious plants. The decoction is then stirred, sugar is added,and the drink fermented in the sun.Pru is presently soldthroughout Cuba as a refreshment, although it is also used asa depurative and hypotensive drink (Volpato and Godınez,submitted for publication), as “it loosens the legs” (aflojalas piernas). Gouania lupuloides, Pimenta dioica, Smilaxdomingensis, and Cinnamomum verum, along with sugar,are the main components of the drink. Like the congenericsGouania polygama(Jacq.) Urban andGouania lupuloides,which are used indistinctly (Roig, 1974; Volpato and Godiez,submitted for publication), Cinnamomum verumis reportedinstead ofCanella winterana(L.) Gaertn (Canellaceae) ascanela(the latter is also known ascúrbana). This fact couldbe explained as the equivalent use as a stomachic of dif-ferent parts (leaves versus stem) of different species, a caseof under-differentiation (Berlin, 1992) due to similar medi-cal properties (both are also used as an abortifacient;Roig,1974; Moreno et al., 1994). Canella winteranawas probablythecanelaused by Cuban aboriginal peoples at the time of
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the arrival of Columbus, who brought to the island samplesof Europeancanela (cinnamon) during his first voyage in1492 (Esquivel and Hammer, 1992), and both species are atpresent cultivated in Eastern Cuban home gardens (Esquivelet al., 1992).
3.4. Cultural aspects and ecological implications
The provinces of Santiago de Cuba and Guantánamo,where our field research was carried out, roughly correspondto the Cuban Eastern Sub-Province ofBorhidi (1991), alsocalledOriente de Cuba, and include the Nipe-Sagua BaracoaMassif and the Sierra Maestra Mountains, considered as themost important centers for the development and evolution ofthe Cuban flora (Capote et al., 1989; Del Risco, 1999). To-gether with western Hispaniola (Haiti), Eastern Cuba is themost prominent center of speciation in the Antilles and it hasone of the richest floras in the world (Borhidi, 1991). Morethan 3000 vascular plant species (almost half of Cuban flora)are present, of which more than 1500 are strictly endemic(Borhidi, 1991; López et al., 1992, 1994a, 1994b, 1995).Nevetheless, this richness in plant species contributes littleto the medicinal flora of herbal mixtures: more than 60%of the 170 species are reported as cultivated in traditionalhome gardens (conucos) of Eastern Cuba (cf.Esquivel et al.,1992; Castiñeiras et al., 2000). Among species collectedfrom the wild, 80% occur in disturbed or managed habitats(roadsides, gardens, plantations, second growth forests), andare gathered in the immediate surroundings of villages andcommunities. Although 51% of the Cuban flora is endemicto the island (Capote et al., 1989), only 4.8% of the speciesused in mixtures are Cuban endemisms, and five per cent ofthe total taxa are from primary forests (i.e.Oxandra lance-olata, Colubrina elliptica, Diospyros grisebachii, Thouiniaelliptica, Swietenia mahagoni). Moreover, the estimatedprimary forest areas covered about 90% of Cuban territoryin the early XVI century, whereas they presently cover onlyabout 20% (Ricardo et al., 1995; Del Risco, 1999) and aremainly restricted to geographically-isolated regions, whereforaging would be a much higher-cost activity. Accordingto the classification of Cuban synanthropic flora byRicardoet al. (1995), about 70% of species used in Eastern Cubato prepare traditional herbal mixtures are synanthropicspecies, of which 30% are Old World exotics of eitherAsian, African, or European origin, some (i.e.Psidiumguajava, Capsicum frutescens) introduced by Amerindiansin the pre-colonial period. Many of the species (i.e.Cissussicyoides, Lepidium virginicum, Petiveria alliacea, Solanumtorvum, Stachytarpheta jamaicensis) are present as weedsin Cuban plantations (cf.Gutte, 1994). The Cuban plantpharmacopoeia is cultivated, exotic, and synanthropic, andthus is highly representative of the environmental and cul-tural changes the island has witnessed since 1492. Actually,managed and disturbed landscapes and home gardens arealso the most important medicinal foraging places to manynative groups of America and worldwide (Alcorn, 1981;
Balick and Mendelsohn, 1992; Grenand, 1992; Frei et al.,2000; Stepp and Moerman, 2001). Alcorn (1981), Grenand(1992), Voeks (1996)andFrei et al. (2000), among others,also confirmed that species originating from forest habitatsare often brought nearby the house because they have beenregarded as useful resources.Ceiba pentandraand Roys-tonea regiaare emerging species in mesophylous semide-ciduous and in subperennifolius forests (Del Risco, 1999),and are also planted near the villages and in home gardensfor their material and spiritual value, the latter also beingnow very common in disturbed areas (Henderson et al.,1995). Here these species can be exploited without walkingfar from the community; having easy access to the resourcesis a factor with much influence in traditional medicinalsystems worldwide, and could be particularly importantfor herbal mixtures, due to the number of plant speciesused.
Plant selection for medicinal use is an ongoing process,and Cuban patterns of plant exploitation have been shapedby Cuban historical developments, mainly by migration phe-nomena and their cultural implications (Fuentes, 1984b).Like in other New World countries (Voeks, 1996; Balicket al., 2000; Ososki et al., 2002), in Cuba, due to the rapidgenocide of aboriginal peoples upon the arrival of the Span-ish, folk medicines of different ethnic groups evolved intoa cultural legacy as long as healers and people (1) intro-duced their own most salient plants into the new environ-ment, (2) substituted familiar plants with local species on thebasis of morphological and organoleptic similarity, and (3)learned folk uses from people of other medical traditions.Although Spanish, Amerindian, and, to a lesser extent, Asi-atic and Antillean knowledge, much influenced and shapedthe Cuban pharmacopoeia (Guarch, 1978; Fuentes, 1984b;Hammer and Esquivel, 1992; Rivero de la Calle, 1992), froman ethnobotanical point of view, Africa has provided themain cultural contributions to Cuban herbalmateria med-ica (Cabrera, 1954; Ortiz, 1956; Laguerre, 1987; Esquivelet al., 1992; Fuentes, 1992). Historically, because of the sav-age form of capture to which African slaves were submitted,they could only bring with them a few plant species and hadto learn to satisfy their needs from the new environments.Spatially constrained to the master’s properties and tempo-rally limited by the work load, Africans must have experi-mented with familiar congenerics and also completely newtaxa and incorporated into their pharmacopoeia the moreeasily available common plants, as well as learning the usesof local plants from Indians. About 15% of the species usedin mixtures have been brought to Cuba from Africa, oftenalong with their medicinal uses in the place of origin.Co-cos nucifera, the species most frequently cited in mixtures,was brought to Cuba soon after the conquest via Africa(Whitehead, 1984), and the decoction of its root is a maincomponent ofgalonesand is used to treat venereal diseasesthroughout Africa (Neuwinger, 2000). Panicum maximum, aspecies of African origin most cited in various combinationsas febrifuge (FEV02,03,04), is used singly or in mixtures
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to treat malaria and other fevers also in Africa (Neuwinger,2000) and Jamaica (Asprey and Thornton, 1955). A com-mon element in all African cultures that were brought toCuba was the belief in the spirituality of wild vegetation(monte) and weeds (malezas), where ancestral deities andpowerful spirits lived as they were living in Africa (Cabrera,1954). Species such asCeiba pentandra, the holy tree of theclassical Maya, have attained such a spiritual and religiousimportance for Africans that they are considered as sacredplants. Maybe Africans incorporated the tree into their ritesbecause of its resemblance to baobab (Adansonia digitataL.,Bombacaceae), a sacred plant in Equatorial Africa (Fuentes,1984a, 1992). They could have inherited the cult ofCeibafrom the Taıno aboriginal people living in Eastern Cuba atthe time of the conquest. The decoction of the fruit or of thepulped fruit ofAdansonia digitatais drunk in Africa to treatdiarrhoea (Neuwinger, 2000), and the same use has been re-ported for the fruit epicarp ofCeiba pentandrain GAS18.
In spite of African influence within Cuban ethnobotan-ical lore, 60% of the total species are of neotropical orCaribbean origin, and many of them (i.e.Bidens pilosa, Cis-sus sicyoides, Chenopodium ambrosioides, Crescentia cu-jete, Justicia pectoralis, Lippia alba), or their congenerics(i.e. Crescentia alataKunth, Justicia segundaVahl) havecommon folk uses in tropical and middle America and inthe Caribbean (cf.Morton, 1981; Balick and Arvigo, 1993;Duke and Vásquez, 1994; Longuefosse and Nossin, 1996;Frei et al., 1998; Ankli et al., 1999). Common uses couldbe due to coevolutionary processes of utilization of plantsas medicines by ethnic groups of America, or to the com-mon ethnobotanical knowledge of Tainos living in Cuba withmainland groups (Wilson, 1997), or more often to the Span-ish activity of assimilating the uses and introducing plantsthroughout the colonies. What is clear is the cultural andanthropological value of Cuban traditional medicine and thestrong link between ecological patterns of exploitation ofplant resources and social phenomena that have occurred inEastern Cuba since European conquest.
4. Conclusions
Herbal mixtures have played and continue to play a promi-nent role in Cuban popular medicine. They are either com-binations of plant species with common therapeutical usesthat are prepared at household level, or culturally definedformulas with specific denominations employed by tradi-tional healers. From our ethnobotanical findings, 170 plantspecies and more than thirty non-botanical products are usedin 199 different medicinal recipes in Eastern Cuba.Galonesare the formulas with the highest variety of different com-ponents, incorporating at least 66 species. More systematicethnobotanical fieldwork is necessary in other Cuban as wellas Caribbean areas in the field of herbal mixtures, and thereis no reason for medicinal plant research to be restricted tosingle-species preparations and uses.
Plant combinations have both cultural and ecologicaldimensions. The Cuban ethnopharmacopoeias, includingmulti-species formulas, are the result of the legacy withinCuban culture of different ethnic groups, each with a spe-cific herbal knowledge, and of the interactions of this legacywith the local environment.Galonesand miel de güiraare the complex formulas with the richest variety of plantcomponents, and their anthropological and ethnobotanicalaspects should be further investigated because of the rele-vance of these preparations within local culture and medicalsystems and because of the threats that modernization posesto the conservation of traditional Cuban herbalism.
Montane systems of Eastern Cuba possess one of therichest floras of the world. Some endemic medicinal plantsare used in mixtures. However, the Cuban pharmacopoeiais a product of human-derived landscapes rather than beingconcentrated in primary forests and natural habitats. Eth-nic groups that were brought to Cuba drew on the florawith which they were already familiar (Old World domes-ticates, cosmopolitan weeds, pantropical congenerics), andselected medicinal plants also on the basis of morphologicaland organoleptic similarities with already known species.Cuban ethnobiology is of utmost cultural interest within theframework of migration and the cultural loss and replace-ment that characterized the island since the late XVth cen-tury. Ethnobiological research in Cuba should not only takeinto account medicinal uses of the species, but it should paymore attention to the cultural and ecological aspects of theirexploitation and to the ongoing relationships between theseaspects.
Acknowledgements
Special thanks are due to all of the people and healerswho shared with us their knowledge. Many thanks are dueto Dr. Andrea Pieroni and Prof. Patricia Howard for theirvaluable comments and suggestions, and to the technicalstaff of BIOECO, particularly the curator of the Herbarium,Florentino Bermúdez Garcıa, for their help in compilinginformation from herbarium specimens. Thanks are also dueto Davide Trinca for helping with the tables.
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Journal of Ethnopharmacology 90 (2004) 317–321
Studies on the anti-inflammatory and analgesic propertiesof Tithonia diversifolia leaf extract
Victor B. Owoyelea,∗, Caleb O. Wuraolaa, Ayodele O. Soladoyea, Samuel B. Olaleyeba Department of Physiology and Biochemistry, University of Ilorin, P.M.B. 1515, Ilorin, Nigeria
b Department of Physiology, College of Medicine, University of Ibadan, Ibadan, Nigeria
Received 1 May 2003; received in revised form 8 August 2003; accepted 9 October 2003
Keywords: Tithonia diversifolia (Hemsl) A. Gray; Anti-inflammatory; Analgesic; Properties
1. Introduction
Tithonia diversifolia (Hemsl) A. Gray (Compositae)is commonly referred to as Mexican sunflower, or treemarigold. It is a bushy perennial weed commonly found onthe fields, wasteland and road sides of Nigeria (Akobunduand Agyakwa, 1987). The plant is used for ornamentalpurposes or as manure for farming (Thijssen et al., 1993)and for treatment of diabetes mellitus (Takanashi, 1998).In addition, the plant is used for the treatment of stomachpains, indigestion, sore throat and liver pains among theLuo tribe of Kenya (Kokwaro, 1976). Validation of some ofthese folkloric claims have shown thatTithonia divorsifoliacontains bioactive compounds that have anti-inflammatoryactivity (Rungeler et al., 1998), anti-diarrhoeal (Tona et al.,1999), anti-amoebic and spasmolytic activities (Tona et al.,1998, 2000). Some of the bioactive compounds that havebeen isolated from the leaves include sesquiterpenes (Kuoand Chen, 1998), saponins and alkaloids (Tona et al., 2000).
In Nigeria there are oral reports among herbal medicinepractitioners linkingTithonia diversifolia with the treatmentof menstrual pain. Therefore, the present study was under-taken to investigate medicinal properties of the plant relatingto anti-inflammatory and analgesic effects.
Male Wistar rats weighing 215.7±4.0 (range 190–260 g)were used for this study. They were bred and housed inthe animal house, Faculty of Health Sciences, University ofIlorin.
The animal house was well ventilated; the rats were fedwith mouse cubes (Bendel feeds, Ilorin) and had water adlibitum. The animals were randomly divided into groupscomprising five rats each. The research was conducted inaccordance with the ethical rules on animal experimentation,approved by Ethical committee, Faculty of Health Sciences,University of Ilorin.
2.2. Plant materials
The leaves (Tithonia diversifolia) used for this study werecollected in August 2002 from the University of Ilorin or-namental garden and submitted to the Botany Department,University of Ilorin, for identification by Professor F.A.Oladele. In addition a voucher specimen (FHI 106521) hasalso been deposited in the Herbarium of Botany Depart-ment, University of Ilorin and Forestry Research instituteof Nigeria (FRIN), Ibadan. The shade-dried leaves were re-duced to powdery form and 200 g of the powdered samplewas extracted with 2.5 l of methanol (analytical grade) for 3
318 V.B. Owoyele et al. / Journal of Ethnopharmacology 90 (2004) 317–321
days. The macerated mixture was filtered and evaporated ina carefully regulated water bath maintained at 45◦C to yielda green solid extract weighing 8 g. The extract was stored ina refrigerator at 4◦C and dilutions of the extract were madein normal saline to determine their effects.
2.3. Carrageenan-induced paw oedema
Oedema was induced by injecting 0.1 ml of 1% (w/v)carrageenan suspension into the subplantar region of theright hind paw of the rats according to the method de-scribed byWinter et al. (1962). The test groups (B–D) ofrats were treated orally with 50, 100 or 200 mg/kg of theextract, respectively, 1 h before carrageenan injection. Thecontrol group (A) received 10 ml/kg saline and the referencegroup (E) received 5 mg/kg indomethacin (Strides, Belgium)orally.
Measurement of paw size was carried out by wrapping apiece of cotton thread round the paw and the length of thethread corresponding to the paw circumference was deter-mined using a meter rule (Hess and Milonig, 1972; Olajideet al., 2000).
Measurement was done immediately before and 1–5 h fol-lowing carrageenan injections. The inhibitory activity wascalculated according to the following formula (Olajide et al.,2000):
Percentage inhibition
= (Ct − Co)control− (Ct − Co)treated
(Ct − Co)control× 100
where Ct is the paw circumference at timet, Co is thepaw circumference before carrageenan injection,Ct −Co isoedema, (Ct − Co)control is oedema or paw size after car-rageenan injection to control rats at timet.
In practice carrageenan activity is maximum at 3 h and theeffect of the extract at that time is accepted as the optimuminhibitory effect.
2.4. Cotton pellet granuloma in rats
A sterilized cotton pellet weighing 30 mg was introducedsubcutaneously into the groin region of rats according to themethod ofMossa et al. (1995). The test groups of animals(B–D) were treated orally with 50, 100 or 200 mg/kg of theextract, respectively, daily for 4 days. Animals in the con-trol and reference groups (A and E) received 0.9% saline(10 ml/kg, p.o.) and indomethacin (5 mg/kg, p.o.), respec-tively. The animals were sacrificed on the fifth day after anoverdose of ether. The pellets surrounded by granuloma tis-sue (five animals per group) were dissected out carefullyand dried at 60◦C to a constant weight. Mean weight of thegranuloma tissue formed in each group was obtained andthe percentage inhibition was expressed by comparing themean weight in the test groups with the mean weight in thecontrol group.
2.5. Hot plate latency assay in rats
The hot plate latency assay was based on the method ofEddy et al. (1950). Briefly in this method rats in groups B,C and D were given extracts ofTithonia diversifolia orallyafter 12 h fast. The dosages were 50, 100 and 200 mg/kg forthe rats in groups B, C and D, respectively. The rats in groupsA and E were given doses of normal saline (10 ml/kg) andindomethacin (5 mg/kg), respectively. A rat from group Bwas placed on the hot plate after the extract had been givenand the reaction time for the animal to lick the paw or jumpfrom the hot plate was taken as the latency (s). This wasalso repeated at 60 and 90 min from the time the extractwas given. The whole procedure was repeated for all therats in the group and the average of the latency was deter-mined from the five rats in the group. The mean latency foreach group (A, C–E) was determined using the same pro-cedure. The temperature of the hot plate was maintained at55± 2 ◦C.
2.6. Formalin-induced paw licking in rat
The formalin-induced paw licking was studied in rats(groups A–E) using the method ofHunskaar and Hole(1997). In this method, 100�l of 3% formalin was injectedinto the subcutaneous tissue on the plantar surface of the lefthind paw of rats 1 h after oral administration of the extracts,indomethacin or normal saline. The rats in groups B–D weregiven oral doses of the extracts 50, 100 and 200 mg/kg, re-spectively, 1 h before formalin injection. The rats in groupsE and A were given oral doses of indomethacin (5 mg/kg)and normal saline (10 ml/kg), respectively, 1 h before theinjection. In each rat the time spent on licking the injectedpaw was observed as soon as the injection was given (i.e.early phase, 0–5 min post-injection) and in the late phase(20–30 min post-injection) after injection. The mean of thetime spent on licking the injected paw in each group wasdetermined (Table 4).
2.7. Statistical analysis
In this study recorded values are mean±S.E.M. Statisticalanalysis was by unpaired comparison using the studentt-test,P-values<0.05 were accepted as significant.
3. Results
3.1. Carrageenan-induced paw oedema
Using the oral doses ofTithonia diversifolia the resultsshow a dose-dependent decrease in the size of the oedemafrom 4.6±0.9 mm to 0.4±0.4 mm. This effect correspondedwith the maximum effect ofTithonia diversifolia at 3 h. Theresponses at 3 and 5 h are shown inTable 1.
V.B. Owoyele et al. / Journal of Ethnopharmacology 90 (2004) 317–321 319
Table 1Effects of the methanol extract ofTithonia diversifolia leaves on carrageenan-induced paw oedema in rats
∗P < 0.05 compared with control; student’st-test.a Each value is the mean± S.E.M. of five rats.
3.2. Cotton pellet granuloma
In this study the oral doses ofTithonia diversifolia extractshow a dose-dependent reduction in the granuloma tissueformation from 57.6 ± 4.5 mg to 31.8 ± 2.9 mg (Table 2).
3.3. Hot plate test
The results from this study show that at 30 min the oraldoses ofTithonia diversifolia extract increased the reactiontime from 6.0±0.2 s to 8.3±0.8 s. Similarly, at 60 and 90 minthe reaction times were significantly increased. However, theincrease was not dose-dependent (Table 3).
3.4. Formalin-induced paw licking in rats
In the present study, oral doses ofTithonia diversifoliaextract decreased the time spent on licking (licking time)
Table 3Effects of the methanol extract ofTithonia diversifolia leaves on hot plate test in rats
∗P < 0.05; +P < 0.001 compared with control; student’st-test.a Each value is the mean± S.E.M. of five rats.
from 114.2 ± 7.0 s to 27.4 ± 5.2 s in the early phase afterinjection. Similarly in the late phase after injection lickingtime was reduced from 68.8±9.8 s to 10.8±3.1 s (Table 4).
4. Discussion and conclusion
The anti-inflammatory effect and analgesic properties ofTithonia diversifolia extract were investigated in the presentstudy. For the anti-inflammatory effect it is important toestimate the activities of the extract in the acute phase ofinflammation as well as in the chronic phase of inflamma-tion. Accordingly the carrageenan test was selected becauseof its sensitivity in detecting orally active anti-inflammatoryagents particularly in the acute phase of inflammation(DiRosa et al., 1971; DiRosa, 1972). The cotton pelletgranuloma on the other hand, is a model of chronic inflam-mation (Ismail et al., 1997) and the dry weight has been
320 V.B. Owoyele et al. / Journal of Ethnopharmacology 90 (2004) 317–321
shown to correlate with the amount of granulomatous tissueformed (Swingle and Shideman, 1972).
The result obtained from using the two models show thatTithonia diversifolia extract can effectively reduce inflam-mation in both the acute and chronic phases (Tables 1 and2). This result provides a scientific basis for the practiceof using Tithonia diversifolia extracts in the treatment ofwounds as reported by some authors (Rungeler et al., 1998).In addition,Rungeler et al. (1998)using different modelsfound that the compound extracted fromTithonia diversifo-lia leaves showed anti-inflammatory effects. In their expla-nation based on the models used, anti-inflammatory effectswere confirmed using transcription factor and reactions ofthe arachidonic acid pathways. These authors later showedthat the active principles mediating the anti-inflammatoryeffects were diversifolin, diversifolin methyl ether and tiro-tundin.
The analgesic properties were also studied using sensitivemodels that could provide different grades of noxious stimuli(in thermal stimulus and chemically induced tissue damage).In the present study the thermal test was selected because ofseveral advantages including the sensitivity to strong anal-gesics and limited tissue damage. Furthermore, the formalintest was selected because of several advantages including theability to mimic human clinical pain conditions, sensitivityto mild analgesics, production of tonic stimulus and sensi-tivity to non-steroidal anti-inflammatory drugs (Prado et al.,1990; Tjølsen et al., 1992; Santos et al., 1997; Hunskaar andHole, 1997). The observed effects in this study (Tables 3 and4) have shown thatTithonia diversifolia can significantly in-hibit the responses to thermal stimulus and formalin-inducedpain. Although the inhibition of formalin-induced pain wasdose-dependent and the effect of thermal stimulus was notdose-dependent. However, it is not unusual for variation ofthis nature to occur. For example there are reports where re-sponses to formalin test were strong while there were no sig-nificant responses to thermal test (Prado et al., 1990; Adairet al., 1995). Therefore, it is concluded that theTithonia di-versifolia extract is capable of inhibiting non-inflammatoryreactions as well as inflammatory pain.
The clinical applications of these findings must await fur-ther studies. Nevertheless studies are in progress to iden-tify the active ingredients inTithonia diversifolia leavesresponsible for anti-inflammatory and analgesic activities(Rungeler et al., 1998; Kuo and Chen, 1998; Tona et al.,2000). Although the mechanism involved was not deter-mined in the present study. This is likely to be the focus ofanother study.
Acknowledgements
The authors are grateful to Dr. A.O. Olajide (Pharma-cology Department, University of Ibadan, Ibadan), Mr. J.L.Fwangle, Miss M.O. Sunmola, Mr. A.U. Akuapa and Mrs.V.O. Adegboyega (Department of Physiology and Biochem-
istry, University of Ilorin, Ilorin) for technical and secretarialassistance.
References
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Akobundu, I.O., Agyakwa, C.W., 1987. A Handbook of West AfricanWeeds. International Institute for Tropical Agriculture, Nigeria,pp. 194–195.
DiRosa, M., 1972. Biological properties of carrageenan. Journal of Phar-macy and Pharmacology 24, 89–102.
DiRosa, M., Giroud, J.P., Willoughby, D.A., 1971. Studies of the acuteinflammatory response induced in rats in different sites by carrageenanand turpentine. Journal of Pathology 104, 15–29.
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Hess, S.M., Milonig, R.C., 1972. Assay for anti-inflammatory drugs.In: Lepow, I.H., Ward, P.A. (Eds.), Inflammation, Mechanisms andControl. Academic Press, New York, pp. 1–2.
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Olajide, O.A., Awe, S.O., Makinde, J.O., Ekhelar, A.I., Olusola, A.,Morebise, O., Okpako, D.T., 2000. Studies on the anti-inflammatory,antipyretic and analgesic properties ofAlstonia boonei stem bark.Journal of Ethnopharmacology 71, 179–186.
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Rungeler, P., Lyss, G., Castro, V., Mora, G., Pahl, H.L., Merfort, I., 1998.Study of three sesquiterpene lactones fromThitonia diversifolia on theiranti-inflammatory activity using the transcription factor MF-Kappa Band enzymes of the arachidonic acid pathway as a target. Planta Medica64, 588–593.
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Journal of Ethnopharmacology 90 (2004) 323–329
Effect of Retama raetam on lipid metabolism in normaland recent-onset diabetic rats
M. Maghrania, A. Lemhadria, N-A. Zeggwagha, A. El Amraouia,M. Halouib,1, H. Jouada, M. Eddouksa,∗
a UFR Physiology of the Nutrition and Endocrinian Pharmacology, BP 688, Boutalamine, Errachidia, Moroccob Inserm U460, CHU Bichat, 16 rue Henri Huchard, Paris 75018, France
Received 5 May 2003; received in revised form 2 June 2003; accepted 9 October 2003
Abstract
The purpose of this study was to examine the effect of single and repeated oral administration of the aqueous extract ofRetama raetam(Forssk) Webb (RR) (20 mg/kg) on lipid metabolism in normal and streptozotocin-induced diabetic rats. In normal rats, the aqueous extractof RR induced a significant decrease of the plasma triglycerides concentrations one week after repeated oral administration (P < 0.05). Thisreduction was maintained two weeks after once daily repeated oral administration (P < 0.05). A significant decrease of plasma cholesterollevels was also observed one week (P < 0.05) and two weeks (0.05) after repeated oral administration.
In diabetic rats, RR treatment caused a significant decrease of plasma triglycerides levels after a single (P < 0.05) and repeated (P < 0.001)oral administration. A significant decrease of cholesterol levels was observed four hours after a single oral administration of the RR aqueousextract (P < 0.05). One week after repeated oral administration of RR aqueous extract, the plasma cholesterol levels were significantlydecreased (P < 0.05) and still dropped after two weeks (P < 0.005).
Keywords: Retama raetam; Cholesterol; Triglycerides; Body weight; Aqueous extract and diabetic rats
1. Introduction
From the beginning of the last century, evidence of thelipid lowering properties of medicinal plants has been accu-mulating (Kritchevsky, 1995). The importance of such in-vestigations is confirmed in obesity, diabetes mellitus, heartfailure and atherosclerosis treatment (Ross, 1986). Manyscientists have evoked the role of medicinal plants in thecontrol of plasma cholesterol concentrations and the reduc-tion of morbidity and mortality due to vascular diseasesaround the world. The influence of diabetes mellitus on lipidmetabolism is well established. The association of hyper-
glycemia and alteration of lipidic parameters present a majorrisk of cardio-vascular diseases in diabetic patients (Jensenet al., 1988; Motta et al., 2001).
Retama raetam (RR), locally named as “R’tm”, is a wildplant belonging to the Fabaceae family. It is common toNorth and East Mediterranean region and the Sinai Penin-sula (Boulos, 1999; Mittler et al., 2001). In Morocco, it islargely located in desert regions and Middle Atlas. The plantflowers from April to May. The molecular and biochemicalmechanisms associated with dormancy and drought toler-ance in this desert plant have been elucidated (Pnueli et al.,2002; Mittler et al., 2001).
According to a recent ethnobotanical survey in south-eastern region of Morocco (Tafilalet), RR is prescribed bytraditional herbal healers for diabetes control and treatment(Eddouks et al., in press). RR was also described for treat-ment of hypertension (Eddouks et al., in press). The scientificconfirmation of this traditional practice was demonstrated in
324 M. Maghrani et al. / Journal of Ethnopharmacology 90 (2004) 323–329
both normal and streptozotocin (STZ) rats (Maghrani et al.,2003a). In addition, the underlying mechanism of the hy-poglycaemic effect of RR seems to be the stimulation ofglycosuria (Maghrani et al., 2003b).
The present study was undertaken to evaluate the poten-tial cholesterol and triglycerides lowering activity of a singleand repeated oral administration of the RR aqueous extractin normal and STZ rats. The effect of RR extract on bodyweight loss was also determined. Sodium-metavanadate(0.8 mg/kg) was used as a reference drug known by its bothhypolipidic and hypoglycaemic activities (DeFronzo, 1988;Reaven, 1988).
Panel a:
0
0,5
1
1,5
2
2,5
NTRt NTRrr NTRv
Pla
sma tri
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ol/
l)
Panel b:
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0,5
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2
2,5
3
3,5
STZt STZrr STZv
Pla
sma t
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des(
mm
ol/
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***
Fig. 1. Plasma triglycerides levels (mmol/l) after a single oral administration of RR aqueous extract (20 mg/kg) in normal (panel a) and STZ (panel b)rats. Data are expressed as means± S.E.M., n = 6 rats per group.∗P < 0.05; ∗∗P < 0.01 when compared to baseline values (the start of treatment). (�)0 h, ( ) four hours of treatment. NTRt: normal control group, NTRrr: normal RR-treated group, NTRv: normal vanadate-treated group, STZt: diabeticcontrol group, STZrr: diabetic RR-treated group, STZv: diabetic vanadate-treated group.
2. Material and methods
2.1. Plant material
The plant used in this study was collected from its nat-ural habitat, from Tafilalet region (Morocco) in May 2002,and dried with hot air (40–60◦C). The plant was identifiedand authenticated asRetama raetam (Forssk) Webb with as-sistance of Prof. M.Rejdali (Veterinary and Agronomy In-stitute, Rabat, Morocco). Voucher specimen (EM 1a) wasdeposited at the herbarium of the Faculty of Sciences andTechniques Errachidia.
M. Maghrani et al. / Journal of Ethnopharmacology 90 (2004) 323–329 325
2.2. Preparation of the aqueous extract
An aqueous extract was prepared according to the tradi-tional method used in Morocco: 1 g of the dried powderedwhole plant of RR were boiled in 100 ml of distilled wa-ter for 10 min and cooled to room temperature for 15 min.Thereafter, the aqueous extract was filtered using a Milliporefilter (Millipore 0.2 mm, St Quentin en Yvelines, France) toremove particulate matter. The filtrate was lyophilized andthe desired dose (mg of lyophilized aqueous extract of RRper kg body weight) was then prepared and reconstituted in1.5 ml of distilled water. The aqueous extracts were prepared
Panel a:
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NTRt NTRrr NTRv
Pla
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ho
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vels
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Panel b:
0
1
2
3
4
STZt STZrr STZv
Pla
sma c
ho
lest
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(mm
ol/
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*
**
Fig. 2. Plasma cholesterol levels (mmol/l) after a single oral administration of RR aqueous extract (20 mg/kg) in normal (panel a) and STZ (panel b)rats. Data are expressed as means± S.E.M., n = 6 rats per group.∗P < 0.05; ∗∗P < 0.01 when compared to baseline values (the start of treatment). (�)0 h, ( ) four hours of treatment. NTRt: normal control group, NTRrr: normal RR-treated group, NTRv: normal vanadate-treated group, STZt: diabeticcontrol group, STZrr: diabetic RR-treated group, STZv: diabetic vanadate-treated group.
daily, just before administration. The extract yield was 13%.Animals were treated orally, i.e., intubation using a syringeat an overnight fasted state.
2.3. Animals used
Experiments were performed in adult male Wistar ratsweighing from 200 to 230 g. The animals were housed understandard environmental conditions (23±1◦C, with 55±5%humidity and a 12 h light/dark cycle) and maintained withfree access to water and ad libitum standard laboratory diet(70% carbohydrates, 25% proteins, 5% lipids).
326 M. Maghrani et al. / Journal of Ethnopharmacology 90 (2004) 323–329
2.4. Induction of diabetes
Streptozotocin (Sigma, St. Louis, MO, USA) was dis-solved in 0.1 M fresh cold citrate buffer at pH 4.5 beforeuse, and injected intravenously into the tail vein at a doseof 65 mg/kg (Burcelin et al., 1995). After 18 h, the rats withstable fasting blood glucose levels greater than 16 mmol/lwere considered as diabetic and used in the present study.The percentage of response to streptozotocin injectionwas 90%.
2.5. Single oral administration
Normal and diabetic rats were randomly assigned to threedifferent groups containing six rats each. One control group
Panel a:
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Time (days)
Plas
m tr
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* * ** *
Panel b:
0
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0 2 4 7 15Time(days)
Plas
ma
trig
lyce
ride
s(m
mol
/l)
*
****
*****
*
Fig. 3. Plasma triglycerides levels (mmol/l) after a repeated oral administration of RR aqueous extract (20 mg/kg) in normal (panel a) and STZ (panelb) rats. Data are expressed as means ± S.E.M., n = 6 rats per group. ∗P < 0.05; ∗∗P < 0.01 when compared to baseline values (the start of treatment).(�) C: control group; ( ) RR: RR-treated group; ( ) vanadate-treated group.
received distilled water, a second treated group receivedthe aqueous extract of RR at a dose of 20 mg/kg (20 mgof lyophilized aqueous extract of RR per kg body weight)and the third group received a reference drug (vanadate(Na+VO3
−) at a dose of 0.8 mg/kg). For single oral adminis-tration, distilled water (control), vanadate (0.8 mg/kg) or theaqueous extract (20 mg/kg) were administered and plasmacholesterol and triglycerides levels were measured beforeand four hours after RR treatment.
2.6. Repeated oral administration
For repeated oral administration, rats were treated oncedaily at a dose of 20 mg/kg for two weeks and plasma choles-terol and triglycerides levels were followed during this pe-
M. Maghrani et al. / Journal of Ethnopharmacology 90 (2004) 323–329 327
riod. The rats (n = 6 in each group) were treated oncedaily. Blood samples were collected from the tail vein andplasma triglycerides and cholesterol levels are determinedenzymatically by colorimetric specific kits (Randox, UK),respectively. The kits used in this study for substrates anal-ysis were specific for both human and rat blood samples atthe same percentage.
2.7. Statistical analysis
Data were expressed as mean ± S.E.M. The statisticalanalysis was performed by the Student’s t-test. The valueswere considered significantly different when the P-value wasless than 0.05 in comparison to baseline values (startingvalues).
Panel a:
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Time (days)
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**
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Time (days)
Plas
ma
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***
****
Fig. 4. Plasma cholesterol levels (mmol/l) after a repeated oral administration of RR aqueous extract (20 mg/kg) in normal (panel a) and STZ (panel b)rats. Data are expressed as means ± S.E.M., n = 6 rats per group. ∗P < 0.05; ∗∗P < 0.01 when compared to baseline values (the start of treatment). (�)C: control group; ( ) RR-treated group; ( ) vanadate-treated group.
3. Results
3.1. Single oral administration
In normal rats, no significant changes of both plasmacholesterol and triglycerides concentrations four hours af-ter a single administration of RR (20 mg/kg) were noted(Figs. 1a and 2a). In vanadate-treated group, the plasmacholesterol and triglycerides concentrations were signifi-cantly decreased over four hours of a single oral dose ofvanadate (0.005) (Figs. 1a and 2a).
In STZ rats, the aqueous extract of RR caused a sig-nificant reduction of plasma triglycerides (P < 0.05) andcholesterol (P < 0.05) levels four hours after RR treat-ment (Figs. 1b and 2b). Vanadate (0.8 mg/kg) reduced both
328 M. Maghrani et al. / Journal of Ethnopharmacology 90 (2004) 323–329
the plasma cholesterol (P < 0.01) and triglycerides levels(P < 0.01) four hours after RR administration in diabeticrats (Figs. 1b and 2b).
3.2. Repeated oral administration
In normal rats, a significant reduction in both plasmatriglycerides and cholesterol levels was observed inRR-treated group from the first to the second week of RRtreatment (P < 0.05) (Figs. 3a and b). After two weeks oftreatment, vanadate caused a significant decrease of plasmatriglycerides (P < 0.01) (Fig. 3a) and cholesterol levels(P < 0.05) (Fig. 4a).
In diabetic rats, RR extract decreased significantly theplasma cholesterol levels from the fourth day (P < 0.05);the most significant effect was reached at the second week
Panel a:
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y w
eigh
t(g) *****
***
***
** *
Fig. 5. Effect of RR aqueous extract treatment (20 mg/kg) on body weight (g) in normal and diabetic rats. Data are expressed as means ± S.E.M., n = 6rats in each group. ∗P < 0.05; ∗∗P < 0.01. ∗∗∗P < 0.001 when compared to baseline values (the start of treatment). (�) C: control group; ( ) RR-treatedgroup; ( ) vanadate-treated group.
of treatment (P < 0.001) (Fig. 3b). The plasma cholesterollevels were increased from the first week (P < 0.05) to thesecond week (P < 0.01) after RR treatment (P < 0.001)(Fig. 4b). Daily vanadate administration (0.8 mg/kg) fortwo weeks produced a statistically significant decrease inboth plasma cholesterol and triglycerides concentrations(Figs. 3b and 4b).
3.3. Body weight loss
A significant decrease on body weight was observed indistilled water treated group from the second day of STZinjection. RR aqueous extract (20 mg/kg) caused a signifi-cant weight loss two weeks after once daily repeated oraltreatment only in STZ rats (Fig. 5b) but not in normal rats(Fig. 5a). Vanadate caused also a significant decrease on
M. Maghrani et al. / Journal of Ethnopharmacology 90 (2004) 323–329 329
body weight in both normal and STZ rats after repeated oraladministration (Fig. 5).
4. Discussion
The aim of this study was to test the effect of RR aqueousextract on plasma cholesterol and triglycerides concentra-tions in normal and STZ-induced diabetic rats. We have usedthe leaves of this plant based on previous ethnobotanical in-formation in Tafilalet region (Eddouks et al., in press). Ac-cording to this survey, RR was largely used in diabetes phy-totherapy and control. We have previously reported that RRexhibited a hypoglycaemic activity in STZ rats (Maghraniet al., 2003a), the mechanism involved in this hypoglycaemiceffect seems to be the stimulation of glycosuria (Maghraniet al., 2003b).
The results demonstrated that aqueous extract of RRinduced a significant decrease of plasma cholesterol andtriglycerides levels in STZ diabetic rats for short term (sin-gle) and long term (repeated) administrations. However,in normal rats, only the long-term treatment had caused asignificant drop of lipidic parameters. Vanadate treatmentcaused a significant decrease of both plasma cholesteroland triglycerides levels in normal and STZ rats after singleas well as repeated oral administration. The plasma triglyc-erides levels were initially increased in STZ rats becausethe lipolysis was stimulated by concomitant insulinopenicstate. Recent-onset insulinopenia in STZ-diabetic rats isassociated with lipid overproduction in the basal (hypergly-caemic) state (Burcelin et al., 1995).
The main constituents of RR are flavonoids (Kassem et al.,2000). Some of them seem to be responsible for poisoningof livestock by ingestion of RR (El Bahri et al., 1999). Inour study, no lethality or behaviour change were noted atleast for the dose and duration used.
We have previously demonstrated that RR did not affectinsulin secretion in both normal and STZ rats (Maghraniet al., 2003b). It seems then that RR extract reduced plasmacholesterol and triglycerides levels without stimulating in-sulin secretion.
The RR aqueous extract caused a weight loss in STZ rats.This effect could be explained directly by the lipid loweringactivity of the extract and/or its influence of rat appetite(Trejo-Gonzàlez et al., 1996) or indirectly by influencingvarious lipidic regulation systems.
We conclude that aqueous extract of RR exhibited longterm cholesterol and triglycerides lowering activities in bothnormal and STZ diabetic rats. This data supporte its use inthe treatment of cardiac diseases by the Moroccan popu-lation. This activity is concomitant with body weight lossin type 1 diabetes mellitus. The underlying mechanism ofthis pharmacological effect seems to be independent of in-sulin secretion. Additionally, precise molecular mechanism
and active substance(s) need to be determined in further ex-periments. Such active principle(s) could be important inatherosclerosis and cardiac diseases therapy and control.
Acknowledgements
We would like to extend our thanks to the “Comitéinter universitaire Maroco-Français, Action integrée N◦MA/03/83” , for supporting this work.
References
Boulos, L., 1999. Flora of Egypt, vol.1. Azollacea-Oxalidaceae. Cairo,Egypt: Al-Hadara Publishing, pp. 258–259.
Burcelin, R., Eddouks, M., Maury, J., Kande, J., Assan, R., Girard, J.,1995. Excessive glucose production, rather than insulin resistance,accounts for hyperglycaemia in recent-onset streptozotocin-diabeticrats. Diabetologia 38, 283–290.
DeFronzo, R.A., 1988. Lilly lecture 1987: the triumvirate: B-cell, muscle,liver: a collusion responsible for NIDDM. Diabetes 37, 667–687.
Eddouks, M., Maghrani, M., Lemhadri, A., Jouad, H., in press. Phytother-apy in Tafilalet region. Revue of Tafilalet Association. Fifth MedicalCaravan.
El Bahri, L., Djegham, M., Bellil, H., 1999. Retama raetam W: a poisonousplant of North Africa. Veterinary and Human Toxicology 41, 33–35.
Jensen, T., Stender, S., Deckert, T., 1988. Abnormalities in plasma con-centrations of lipoprotein and fibrinogen in type 1 (insulin-dependent)diabetic patients with increased urinary albumin excretion. Diabetolo-gia 31, 142–145.
Kassem, M., Mosharrafa, S.A., Saleh, N.A., Abdel-Wahab, S.M., 2000.Two flavonoids from Retama raetam. Fitoterapia 71, 649–659.
Kritchevsky, D., 1995. Dietary protein, cholesterol and atherosclerosis: areview of the early history. Journal of Nutrition 125, 589S–593S.
Maghrani, M., Lemhadri, A., Jouad, H., Michel, J.-B., Eddouks, M., 2003a.Effect of the desert plant Retama raetam on glycaemia in normal andstreptozotocin-induced diabetic rats. Journal of Ethnopharmacology 87,21–25.
Maghrani, M., Lemhadri, A., Zeggwagh, N.A., Jouad, H., Haloui, M.,Eddouks, M., 2003b. Glucose lowering activity of Retama raetam instreptozotocin-induced diabetic rats. Phytotherapy Research. In press.
Mittler, R., Merquiol, E., Hallak-Herr, E., Rachmilevitch, S., Kaplan,A., Cohen, M., 2001. Living under a “dormant” canopy: a molecularacclimation mechanism of the desert plant Retama raetam. The PlantJournal 25, 407–416.
Motta, M., Giugno, I., Bosco, S., Pistone, G., Ruello, P., Maugeri, D.,Malaguarnera, M., 2001. Serum lipoprotein(a) changes in acute my-ocardial infraction. Panminevra Medicine 43, 77–80.
Pnueli, L., Hallak-herr, E., Rozenberg, M., Cohen, M., Goloubinoff, P.,Kaplan, A., Mittler, R., 2002. Molecular and biochemical mechanismsassociated with dormancy and drought tolerance in the desert legumeRetama raetam. The Plant Journal 31, 319–330.
Reaven, G.M., 1988. Banting lecture 1988: role of insulin resistance inhuman disease. Diabetes 37, 1595–1607.
Ross, R., 1986. The pathogenesis of atherosclerosis. New England Journalof Medicine 314, 488–500.
Trejo-Gonzàlez, A., Gabriel-Ortiz, G., Puebla-Pérez, A.M., Huizar-Contreras, M.D., Munguia-Mazariegos, M.D.R., Mejia-Arregum, S.,Calva, E., 1996. A purified extract from prickly pear cactus (Opunticafuliginosa) controls experimentally induced diabetes in rats. Journal ofEthnopharmacology 55, 27–33.
Journal of Ethnopharmacology 90 (2004) 331–337
Effects of an aqueous extract ofTriticum repens on lipidmetabolism in normal and recent-onset diabetic ratsM. Maghrania, A. Lemhadria, N.-A. Zeggwagha, M. El Amraouia,
M. Halouib, H. Jouada, M. Eddouksa,∗a UFR PNPE, BP 21, Errachidia, Morocco
b Inserm U460, CHU Bichat, 16 rue Henri Huchard, 75018 Paris, France
Received 5 May 2003; received in revised form 19 June 2003; accepted 9 October 2003
Abstract
The aim of this study was to demonstrate the effects of single and repeated oral administration of the aqueous rhizomes extract ofTriticumrepens (TR) (20 mg/kg) on lipid metabolism in normal and streptozotocin-induced diabetic rats. In normal rats, the aqueous extract of TRinduced a significant decrease in the plasma triglycerides concentrations 4 days (P < 0.05) and 1 week after repeated oral administration(P < 0.05). This reduction was abolished 2 weeks after once daily repeated oral administration. A significant decrease of plasma cholesterollevels was observed only 1 week (P < 0.05) after repeated oral administration.
In diabetic rats, TR treatment caused a significant decrease in plasma triglycerides levels after a single (P < 0.01) and repeated (P < 0.001)oral administration. A strong decrease in cholesterol level was observed 6 h after a single oral administration of the aqueous extract TR(P < 0.001). Four days after repeated oral administration of TR aqueous extract, the plasma cholesterol level was significantly decreased(P < 0.05) and still dropped after 2 weeks (P < 0.001).
On other hand, the repeated oral administration of aqueous TR extract caused a significant decrease in body weight 2 weeks after repeatedoral treatment in diabetic rats (P < 0.05).
Keywords: Triticum repens; Cholesterol; Triglycerides; Body weight; Aqueous extract and diabetic rats
1. Introduction
The influence of diabetes mellitus on lipid metabolism iswell established. The association of hyperglycaemia and al-teration of lipid parameters present a major risk of cardiovas-cular diseases in diabetic patients (Jensen et al., 1988; Mottaet al., 2001). Diabetes mellitus is a disease with profound ef-fects on lipid metabolism. Insulin affects lipid metabolism,e.g. by inhibition of the activity of lipoprotein lipase, there-fore it decreases the mobilization of free acids from periph-eral fat depots. On other hand, it stimulates the synthesisof fatty acids in the liver, adipose tissue and intestine. Thediabetogenic effect of streptozotocin (STZ) is the result ofirreversible damage to pancreatic� cells. The STZ-induced
diabetic animal is thus considered as an animal model ofhyperlipidemia (Burcelin et al., 1995).
Triticum repens P. Beauv. (TR), locally named as “N’jmL’bouri or Outara” is a spontaneous plant belonging to theGramin family. By reviewing the current literature, we knowof no previous research on the pharmacological propertiesof this plant. However, according to a recent ethnobotan-ical survey in south-eastern region of Morocco (Tafilalet),TR is prescribed by traditional healers for diabetes control(Eddouks et al., 2003).
The present study was undertaken to evaluate the po-tential cholesterol and triglycerides lowering activity of asingle and repeated oral administration of the aqueous TRextract in normal and STZ-induced diabetic rats. The ef-fect of TR extract on body weight loss was also monitored.Sodium-metavanadate (0.8 mg/kg) was used as a referencedrug known by its both hypolipidemic and hypoglycaemicactivities.
332 M. Maghrani et al. / Journal of Ethnopharmacology 90 (2004) 331–337
2. Material and methods
2.1. Plant material
The plant used in this study was collected from its nat-ural habitat, from Tafilalt region (Morocco) in May 2002,and dried under hot air (40–60◦C). The plant was identi-fied and authenticated asTriticum repens with assistance ofProf. M. Rejdali (Veterinary and Agronomy Institute, Ra-bat, Morocco). Voucher specimen (EM 3a) was depositedat the herbarium of the Faculty of Sciences and TechniquesErrachidia.
2.2. Preparation of the aqueous extract
An aqueous extract was prepared according to the tradi-tional method used in Morocco: 1 g of the dried powderedrhizomes of TR were boiled in 100 ml of distilled water for10 min and cooled to room temperature for 15 min. There-
0
0,7
1,4
2,1
0 6
Time(hours)
Tri
glyc
erid
esle
vels
(m
mol
/l)
0
1,75
3,5
0 6
Time(hours)
Tri
glyc
erid
es le
vels
(m
mol
/l)
****
(a)
(b)
Fig. 1. Plasma triglycerides levels (mmol/l) after a single oral administration of TR aqueous extract (20 mg/kg) in normal (Panel a) and STZ (Panel b)rats. Data are expressed as means ± S.E.M., n = 6 rats per group. ∗∗P < 0.01 when compared to baseline values (the start of treatment). (�) controlgroup; ( ) TR-treated group; (�) vanadate-treated group.
after, the aqueous extract was filtered using a Milliporefilter (Millipore 0.2 mm, St. Quentin en Yvelines, France)to remove particulate matter. The filtrate was lyophilizedand the desired dose (milligrams of lyophilized aqueousextract of TR per kilogram body weight) was then pre-pared and reconstituted in 1.5 ml of distilled water. Theaqueous extracts were prepared daily, just before adminis-tration. The extract yield was 14%. Animals were treatedorally, i.e. intubation using a syringe at an overnight fastedstate.
2.3. Animals used
Experiments were performed in adult male Wistar ratsweighing from 200 to 230 g. The animals were housed understandard environmental conditions (23±1 ◦C, with 55±5%humidity and a 12-h light/dark cycle) and maintained withfree access to water and ad libitum standard laboratory diet(70% carbohydrates, 25% proteins, 5% lipids).
M. Maghrani et al. / Journal of Ethnopharmacology 90 (2004) 331–337 333
2.4. Induction of diabetes
Streptozotocin (Sigma, St. Louis, MO, USA) was dis-solved in 0.1 M fresh cold citrate buffer at pH 4.5 beforeuse, and injected intravenously into the tail vein at a dose of65 mg/kg (Burcelin et al., 1995). After 18 h, the rats with sta-ble fasting blood glucose levels greater than 16 mmol/l wereconsidered as diabetic and used in the present study. Thepercentage of response to streptozotocin injection was 90%.
2.5. Single oral administration
Normal and diabetic rats were randomly assigned to threedifferent groups containing six rats each. One control groupreceived distilled water, a second treated group received theaqueous extract of TR at a dose of 20 mg/kg (20 mg oflyophilized aqueous extract of TR per kilogram body weight)and the third group received a reference drug (vanadate
0
1,2
2,4
3,6
0 6
Time (hours)
Cho
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ls (
mm
ol/l)
0
1
2
3
4
0 6
Time (hours)
Cho
lest
erol
leve
ls (m
mol
/l)
*** **
(a)
(b)
Fig. 2. Plasma cholesterol levels (mmol/l) after a single oral administration of TR aqueous extract (20 mg/kg) in normal (Panel a) and STZ (Panel b)rats. Data are expressed as means ± S.E.M., n = 6 rats per group. ∗∗P < 0.01; ∗∗∗P < 0.001 when compared to baseline values (the start of treatment).(�) control group; ( ) TR-treated group; (�) vanadate-treated group.
(Na+VO3−) at a dose of 0.8 mg/kg). For single oral admin-istration, distilled water (control), vanadate (0.8 mg/kg) orthe aqueous extract (20 mg/kg) was administered and plasmacholesterol and triglycerides levels were measured beforeand 6 h after TR treatment.
2.6. Repeated oral administration
For repeated oral administration, rats were treated oncedaily at a dose of 20 mg/kg for 2 weeks and plasma choles-terol and triglycerides levels were followed during this pe-riod. The number of rats was six in each group (n = 6).Blood samples were collected from the tail vein and plasmatriglycerides and cholesterol levels are determined enzymat-ically by colorimetric technique using specific kits (Randox,UK), respectively. The kits used in this study for substratesanalysis were specific for both human and rat blood samplesat the same percentage.
334 M. Maghrani et al. / Journal of Ethnopharmacology 90 (2004) 331–337
2.7. Statistical analysis
Data were expressed as mean ± S.E.M. The statisticalanalysis was performed by the Student’s t test. The valueswere considered significantly different when the P valuewas less than 0.05 in comparison to baseline values (startingvalues).
3. Results
3.1. Single oral administration
In normal rats, no significant changes of both plasmacholesterol and triglycerides concentrations after a sin-gle administration of TR (20 mg/kg) were noted (Figs. 1a
0
1,1
2,2
0 2 4 7 15
Time (days)
Tri
glyc
erid
es le
vels
(m
mol
/)
* *
*
* *
0
1,2
2,4
3,6
0 2 4 7 15
Time (days)
Tri
glyc
erid
es le
vels
(m
mol
/)
*
*****
******
*
* *
(a)
(b)
Fig. 3. Plasma triglycerides levels (mmol/l) after a repeated oral administration of TR aqueous extract (20 mg/kg) in normal (Panel a) and STZ (Panelb) rats. Data are expressed as means ± S.E.M., n = 6 rats per group. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001 when compared to baseline values (the startof treatment). (�) control group; ( ) TR-treated group; (�) vanadate-treated group.
and 2a). In vanadate-treated group, the plasma cholesteroland triglycerides concentrations were significantly de-creased over 6 h of a single oral dose of vanadate (P < 0.01)(Figs. 1a and 2a).
In STZ rats, the aqueous extract of TR caused a significantreduction of plasma triglycerides (P < 0.01) and cholesterol(P < 0.001) levels 6 h after TR treatment (Figs. 1b and 2b).Vanadate (0.8 mg/kg) reduced both the plasma cholesterol(P < 0.01) and triglycerides levels (P < 0.01) 6 h after TRadministration in diabetic rats (Figs. 1b and 2b).
3.2. Repeated oral administration
In normal rats, the aqueous extract of TR induced a sig-nificant decrease of the plasma triglycerides concentrations4 days (P < 0.05) and 1 week after repeated oral adminis-
M. Maghrani et al. / Journal of Ethnopharmacology 90 (2004) 331–337 335
0
1
2
3
0 2 4 7 15
Time (days)
Cho
lest
erol
lev
els
(mm
ol/l
)*
*****
*
0
1,7
3,4
0 2 4 7 15
Cho
lest
erol
leve
ls (
mm
ol/l) **
***
***
****
*
(a)
(b)
Time (days)
Fig. 4. Plasma cholesterol levels (mmol/l) after a repeated oral administration of TR aqueous extract (20 mg/kg) in normal (Panel a) and STZ (Panel b)rats. Data are expressed as means ± S.E.M., n = 6 rats per group. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001 when compared to baseline values (the start oftreatment). (�) control group; ( ) TR-treated group; (�) vanadate-treated group.
tration (P < 0.05) (Fig. 3a). This reduction was abolished 2weeks after once daily repeated oral administration. A sig-nificant decrease of plasma cholesterol levels was observedonly 1 week (P < 0.05) after repeated oral administration(Fig. 4a). After 2 weeks of treatment, vanadate caused a sig-nificant decrease of both plasma triglycerides (P < 0.05)(Fig. 3a) and cholesterol levels (P < 0.001) (Fig. 4a).
In diabetic rats, TR extract decreased significantly theplasma cholesterol levels from the fourth day (P < 0.05);the most significant effect was reached from the first to thesecond week of treatment (P < 0.001) (Fig. 3b). The plasmacholesterol levels were decreased from the fourth day (P <
0.05) to the first (P < 0.01) and second week (P < 0.001)after TR treatment (Fig. 4b). Daily vanadate administration(0.8 mg/kg) for 2 weeks produced a statistically significant
decrease in both plasma cholesterol and triglycerides con-centrations (P < 0.001) (Figs. 3b and 4b).
3.3. Body weight loss
In normal rats, the TR extract caused a slight increase ofbody weight 2 weeks after daily oral administration of TR(20 mg/kg) (P < 0.01) (Fig. 5a). A significant decrease inbody weight was observed in distilled water treated groupfrom the second day of STZ injection. TR aqueous extract(20 mg/kg) caused a significant weight loss 2 weeks afteronce daily repeated oral treatment (P < 0.01) in STZ rats(Fig. 5b). Vanadate caused also a significant decrease inbody weight in both normal and STZ rats after repeated oraladministration (P < 0.001) (Fig. 5).
336 M. Maghrani et al. / Journal of Ethnopharmacology 90 (2004) 331–337
0
100
200
300
0 2 4 7 15Time (days)
Bod
y w
eigh
t (g)
*** ***
***
**
**
0
70
140
210
280
0 2 4 7 15
Time (days)
Bod
y w
eigh
t (g)
********
**** *
(a)
(b)
Fig. 5. Effect of TR aqueous extract treatment (20 mg/kg) on body weight (g) in normal and diabetic rats. Data are expressed as means ± S.E.M., n = 6rats per group. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001 when compared to baseline values (the start of treatment). (�) control group; ( ) TR-treatedgroup; (�) vanadate-treated group.
4. Discussion
In the present study, the effect of aqueous TR extracton plasma cholesterol and triglycerides concentrations wasevaluated in the normal and STZ-induced diabetic rats, amodel of type 1 diabetes mellitus. Until now, TR remainsunknown as a medicinal plant. We have used the rhizomesof this plant based on an ethnobotanical information in Tafi-lalet region (Eddouks et al., 2003). According to this survey,TR was largely used in diabetes phytotherapy and control.Tafilalet region is considered as a great reserve of medicinalplants in which the phytotherapy knowledge is very devel-oped (Eddouks et al., 2002). Diabetes is associated with hy-pertriglyceridaemia (Rodrigues et al., 1986) which is due toincrease in adipose tissue lipolysis in absence of insulin, a
decrease in lipoprotein lipase activity and reduced carnitinelevels. In diabetic state due to absence of insulin sensitivity(Burcelin et al., 1995), there is mobilization of fatty acidsfrom adipose tissues and proteins leading to increase in freefatty acids. In addition, it has been well documented thatrats treated with STZ had increased plasma cholesterol andtriglycerides levels (Shah et al., 1995).
Vanadate was used as a reference drug because it hasbeen reported to be a potent insulin mimetic agent in manycells (DeFronzo, 1988; Reaven, 1988). Administration ofthis compound to diabetic animals normalized blood glu-cose concentration and reduced the triglycerides levels(Kashiwagi et al., 1983; Brichard and Henquin, 1995).
The results demonstrated that aqueous extract of TRinduced a significant decrease of plasma cholesterol and
M. Maghrani et al. / Journal of Ethnopharmacology 90 (2004) 331–337 337
triglycerides levels in STZ-diabetic rats for both short term(single) and long term (repeated) administrations. However,in normal rats, only the long term treatment had caused asignificant drop of lipidic parameters. Vanadate treatmentcaused a significant decrease of both plasma cholesteroland triglycerides levels in normal and STZ rats after shortterm as well as long term treatment. The plasma triglyc-erides levels were initially increased in STZ rats becausethe lipolysis was stimulated by concomitant insulinopenicstate. Recent-onset insulinopenia in STZ-diabetic rats isassociated with lipid overproduction in the basal (hypergly-caemic) state (Burcelin et al., 1995).
Some studies have reported a similar lipid lowering ac-tivity of some medicinal plants (Ram et al., 1997; Sharmaet al., 1997). Parallely we have previously reported a hypoc-holesterolaemic activity of Spergularia purpurea in normaland STZ rats (Jouad et al., 2003).
The underlying mechanism of the lipid lowering activityof TR could be the inhibition of lipid absorption due tothe presence of saponins and tannins in the aqueous extract(Dwivedi and Agarwal, 1994; Vaidya, 1994; Ram et al.,1997) and/or inhibition of cholesterol-esterase, activation offatty acids synthase, acetyl-CoA carboxylase and productionof triglycerides precursors such as acetyl-CoA and glycerolphosphate.
The TR aqueous extract caused a weight loss in STZ rats.This effect could be explained directly by the lipid loweringactivity of the extract and/or its influence of rat appetite(Trejo-Gonzàlez et al., 1996) or indirectly by influencingvarious lipidic regulation systems.
Other advanced toxicological investigations are requiredto precise eventual TR toxicity in different organs and tis-sues. After such investigations, aqueous TR extract could beused in human healthcare system, especially in the treatmentof hypercholesterolaemia associated with diabetes, obesityand cardiovascular diseases.
We conclude that aqueous extract of TR exhibited longterm cholesterol and triglycerides lowering activities in bothnormal and STZ-diabetic rats and confirms its use in Moroc-can phytomedicine. This activity is concomitant with bodyweight loss in type 1 diabetes mellitus. In this study, the pe-riod of plant collection, the dose and duration of TR treat-ment were respected according to Moroccan traditional us-age. Further experiments dealing with the effect of seasonalas well as geographical variability of the plant material arestill to be performed. Additionally, precise molecular mech-anism and active substance(s) need to be determined in fur-ther experiments. Such active principle(s) could be preciousin obesity, atherosclerosis and cardiac diseases therapy andcontrol.
Acknowledgements
We extend our thanks to the “Comité Inter UniversitaireMaroco-Français, Action integrée no. MA/03/83” for sup-
porting this work and to Mr. Moussaoui Bassidi for his as-sistance.
References
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DeFronzo, R.A., 1988. Lilly lecture 1987: the triumvirate: B-cell, mus-cle, liver: a collusion responsible for NIDDM. Diabetes 37, 667–687.
Dwivedi, S., Agarwal, M.P., 1994. Antianginal and cardioprotective ef-fects of Terminalia arjuna, an indigenous drug in coronary arterydisease. Journal of Association of Physicians of India, 42, 287–289.
Eddouks, M., Maghrani, M., Lemhadri, A., Ouahidi, M.-L., Jouad, H.,2002. Ethnopharmacological survey of medicinal plants used for thetreatment of diabetes mellitus, hypertension and cardiac diseases in thesouth-east region of Morocco (Tafilalet). Journal of Ethnopharmacology82, 97–103.
Eddouks, M., Maghrani, M., Lemhadri, A. Jouad, H., 2003. Phytother-apy in Tafilalet region. Revue of Tafilalet Association. Fifth MedicalCaravan (in press.)
Jensen, T., Stender, S., Deckert, T., 1988. Abnormalities in plasma con-centrations of lipoprotein and fibrinogen in type 1 (insulin-dependent)diabetic patients with increased urinary albumin excretion. Diabetolo-gia 31, 142–145.
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Kashiwagi, M., Verso, A., Andrews, J., Vasquez, B., Reaven, G., Foley,J.E., 1983. In vitro insulin resistance of human adipocytes isolated fromsubjects with non-insulin dependent diabetes mellitus. The Journal ofClinical Investigation 72, 1246–1254.
Motta, M., Giugno, I., Bosco, S., Pistone, G., Ruello, P., Maugeri, D.,Malaguarnera, M., 2001. Serum lipoprotein(a) changes in acute my-ocardial infraction. Panminevra Medica 43, 77–80.
Ram, A., Lauria, P., Gupta, R., Kumar, P., Sharma, V.N., 1997. Hypoc-holesterolemic effects of Terminalia arjuna tree bark. Journal ofEthnopharmacology 55, 165–169.
Reaven, G.M., 1988. Banting lecture 1988: role of insulin resistance inhuman disease. Diabetes 37, 1595–1607.
Rodrigues, B., Goyal, R.K., McNeil, J.H., 1986. Effects of hydralazine onSTZ induced diabetic rats: prevention of hyperlipidaemia and improve-ment in cardiac function. Journal of Pharmacology and ExperimentalTherapeutics 237, 299–307.
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Journal of Ethnopharmacology 90 (2004) 339–346
Immunomodulatory and antitumor activity ofPiper longum Linn. and piperine
E.S. Sunila, G. Kuttan∗Amala Cancer Research Centre, Amalanagar, Thrissur 680 553, Kerala, India
Received 9 January 2003; received in revised form 12 June 2003; accepted 13 October 2003
One of the major drawbacks of the current cancer thera-peutic practices, such as chemotherapy and radiation ther-apy, is suppression of immune system (Devasagayam andSainis, 2002). A wide variety of compounds are capable ofpotentiating immune responses. Classical adjuvants of bac-terial origin, such as Bacillus Calmette Guerin (BCG), havebeen shown to exert therapeutic effects in the treatment ofcancer. But the effect is limited due to a number of unde-sirable side effects in host, like liver dysfunction, inductionof hepatic granuloma, enhancement of tumor growth, whenlarge doses of BCG are administered (Ribi et al., 1981).Chemical agents, such as levamisole and interferons, werewidely used to treat cancer in the mid 1970s to early 1980s(Gomi et al., 1983). Despite the immunological effects, ad-juvant levamisole (Stevenson et al., 1991) treatment of lev-amisole alone or in combination with interferon (Kirkwoodand Ernstoff, 1990) showed no significant clinical benefit.Cytokines play a critical role in the induction and effec-
∗ Corresponding author. Fax:+91-487-307868.E-mail address: [email protected] (G. Kuttan).
tor functions of both humoral and cell-mediated immuneresponses. The immunomodulating property of IL-2, IL-4,IL-7, etc., promoted their use in the treatment of cancerpatients. But their unique and diverse side effects, such ascardiovascular toxicity, pulmonary toxicity, hematologicaltoxicity, etc., made limitations in their use (Ognibene et al.,1988; Rosenberg et al., 1994). Immunomodulators, whichcan be used for long period without or less side effects, areappreciable in the cancer therapy.
Several medicinal herbs have shown to promote immu-nity in different ways. They have shown to augment spe-cific cellular and humoral immune response (Duke, 1985).We have reported the immunomodulatory activity of someplants, such asViscum album (Kuttan and Kuttan, 1992),Tinospora cordifolia (Mathew and Kuttan, 1999), Withaniasomnifera (Davis and Kuttan, 2000), etc.
Piper longum Linn., an important medicinal plant, is usedin traditional medicine by many people in Asia and Pacificislands especially in Indian medicine (Guido et al., 1998).Piper longum is a component of medicines reported as goodremedy for treating gonorrhea, menstrual pain, tuberculo-sis, sleeping problems, respiratory tract infections, chronicgut related pain, and arthritic conditions (Singh, 1992).Other reported beneficial effects ofPiper longum include
340 E.S. Sunila, G. Kuttan / Journal of Ethnopharmacology 90 (2004) 339–346
analgesic and diuretic effects, relaxation of muscle tension,and alleviation of anxiety (Singh and Blue Menthal, 1997).Piperine was the first amide isolated from piper species andwas reported to display central nervous system depression,antipyretic, and anti-inflammatory activity (Virinder et al.,1997). Piperine is a potent inhibitor of the mixed functionoxygenase system and non-specific inhibition of P450 isoen-zymes (Atal et al., 1985). Constituents of piper species haveinhibitory activity on prostaglandin and leukotriene biosyn-thesis in vitro (Stohr et al., 2001). However, no study on theimmunomodulatory and antitumor activity ofPiper longumand piperine has been reported. In order to verify the anecdo-tal claims thatPiper longum and piperine has numerous phy-tochemical benefits, we have investigated the immunomodu-latory and antitumor activities of the plant and its derivative.
2. Materials and methods
2.1. Animals
Balb/c mice were taken from Amala Cancer ResearchCentre breeding section. Swiss albino mice were purchasedfrom National Institute Of Nutrition, Hyderabad, India. Theanimals were kept in air-controlled room, fed with normalmice chow (Sai Feeds, India) and water ad libitum.
All the animal experiments were performed according tothe rules and regulations of the Animal Ethical Committee,Government of India.
2.2. Cells
L929 cells were procured from National Facility for An-imal Tissue and Cell Culture, Pune, India. Dalton’s lym-phoma ascites (DLA) and Ehrlich ascites carcinoma (EAC)cells were initially procured from Adayar Cancer Institute,Chennai, India. Sheep red blood cells (SRBC) was collectedfrom local slaughter house in Alsever’s solution.
Minimum Eagle’s medium was purchased from Hi-Media,Mumbai, India. Piperine was purchased from Sigma Chem-icals, USA. para-Rosaniline and�-napthyl acetate wereobtained from Loba Chemie, Bombay. Harri’s hematoxylinwas purchased from Glaxo (Bombay, India). All otherchemicals used were analytical reagent grade.
2.3. Drug preparation
Source—AuthenticatedPiper longum was obtained fromAmala Ayurvedic Centre.
2.3.1. Alcoholic extract100 g of dried fruit powder was stirred overnight in 70%
methanol (1 l), centrifuged at 10,000 rpm (7275 g) for 10 minat 4◦C, and supernatant was collected. Methanol was re-moved in vacuum and the yield obtained was 26%. Theextract was resuspended in PBS (pH 7.2). Phytochemical
analysis of the extract showed the presence of alkaloid. Thecrude extract ofPiper longum contains 3–8% of piperineJames (1999).
2.3.2. PiperinePiperine was suspended in 1% gum acasia and used for
the experiments.
2.4. Drug administration
In all the immunological parameters the Balb/c mice(4–6 weeks old) were used. The animals were treatedwith five doses of alcoholic extract (10 mg/dose/animal) ofPiper longum and piperine (1.14 mg/dose/animal) intraperi-toneally for 5 consecutive days.
2.5. Immunomodulatory activity of Piper longum andpiperine
2.5.1. Determination of the effect of Piper longum andpiperine on hematological parameters
Two groups of Balb/c mice (6 mice/group; 4–6 weeksold) were treated with five doses ofPiper longum extractand piperine as explained earlier. Blood was collected fromthe caudal vein and parameters, such as total WBC count(hemocytometer), differential count (Leishman’s stain),hemoglobin content (cyanmethemoglobin method) (Drabkinand Austin, 1932) as well as body weight were recordedprior to the drug treatment and every third day for 1 month.
2.5.2. Determination of the effect of Piper longum andpiperine on the bone marrow cellularity and �-esterasepositive cells
Bone marrow cellularity was determined by the methodof Sredni et al. (1992). The animals were divided in tothree groups (6 mice/group). Group I was untreated con-trol; groups II and III were treated with alcoholic extract ofPiper longum and piperine intraperitoneally for 5 consec-utive days. The animals were sacrificed 24 h after the lastdose of drug treatment. Bone marrow cells from femur wascollected and made into single cell suspension and the cellnumber was determined using hemocytometer.
The number of�-esterase positive cells was determined bythe azodye coupling method (Bancroft and Cook, 1984). Asmear of bone marrow cells from the above preparation wasmade on clean glass slides, air-dried, stained with�-naphthylacetate andpara-rosaniline hydrochloride, counter stainedwith hematoxylin and observed using light microscope under100×. The number of�-esterase positive cells was expressedout of 4000 cells.
2.5.3. Determination of the effect of Piper longum andpiperine on circulating antibody titer
In this study three groups of Balb/c mice (6 mice/group20–25 g body weight) were used. Group I was kept as un-treated control, groups II and III animals were pretreated
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with alcoholic extract ofPiper longum and piperine. All theanimals were immunized with SRBC (2.5×108 cells/animal,i.p.) after the last dose of drug. Blood was collected fromthe tail vain, serum separated, and heat inactivated at 56◦C.Antibody titer was conducted by hemagglutination accord-ing to the method ofSingh et al. (1984)using SRBC as theantigen in 96-well round-bottom titer plates.
2.5.4. Determination of the effect of Piper longum andpiperine on plaque forming cells in spleen
Three groups of Balb/c mice (7 mice/group; 4 weeks old)were used in this study. Groups I and II were treated withfive doses of alcoholic extract ofPiper longum and piperine,respectively. Group III was kept as untreated control. All theanimals were immunized with SRBC (2.5×108 cells/animal,i.p.) after the last dose of drug. The animals were sacri-ficed on different days, spleen was processed to single cellsuspension, and used for the determination of the antibodyproducing cells by Jerne’s plaque assay (Jerne and Nordin,1963).
2.6. Antitumor activity of Piper longum and piperine
2.6.1. Determination of the in vitro cytotoxic activity ofPiper longum and piperine to DLA and EAC cell
DLA and EAC cells (1× 106) were incubated with var-ious concentrations (100–500�g/ml) of Piper longum ex-tract and piperine in a final volume of 1 ml for 3 h at 37◦C.After incubation the viability of cells were determined bythe trypan blue dye exclusion method (Talwar, 1974).
2.6.2. Determination of the cytotoxicity of Piper longumand piperine to L929 cells in culture
Cytotoxicity of the extract ofPiper longum and piper-ine was determined using L929 cells. Cells were seeded in96-well flat-bottom plates (5000 cells/well) and allowed toadhere for 24 h at 37◦C with 5% CO2 atmosphere. Dif-ferent concentrations ofPiper longum extract and piperine(25–100�g/ml) were added and incubated further for 48 h.Before 4 h of the completion of incubation, 20�l of MTT(5 mg/ml) was added (Cole, 1986; Campling et al., 1991).Percentage of dead cells was determined using an ELISAplate reader set to record absorbance at 570 nm.
2.6.3. Determination of the effect of Piper longum andpiperine on solid tumor development
Solid tumor was induced by injecting DLA cells(1 × 106 cells/animal) subcutaneously to the right hindlimbs of three groups (6 mice/group) of Swiss albino mice.Groups I and II animals were treated with alcoholic ex-tract of Piper longum (10 mg/dose/animal) and piperine(1.14 mg/dose/animal), respectively, for 10 days. GroupIII animals were kept as untreated control. The radii ofdeveloping tumor were measured using vernier calipersat 3 days intervals for 1 month and tumor volume wascalculated using the formulaV = 0.4ab2, where ‘a’ and
‘b’ represent the major and minor diameter, respectively(Atia and Weiss, 1966). This was compared with untreatedcontrol.
2.6.4. Determination of the effect of Piper longum andpiperine on the survival of ascites tumor bearing animals
Three groups (6 mice/group) of Swiss albino mice wereinduced ascites tumor by injecting 1× 106 cells/animalsto the peritoneal cavity. Group I was inoculated with EACcells alone and kept as untreated control. Groups II andIII received alcoholic extract ofPiper longum (10 mg/dose/animal, i.p.) and piperine (1.14 mg/dose/animal), re-spectively, for 10 consecutive days. The death pattern ofanimals due to tumor burden was noted and the percentageof increase in life span due to tumor burden was noted andthe percentage of increase in life span was calculated usingthe formula((T − C)/C) × 100 were ‘T’ and ‘C’ repre-sent the number of days that treated and control animalssurvived, respectively.
2.7. Statistical analysis
Results were expressed as the mean±standard deviation.Statistical evaluation was done by Student’st-test.
3. Results
3.1. Immunomodulatory activity of Piper longum andpiperine
3.1.1. Effect of Piper longum and piperine onhematological parameters
Administration of the alcoholic extract ofPiper longumand its component piperine was found to increase the totalWBC count in Balb/c mice (Fig. 1). The maximum increasein the animals treated with alcoholic extract (142.8%) andpiperine (138.9%) were observed on 15th day after drug ad-ministration. There was no appreciable change in the differ-ential count, body weight, and Hb content after the admin-istration ofPiper longum as well as piperine.
3.1.2. Effect of Piper longum and piperine on the bonemarrow cellularity and �-esterase positive cells
The effect of Piper longum extract and piperine onthe bone marrow cellularity and�-esterase positive cellsis given in Table 1. Administration of alcoholic extractof Piper longum (20.5 × 106 cells/femur and piperine)(24.03×106 cells/femur) showed a significant enhancementin the bone marrow cellularity compared to the normalcontrol (14.37 cells× 106 cells/femur) animals.
Moreover the number of�-esterase positive cells was alsofound to be increased significantly in these animals (alco-holic extract 1398 cells/4000 bone marrow cells and piper-ine 1236.6 cells/4000 bone marrow cells) compared to thenormal animals (779.5 cells/4000 bone marrow cells).
342 E.S. Sunila, G. Kuttan / Journal of Ethnopharmacology 90 (2004) 339–346
Fig. 1. Effect ofPiper longum and piperine on total WBC count.
3.1.3. Effect of Piper longum and piperine on circulatingantibody titer
The enhancement of total antibody production by the ad-ministration of alcoholic extract ofPiper longum and piper-ine is shown inFig. 2. The maximum antibody titer valueof 256 for alcoholic extract ofPiper longum and 128 forpiperine-treated animals was observed on 18th day after im-munization.
3.1.4. Effect of Piper longum and piperine on plaqueforming cells (PFC) in spleen
The effect ofPiper longum extract and piperine on thenumber of plaque forming cells is shown inFig. 3. Themaximum increase in the plaque forming cells in alcoholicextract ofPiper longum (100.3%)- and piperine (71.4%)-
Table 1Effect of Piper longum and piperine on bone marrow cellularity and�-esterase activity
Treatment Bone marrowcellularity(cells/femur)× 106
Number of�-esterasepositive cells/4000 cells
Normal 14.3 ± 0.2 779.5± 57.2Piper longum
(alcoholic extract)29.5 ± 0.8∗ 1398.3± 112.9∗
Piperine 24.0 ± 0.4∗ 1236.6± 108.5∗
Treated animals received five doses of alcoholic extract ofPiper longum(10 mg/dose/animal) and piperine (1.14 mg/dose/animal). Bone marrowcells were collected from femur. Values are the mean± S.D., statisticallysignificant from untreated control.∗P < 0.001.
treated groups were observed on 5th day after the immuni-zation.
3.2. Antitumor activity of Piper longum and piperine
3.2.1. Cytotoxicity of Piper longum and piperine towardsDLA and EAC cells
Alcoholic extract was found to be toxic at a concentrationof 500�g/ml for DLA cells and 200�g/ml for EAC cells.Piperine was cytotoxic towards DLA cells and EAC cells ata concentration of 200�g/ml (Tables 2 and 3).
3.2.2. Cytotoxicity of Piper longum and piperine towardsL929 cells in culture
Extract ofPiper longum and piperine was found to be cy-totoxic towards L929 cells in culture (Table 4). Alcoholicextract of Piper longum was found to be toxic at a con-
Table 2Cytotoxicity of Piper longum extract and piperine to Dalton’s lymphomaascites (DLA) cells
Concentration (�g/ml) Percentage of cytotoxicity
Alcoholic extract Piperine
500 100 100250 50 100100 12 30
DLA cells (106) were incubated with different concentrations(100–500�g/ml) of alcoholic extract and piperine. Percentage of deadcells was determined by trypan blue exclusion method.
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Fig. 2. Effect ofPiper longum and piperine on antibody titre.
Fig. 3. Effect ofPiper longum and piperine on plaque forming cells.
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-0.5
0
0.5
1
1.5
2
2.5
3
3.5
7th 10th 13th 16th 19th 22nd 25th 28th 31st
Days
Tu
mo
r vo
lum
e (c
c)
Control
Alcoholic extract
Piperine
Fig. 4. Effect ofPiper longum and piperine on solid tumor reduction.
Table 3Cytotoxicity of Piper longum extract and piperine to Ehrlich ascitescarcinoma (EAC) cells
Concentration (�g/ml) Percentage of cytotoxicity
Alcoholic extract Piperine
500 100 100250 100 85100 50 15
EAC cells (106) were incubated with different concentrations of(100–500�g/ml) of extract and piperine. Percentage of dead cells wasdetermined by trypan blue exclusion method.
centration of 100�g/ml and piperine was 100% toxic at aconcentration of 50�g/ml.
3.2.3. Effect of Piper longum and piperine on solid tumordevelopment
There was a significant reduction of tumor volume inPiper longum- and piperine-treated animals (Fig. 4). Tumorvolume of control animal was 1.7 cc3 on 22nd day while
Table 4Cytotoxicity of Piper longum extract and piperine to L929 cells in culture
Concentration (�g/ml) Percentage of cytotoxicity
Alcoholic extract Piperine
100 45 10050 0 10025 0 60
L929 cells were incubated with different concentrations (25–100�g/ml) ofalcoholic extract and piperine. Percentage of cytotoxicity was determinedusing MTT assay.
Table 5Effect of Piper longum and piperine on the life span of ascites tumorbearing animals
Treatment Mean survivaldays
Percentage ofincrease in lifespan (% ILS)
Control (tumor alone) 15± 0.8Tumor + alcoholic extract
of Piper longum20.6 ± 1.7∗ 37.3
Tumor + piperine 23.8 ± 2.4∗ 58.8
Animals were treated with five doses ofPiper longum and piperineafter injecting Ehrlich ascites carcinoma (EAC) cells (106 cells/animal).Survival pattern was observed for 30 days. Values are the mean± S.D.,statistically significant from untreated control.∗P < 0.001.
that of alcoholic extract- and piperine-treated animals wasonly 0.4 cc3, respectively, on the same day.
3.2.4. Effect of Piper longum and piperine on the survivalof ascites tumor bearing animals
Life span of ascites tumor bearing mice treated with alco-holic extract ofPiper longum and piperine was found to besignificantly increased (Table 5). Control animals survivedonly 15 days after the tumor induction while the alcoholicextract-treated animals survived 20 days with an increase inlife span of 30%. Piperine treatment was found to be moreeffective showing 58% increase in the life span of the tumorbearing animals.
4. Discussion
The main objective of this study was to focus on theimmunomodulatory and antitumor activity of alcoholic
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extract of Piper longum and its component piperine. Im-munoregulation is a complex balance between regulatoryand effector cells, and any imbalance in immunologicalmechanism can lead to pathogenesis (Steven et al., 1985).Immunosuppression is one of the main obstacles in theconventional cancer treatment such as chemo- and radio-therapy.
Presently different types of immunomodulators are avail-able. Depending upon their sources they can be divided intonatural and synthetic immunomodulators. These can allevi-ate the side effects, which is the major problems in otherconventional therapies.
There are reports that extracts from plants, such asVis-cum album (Kuttan and Kuttan, 1992), Withania somnifera(Davis and Kuttan, 2000), Tinospora cordifolia (Mathewand Kuttan, 1999), etc., could stimulate the immunity in nat-ural as well as tumor bearing animals. They have also shownto alleviate the immunosuppression induced by chemo-and radiotherapy (Praveen Kumar et al., 1999). We alreadyreported that the piperine could inhibit the metastasis in-duced by B16F10 melanoma cells (Pradeep and Kuttan,2002).
The dosage ofPiper longum extract and piperine was se-lected on the basis of cytotoxicity. 10 mg/dose/animal forPiper longum extract and 1.14 mg/dose/animal for piperineis the lowest concentration with maximum activity. Admin-istration of Piper longum extract and piperine showed in-creased number of total WBC count. This indicates theycan stimulate the hemopoietic system. The differential countshows the drug did not alter the ratio of different WBCtypes.
Bone marrow serves as the major source of all blood cells,including lymphocytes. Administration ofPiper longum ex-tract and piperine showed an increase in bone marrow cel-lularity and�-esterase positive cells indicating its effect onstem cell proliferation.
Extract of Piper longum and piperine was found to in-crease the circulating antibody titer and antibody formingcells indicating its stimulatory effect on the humoral arm ofimmune system. Administration of this drug could also sig-nificantly inhibit the growth of solid tumor induced by DLAcells and ascites tumor induced by EAC cells.
Immunomodulators may activate cytotoxic effector cells,such as cytotoxic T lymphocytes, natural killer (NK) lym-phocytes, macrophages, and activated neutrophils (Fidlerand Poste, 1985). Use of chemotherapy plus target-specificimmunomodulators hold a reasonable promise for clinicalutility in future.
Immunomodulatory activity ofPiper longum and piper-ine may be due to the combined action of humoral andcell-mediated immune responses. Hence, the results in-dicated that thePiper longum and piperine could act asa non-toxic immunomodulator which posses antitumorproperty also. The exact mechanism of action respon-sible for the tumor reducing activity has to be studiedfurther.
Acknowledgements
The authors sincerely thank Dr. Ramdasan Kuttan, Re-search Director, Amala Cancer Research Centre, for his valu-able suggestions.
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Pradeep, C.R., Kuttan, G., 2002. Effect of piperine on the inhibition oflung metastasis induced by B16F-10 melanoma cells in mice. ClinicalExperimental Metastasis 19, 703–708.
Praveen Kumar, V., Kuttan, R., Kuttan, G., 1999. Effect of Rasayanasin cell mediated immune responses in tumour bearing mice. IndianJournal of Experimental Biology 37, 23–26.
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Venomous snakebite is an important medical problem inThailand since the economic activities of the country aremainly agricultural and the area is abundant of snakes. In1994, a national survey of snakes responsible for snakebitein Thailand showed that 70% of them are venomous species,the most common species beingCalloselasma rhodostoma(Malayan pit viper (MPV), 38%), white-lipped green pitviper (27%) and Russell’s viper (14%) (Viravan et al., 1992).Venom of these vipers can seriously damage the affectedtissue and haemostatic system. Symptoms of local pain, localbleeding, inflammation and complications including localwound necrosis are usually the main problems at the bitesite (Reid et al., 1963; Bjarnason and Fox, 1994; Markland,1997, 1998).
Eclipta prostrata L. (Asteraceae) is a pantropical and sub-tropical plant used as an anti-venom against snakebite inChina and in Brazil (Mors, 1991). The plant grows abun-
dantly throughout Thailand, but there was no report onits use against Thai snakes. Several compounds, includ-ing wedelolactone (1), were identified as its constituents(Bhargava et al., 1972; Wagner and Fessler, 1986; Wagneret al., 1986; Mors et al., 2000). Wedelolactone was found toexert anti-venom activities against South American crotalidvenoms ofCrotalus durissus terrificus (Mors et al., 1989),Bothrops jararaca, Bothrops jararacussu andLachesis muta(Melo et al., 1994), Crotalus viridis viridis andAgkistrodoncontortrix (Melo and Ownby, 1999). It was therefore inter-esting to study the possibility of usingEclipta prostrata ex-tracts for the topical treatment of local tissue damage causedby MPV venom in Thailand. In this study, the aerial part ofthe plant was extracted with methanol, then partitioned withethyl acetate and butanol. The major constituent of the bu-tanolic extract was identified and the ability of the extract toinhibit enzymatic and lethal activities of MPV venom wasdetermined both in vitro and in vivo.
2. Material and methods
2.1. Plant material
The aerial part of Eclipta prostrata was collectedfrom Nonthaburi Thailand and identified by one of us
348 P. Pithayanukul et al. / Journal of Ethnopharmacology 90 (2004) 347–352
(R. Bavovada). A voucher specimen (no. P.B. 20005) hasbeen deposited at the Museum of Natural Medicine, Fac-ulty of Pharmaceutical Sciences, Chulalongkorn University,Bangkok, Thailand.
2.2. Plant extraction and identification of majorconstituent
Fresh aerial part ofEclipta prostrata was extracted withmethanol in a Soxhlet apparatus for 48 h. The solvent wasremoved under reduced pressure to yield a viscous masswhich was further suspended in water and heated at 80◦C.The aqueous suspension was centrifuged at 2000 rpm for10 min, then the aqueous supernatant was partitioned withethyl acetate and butanol, respectively. The butanolic extract(BE) was subjected to a silica gel (E. Merck) quick column(6.5 cm i.d.× 4 cm, 30 g), using ethyl acetate/MeOH= 5:1as eluting solvent. Five major fractions (A–E) were col-lected. Fraction C yielded demethylwedelolactone (2) as paleyellow needles from MeOH. The compound was identifiedby comparison of its mass spectrum and NMR spectral datawith literature (Wagner et al., 1986). Fraction D was washedwith cold MeOH (4◦C) to yield the purified butanolic ex-tract (PBE).
2.3. Standardization
Calibration curve of demethylwedelolactone (Figs. 1 and2) was prepared by plotting several concentrations of com-pound2 in the range of 1.25–20�g/ml in MeOH againsttheir UV absorbances at 349 nm. Two milligrams each ofBE and PBE were dissolved in MeOH to make 6 ml of stocksolutions. A portion (0.2 ml) of these was then diluted withMeOH to the ratio of 1:20 in order to make sample solutionsof BE and PBE and their demethylwedelolactone contentswere calculated.
0.000
0.200
0.400
0.600
0.800
1.000
0 5 10 15 20
Concentrations of demethylwedelolactone (mcg/ml)
Abs
orba
nce
at 3
49 n
m
Fig. 1. Calibration curve of demethylwedelolactone at UV absorbance349 nm.
Fig. 2. Structures of wedelolactone; R= CH3 (1) and demethywedelo-lactone; R= H (2).
2.4. Animals and venom
Swiss albino mice of both sexes weighing about 18–20 gand about 25–30 g were used for the lethal effect and hemor-rhagic activity assays, respectively. Lyophilized MPV venomwas provided by Queen Saovabha Memorial Institute, ThaiRed Cross Society, Thailand.
2.5. Anti-snake venom activity
2.5.1. Neutralization of lethalityThe median lethal dose (LD50) of MPV venom was deter-
mined according to the method developed byTheakston andReid (1983). The venom in 0.2 ml of physiological salinewas injected into the tail vein of mice. Duplicated experi-ment was carried out in five mice at each venom dose. TheLD50 was calculated with the confidence limit at 95% prob-ability by the analysis of deaths occurring within 24 h ofvenom injection (Reed and Muench, 1938). The anti-lethalpotentials of BE and PBE were determined against 2LD50of MPV venom in six mice for each test concentration ofthe extracts, which was dissolved in either 20% aqueouspolyethylene glycol 400 (PEG 400) or a mixture of wa-ter/DMSO/EtOH/PEG 400 (55:5:20:20). The test solutionwas pre-incubated with the venom at 37◦C for 1 h and cen-trifuged at 10,000× g for 10 min before 0.2 ml of the su-pernatant were injected through the tail vein of mice. Eachexperiment was done in triplicate. The solvents, the extractsolutions, and the venom alone were used as controls.
2.5.2. Anti-hemorrhagic activityHemorrhagic activity was performed according to the
method ofKondo et al. (1960)and modified methods ofGutierrez et al. (1985)andOtero et al. (2000). The minimumhemorrhagic dose (MHD) of MPV venom was determinedby injecting intradermally 0.04 ml of different amount ofvenom (2.0–32.0�g in physiological saline) into two sepa-rately marked positions on the shaved dorsal skin of unanes-thesized mice. After 1.5 h, mice were killed with an overdoseof chloroform, the dorsal skin was removed and the diame-ter of the hemorrhagic lesion on the inner surface of the skinwas measured using calipers and background illumination.
P. Pithayanukul et al. / Journal of Ethnopharmacology 90 (2004) 347–352 349
The mean diameter of the hemorrhagic lesion was calcu-lated for each venom dose. Dose–response curve betweenthe diameter of the hemorrhagic lesion and the venom dosewas plotted and the dose causing 10-mm diameter recordedas MHD. This experiment was done in triplicate. TheBE (5–50 mg/ml) and PBE (2.5–30 mg/ml) test solutionswere prepared in 20% aqueous PEG 400 or water/DMSO/EtOH/PEG 400 (55:5:20:20). Anti-hemorrhagic potentialof the test solutions was determined against 3MHD of thevenom. Four mice were used for each tested dose and theexperiments were done in triplicate. Each 0.1 ml of the testsolution was pre-incubated with 0.1 ml of MPV venom solu-tion at 37◦C for 1 h and centrifuged at 10,000×g for 10 min.An aliquot (0.04 ml) of the supernatant containing 3MHDof venom was then injected intradermally into the markedpositions on the shaved dorsal skin of unanesthesized mice.After the mice were killed, the diameters of the hemorrhagiclesions were measured. The anti-hemorrhagic activities ofBE and PBE were calculated as percent inhibition. Thevenom and the extract solutions were used as controls.
2.5.3. Anti-proteolytic activityProteolytic activity of MPV venom was measured by mod-
ifying the methods ofKunitz (1947)andTan et al. (1986).Two milliliters of 1% casein in 0.25 M sodium phosphatebuffer pH 7.75 and 0.1 ml of venom (10–2000�g) in phys-iological saline were incubated for 1 h at 37◦C. The undi-gested casein was precipitated and the reaction terminated byadding 2 ml of 5% trichloroacetic acid. After centrifugationat 10,000× g for 10 min, the absorbance of the supernatantwas measured at 280 nm. One unit of proteolytic activity wasdefined as an increase of 0.001 absorbance units at 280 nm/h.The initial proteolytic dose of MPV venom was obtainedfrom the plot between proteolytic activity and the venomdoses. Solutions of 5–25 mg/ml BE and PBE were evalu-ated for their anti-proteolytic potentials against MPV venom.Each 0.05 ml of the test solution was pre-incubated for 1 h at37◦C with an equal amount of the venom solution (1 mg/ml)before the mixture was subjected to proteolytic activity eval-uation. The experiments were performed three times.
2.5.4. Anti-phospholipase A2 activityPhospholipase A2 activity was measured using an indirect
hemolytic assay on agarose–erythrocyte–egg yolk gel plateto define the minimum indirect hemolytic dose (MIHD). Themethods described byGutierrez et al. (1988)andOtero et al.(2000)was followed. The minimum indirect hemolytic dose(MIHD) of MPV venom (1.8�g) was the dose that inducedhemolysis halo having the diameter of 20 mm after incuba-tion for 20 h at 37◦C. Both BE and PBE at 2.5–30 mg/mlwere tested against one MIHD of MPV venom. Test solu-tions and MPV venom, 0.05 ml each, were pre-incubatedfor 1 h at 37◦C. After centrifugation at 10,000× g for10 min, the supernatant was tested for phospholipase A2activity. The anti-phospholipase A2 potential of the ex-tracts was expressed as percent inhibition of the enzyme
activity, in which 100% inhibition should produce no clearzone.
2.6. Statistical analysis
The survival rate and the inhibition of hemorrhagic ac-tivity in mice that received mixtures of venom and extractwere compared at different time intervals with the controls(receiving either venom or extract alone). Analysis of vari-ance (ANOVA) was used to determine the significance (P <
0.05) of the data obtained in all experiments.
3. Results
3.1. Neutralization of lethality
The LD50 of MPV venom was 129± 7.74�g per mouse.From Fig. 3, it can be seen that only the butanolic extractof Eclipta prostrata at dose of 2.5 mg per mouse demon-strated 100% neutralization against 2LD50 of MPV venom(P < 0.05 compared to the control group of venom alone).The increase in the amount of BE to 5.0 mg per mousesignificantly diminished the survival rate of mice to 39%(P < 0.05). Fig. 4 demonstrated that the amount of PBEbetween 1.5 and 4.5 mg per mouse exhibited partial pro-tection against lethality of the venom, with mean survivalrate against 2LD50 of MPV venom in the range of 50–58%within 24 h (P < 0.05 compared to venom alone). Both BEand PBE alone produced no lethal effect at the tested doses.
3.2. Anti-hemorrhagic activity
The MHD of MPV venom was determined as 17�g, whilethe venom at 3MHD induced hemorrhagic lesion having thediameter of 19.67±0.03 mm.Fig. 5demonstrated the reduc-tion in diameter of hemorrhagic lesion to 15.50–14.00 mmwhen 5–50 mg/ml of BE was pre-incubated with the venom.This indicated partial protection (up to 28% atP < 0.01)against hemorrhagic activity of MPV venom by butano-lic extract of Eclipta prostrata. On the other hand, PBE
0
20
40
60
80
100
120
0 2 4 6
Doses of BE (mg/mouse)
% S
urvi
val r
ate
of m
ice
Fig. 3. Percent survival rate of mice receiving 2LD50 of MPV venomwith BE. ( ) BE with 2LD50 of MPV venom. (�) Control, BE.
350 P. Pithayanukul et al. / Journal of Ethnopharmacology 90 (2004) 347–352
0 .00
20.00
40.00
60.00
80.00
100 .00
120 .00
0 1 2 3 4 5
Doses of PBE (mg/mouse)
% S
urvi
val r
ate
of m
ice
Fig. 4. Percent survival rate of mice receiving 2LD50 of MPV venomwith PBE. (�) PBE with 2LD50 of MPV venom. (�) Control, PBE.
0
5
10
15
20
25
0 5 25 50
Concentrations of BE (mg/ml)
Hem
orrh
agic
dia
met
er (m
m)
0
10
20
30
40
50
60
70
80
% I
nhib
itio
n of
he
mor
rhag
ic a
ctiv
ity
Fig. 5. Inhibition of hemorrhagic activity (%) of MPV venom by BE. ()Hemorrhagic diameter. (�) Percent inhibition of hemorrhagic activity.
(Fig. 6) was able to significantly (P < 0.05) diminish thediameter of hemorrhagic lesion induced by 3MHD of MPVvenom (21.77 ± 1.50 mm) to 16.08–7.17 mm at doses be-tween 2.5 and 30 mg/ml. The percent inhibition of hemor-
0
5
10
15
20
25
0 2.5 5 10 20 30Concentrations of PBE (mg/ml)
Hem
orrh
agic
dia
met
er (m
m)
0
10
20
30
40
50
60
70
80
% I
nhib
itio
n of
he
mor
rhag
ic a
ctiv
ity
Fig. 6. Inhibition of hemorrhagic activity (%) of MPV venom by PBE. ()Hemorrhagic diameter. (�) Percent inhibition of hemorrhagic activity.
0
50
100
150
200
250
300
0 5 10 25
Concentrations of BE (mg/ml)
Pro
teol
ytic
act
ivit
y (u
nit)
0 5 10 25
Concentrations of PBE (mg/ml)
Fig. 7. Proteolytic activity of MPV venom after pre-incubation with BEand PBE. (�) BE. ( ) PBE.
rhagic activity by PBE was calculated to be between 26and 57%.
3.3. Anti-proteolytic activity
The initial proteolytic dose of MPV venom obtained fromthe plot between venom doses and their proteolytic activitywas 50�g. Fig. 7 shows that both BE and PBE at all testedconcentrations did not significantly inhibit this activity ofthe venom (P > 0.05) at its initial proteolytic dose.
3.4. Anti-phospholipase A2 activity
MPV venom at the amount of 1.8�g, which producedthe clear zone diameter of 19.67 ± 0.20 mm on agarose–erythrocyte–egg yolk gel plate, was selected as the minimum
0
5
10
15
20
25
0 5 10 25
Concentrations of BE (mg/ml)
Dia
met
er o
f cl
ear
zone
(m
m)
0
10
20
30
40%
Inh
ibit
ion
of
phos
phol
ipas
e A
2 a
ctiv
ity
Fig. 8. Effect of concentrations of BE on the diameter of clear zone onagarose–erythrocyte–egg yolk gel plate and the inhibition of phospholi-pase A2 activity. (�) Diameter of clear zone. (�) Percent inhibition ofphospholipase A2 activity.
P. Pithayanukul et al. / Journal of Ethnopharmacology 90 (2004) 347–352 351
0
5
10
15
20
25
0 2.5 5 10 20 30
Concentrations of PBE (mg/ml)
Dia
met
er o
f cl
ear
zone
(m
m)
0
10
20
30
40
% I
nhib
itio
n of
ph
osph
olip
ase
A 2
act
ivit
y
Fig. 9. Effect of concentrations of PBE on the diameter of clear zoneon agarose–erythrocyte–egg yolk gel plate and the percent inhibitionof phospholipase A2 activity. (�) Diameter of clear zone. (�) Percentinhibition of phospholipase A2 activity.
indirect hemolytic dose (MIHD). Partial inhibition of phos-pholipase A2 activity by BE was observed (Fig. 8) asthe diameters of clear zone were significantly reducedto 16.72–12.92 mm (P < 0.05) when 5–25 mg/ml of theextract were tested. The percent inhibition was 15–34%.Phospholipase A2 activity of MPV venom was also slightlybut significantly (up to 10% atP < 0.05) inhibited by PBE,causing the reduction in the clear zone diameter from aninitial value of 20.22 mm at 1MIHD to 19.78–18.17 mm(Fig. 9).
4. Discussion
Both the butanolic (BE) and PBEs ofEclipta prostratacontained abundant amount of the coumestan demethyl-wedelolactone (2): about 50% in BE and as high as 90%in PBE. Wedelolactone (1), which has been reported as theconstituent with anti-venom activity from this plant, is lesspolar and mostly remained in the ethyl acetate fraction dur-ing the extraction procedure. However, the butanolic extractswere also demonstrated as able to neutralize the lethality ofMPV venom and significantly inhibit the hemorrhagic andphospholipase A2 activities of the venom. Therefore,2 islikely an active constituent in the use of this plant as a treat-ment against snake venoms.
A possible mechanism of action of2 in inhibiting thelethal and enzymatic activities of MPV venom might resultfrom the polyphenolic nature of the compound (Mors et al.,2000). Hydrogen bonding could form between the phenolichydroxyls of 2 and the carbonyl functions in the peptidebonds of the venom proteins, or, on the other hand, betweenthe carbonyl group of2 and NH-function within the venomenzymes.
The inferior ability of PBE, which contains larger per-centage of2 than BE, in neutralizing the lethal effect of
2LD50 of MPV venom suggested that there were other com-ponents of the butanolic extract that might be more activethan2 and were eliminated in the purification process. Theanti-lethal activity of BE, which peaked at 2.5 mg per mouseand appeared to diminish when the dosage was increased upto 5.0 mg per mouse, could reflect the optimum combina-tion between2 and these other active compounds. Increas-ing the dose of BE might also produce more antagonizingeffect against this activity from its other constituents. How-ever, for anti-hemorrhagic activity, purification of BE gavethe extract (PBE) with better activity.
Another factor which might affect the outcome of theseexperiments is the limited solubility of both BE and PBE inphysiological solutions and aqueous systems. Higher dosesof both extracts could not be evaluated due to this problem.
The potential of butanolic extracts ofEclipta prostratato act against MPV venom was therefore demonstrated inits ability to significantly neutralize the lethal and hem-orrhagic effects of the venom. The anti-proteolytic andanti-phospholipase A2 activities of the plant, which werelow or insignificant in these extracts, should be furtherinvestigated in other extracts from this anti-venom plant.
In conclusion, the results from this preliminary studywould be beneficial in developing topical anti-snake venompreparations fromEclipta prostrata extracts to treat localwound effects, including hemorrhagic bullae and musclenecrosis from viper bites.
References
Bhargava, K.K., Krishnaswamy, N.R., Seshadri, T.R., 1972. Demethyl-wedelolactone glucoside fromEclipta alba leaves. Indian Journal ofChemistry 10, 810–811.
Gutierrez, J.M., Avila, C., Rojas, E., 1988. An alternative in vitro methodfor testing the potency of the polyvalent antivenom produced in CostaRica. Toxicon 26, 411–413.
Gutierrez, J.M., Gene, J.A., Rojas, G., Cerdas, L., 1985. Neutralizationof proteolytic and hemorrhagic activities of Costa Rica snake venomsby a polyvalent antivenom. Toxicon 23, 887–893.
Kondo, H., Kondo, S., Ikezawa, H., 1960. Studies on the quantitativemethod for determination of haemorrhagic activity of Habu snakevenom. Japanese Journal of Medical Science & Biology 13, 43–51.
Kunitz, M., 1947. Crystalline soybean trypsin inhibitor II: general prop-erties. Journal of General Physiology 30, 291–310.
Markland, F.S., 1997. Snake venoms. Drugs 54, 1–10.Markland, F.S., 1998. Snake venoms and the hemostatic system. Toxicon
Inhibition of the myotoxic and haemorrhagic activities of crotalidvenoms byEclipta prostrata (Asteraceae) extracts and constituents.Toxicon 32, 595–603.
Melo, P.A., Ownby, C.L., 1999. Ability of wedelolactone, heparin, andpara-bromophenacyl bromide to antagonize the myotoxic effects oftwo crotaline venoms and their PLA2 myotoxins. Toxicon 37, 199–215.
Mors, W.B., do Nascimento, M.C., Parente, J.P., da Silva, M.H., Melo,P.A., Suarez-Kurtz, G., 1989. Neutralization of lethal and myotoxicactivities of South American rattle snake venom by extracts and
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constituents of the plantEclipta prostrata (Asteraceae). Toxicon 27,1003–1009.
Mors, W.B., 1991. Plants active against snake bite. In: Wagner, H.,Farnsworth, N.R. (Eds.), Economic and Medicinal Plant Researh, vol.5, pp. 353–373.
Mors, W.B., do Nascimento, M.C., Pereira, B.M.R., Pereira, N.A., 2000.Plant natural products active against snake bite-the molecular approach.Phytochemistry 55, 627–642.
Otero, R., Nunez, V., Jimenez, S.L., Fonnergra, R., Osorio, R.G.,2000. Snakebites and ethnobotany in the northwest region ofColombia Part II: neutralization of lethal and enzymatic effectsof Bothrops atrox venom. Journal of Ethnopharmacology 71, 505–511.
Otero, R., Nunez, V., Borona, J., Fonnergra, R., Jimenez, S.L., 2000.Snakebites and ethnobotany in the northwest region of Colombia PartIII: neutralization of haemorrhagic effects ofBothrops atrox venom.Journal of Ethnopharmacology 73, 233–241.
Reed, L., Muench, H., 1938. A simple method of estimating 50 percentend point. American Journal of Hygiene 27, 493–497.
Theakston, R.D.G., Reid, H.A., 1983. Development of simple standardassay procedures for the characterization of snake venom. Bulletin ofWorld Health Organization 61, 949–956.
Viravan, C., Looareesuwan, S., Kosakarn, W., Wuthiekanun, V., McCarthy,C.J., Simson, A.F., et al., 1992. A national hospital-based survey ofsnakes responsible for bites in Thailand. Transactions of the RoyalSociety of Tropical Medicine and Hygiene 86, 100–106.
Wagner, H., Fessler, B., 1986. In vitro 5-lipoxygenase hemmung durchEclipta alba extrakte und das coumestan derivat wedelolactone. PlantaMedica 52, 374–377.
Wagner, H., Geyer, B.B., Kisi, Y., Hikino, H., 1986. Coumestans as themain active principles of the liver drugsEclipta alba and Wedeliacalendulacea. Planta Medica 52, 370–374.
Journal of Ethnopharmacology 90 (2004) 353–357
In vitro antimicrobial activity of certain medicinal plantsfrom Eastern Ghats, India, used for skin diseases
A. Jeevan Ram, L. Md. Bhakshu, R.R. Venkata Raju∗Department of Botany, Sri Krishnadevaraya University, Anantapur 515003, India
Received 29 August 2003; received in revised form 8 October 2003; accepted 13 October 2003
Medicinal plants have long been the subject of human cu-riosity and need. Plant-derived products are present in 14of the 15 therapeutic categories of pharmaceutical prepara-tions that are currently recommended by medical practition-ers and they form an important part of the health-care systemin the western world (Phillipson and Anderson, 1989). It isestimated that there are about 2,500,000 species of higherplants and the majority of these have not been examined indetail for their pharmacological activities.
The antimicrobial properties of certain Indian medicinalplants were reported based on folklore information (Dayaland Purohit, 1971; Hook and Thomas, 1995; Reddy, 1995;Suresh et al., 1995; Ahmad et al., 1998; Mehmood et al.,1999; Perumal Samy et al., 1998, 1999), and a few attemptswere made on inhibitory activity against certain pathogenicbacteria and fungi (Taylor et al., 1995).
Infectious diseases, particularly skin and mucosal infec-tions, are common in most of the tribal inhabitants due tolack of sanitation, potable water and awareness of hygienicfood habits. An important group of these skin pathogensare the fungi, among which dermatophytes andCandidaspp., besides certain pathogenic bacteria are the most fre-quent (Caceres et al., 1993; Desta, 1993). Furthermore, inthe last few years, the number of immunosupressed and im-munocompromised patients, who frequently develop oppor-
tunistic systemic and superficial mycoses (Rahalison et al.,1994; Li et al., 1995) such as candidiasis, dermato-mycosis,fungal infections etc., have increased dramatically (Odds,1988; Ribbon, 1988; Diamond, 1991). This is mainly dueto the non-availability of effective antifungal drugs for sys-temic fungal infections and toxicity of available drugs likeamphotericin-B (Maddux and Brarriere, 1980; Saral, 1991).Thus there is an increased need for the development of al-ternative antipathogenic substances. One possible approachis to screen local medicinal plants in search of suitablechemotherapeutic antibacterial and antifungal substances.The herbalists prescribed various preparations of medicinalplants in treating ailments such as itch, eczema, scabiesand skin diseases (Chopra et al., 1992; Behl et al., 1993;Iyengar et al., 1997).
1.1. Study area
The Eastern Ghats cover an area about 75,000 km2
traversing the Coromandel coast between 11◦30′–22◦00′Nlatitudes and 76◦50′–86◦30′E longitudes, show discontin-uous hill ranges with undulated topography. The East-ern Ghats of Andhra Pradesh state passes through Viza-yanagaram, Srikakulam, Visakhapatnam, East and WestGodavari, Khammam, Krishna, Mahabubnagar, Kurnool,Prakasam, Cuddapah, Nellore and Chittoor districts.
1.2. Ethnology of tribes
The Eastern Ghats region of Andhra Pradesh with variousethnic groups and diverse flora offers an excellent oppor-
354 A. Jeevan Ram et al. / Journal of Ethnopharmacology 90 (2004) 353–357
tunity for ethnobotanical studies. There are altogether 550tribal communities all over India. According to 1991 esti-mates (Source: Ministry of Welfare, Government of India,Annual Report, 1992), the scheduled tribes population inthe country is 80.66 million constituting about 9.55% of thecountry’s total population of 844.32 million. It is estimatedthat the predominant tribal areas cover about 15% of thetotal geographical area of the country.
According to 1991 census, the tribal population of AndhraPradesh is 4.75 million, comprising 33 tribes and 60 othersmall tribal communities, which constitutes 7.15% of thetotal population of the state. Of these, 27 tribal communi-ties are confined to the isolated hills and adjacent plains ofEastern Ghats. The major groups among them are Bagatas,Chenchus, Jatapus, Khonds (Samantas), Konda doras,Konda kammaras, Konda reddis, Koyas, Lambadis (Sug-alis), Nuka doras (Muka doras), Porjas (Gadabas), Savarasand Valmikis. All the above tribes are aboriginal exceptLambadis (Sugalis), who have migrated from north-westto southern India and settled in Khammam, Anantapur,Kurnool, Prakasam and Warangal districts (Rama Rao andHenry, 1996).
In the upper Godavari region the tribes commonly foundare Bagatas, Jatapus, Khonds, Konda doras, Konda kam-maras, Nuka doras, Porjas and as Savaras. The Godavari val-ley is inhabited mainly by Konda reddis and Koyas, whileChenchus are the oldest inhabitants in Nallamalais. TheValmikis are found in Godavari valley and other adjoininghills in Eastern Ghats.
2. Materials and methods
2.1. Collection of plant materials
The extensive and intensive explorations conducted in theforests of Eastern Ghats during 1997–2001, based on the in-formation recorded from bush/witch doctors, revealed 350drug-yielding plants, among which 122 species are beingused for skin diseases (Jeevan Ram and Venkataraju, 2001;Jeevan Ram, 2002). The ethnobotanical information regard-ing the drug-yielding plants was recorded using the standardmethods ofSchultes (1960), Croom (1983)and Hamilton(1995). The information on name, part used, purpose, modeof administration, etc. was recorded in the field notebooksas well as audiotapes. The recorded information was crosschecked with other adivasi inhabitants in order to evaluatethe authenticity of the drugs. The sample specimens werecollected in bulk quantities for analysis. Based on the folkevidences regarding the effective utilisation for different skinailments the samples were collected and screened for an-timicrobial properties. The voucher specimens were identi-fied with the help of regional floras (Gamble, 1935; Pullaiahet al., 1997) and the same were deposited at Sri Krishnade-varaya University Herbarium (SKU), Anantapur.
2.2. Preparation of plant extracts
The collected samples were shade dried, powdered (about60 g) and successively extracted with ethanol (250 ml) us-ing a Soxhlet apparatus for 6 h. The extracts were filteredand concentrated under reduced pressure, below 40◦C todryness.
2.3. Microorganisms
The test organismsMicrococcus luteus, Micrococcusroseus, Pseudomonas aeruginosa, Staphylococcus aureus(bacterial strains) andCandida albicans (fungal strain)were obtained from the Institute of Microbial Technology(IMTECH), Chandigarh, India. The test organisms weresub-cultured at 37◦C and maintained on nutrient agar me-dia for bacteria and sabouraud agar medium forCandidaalbicans.
2.4. Antimicrobial assay
The antimicrobial activity of the ethanol extracts of eachsample was evaluated by using disc diffusion method (Baueret al., 1966). Petriplates containing 20 ml of respectivemedia were seeded with selected microbial strains. Fivemilliliters of nutrient broth was inoculated with a loop(6 mm) of bacteria/yeast and incubated at 35◦C for 6 h.One milliliter of broth was taken at 0.6 optical density (atlog phase, ca. 108 cells/ml) and inoculated the nutrient agar(sterile) and transferred to 180 mm× 20 mm petri dishes.The sterile Whatmann No. 1 filter paper discs of 6 mmdiameter were impregnated with 1000–5000�g of concen-trated plant extracts and placed on the surface of the freshlyinoculated medium. Standard antibiotic discs viz., Ampi-cillin, Tetracycline (30�g/disc) obtained from Hi-Media,Mumbai, were used as positive controls. Ethanol and wateralone served as negative controls. The assessment of an-timicrobial activity was based on measurement of inhibitionzones formed around the discs. The media were incubatedfor 24 h at 37◦C and the diameters of the inhibition zoneswere recorded. Three independent trials were conducted foreach concentration.
3. Results
Twenty-three species belonging to 15 families are reportedin Table 1with their part used, medicinal uses and mode ofpreparation/administration. The ethanolic extracts exhibiteddifferent degrees of inhibitory activity against all the testedhuman pathogenic microorganisms at different concentra-tions (Table 2).
The maximum inhibitory effect was exhibited by the ex-tracts ofAcanthospermum hispidum, Andrographis panic-ulata, Aristolochia indica, Plumbago zeylanica, Sphaeran-thus indicus andPentanema indicum.
A. Jeevan Ram et al. / Journal of Ethnopharmacology 90 (2004) 353–357 355
Table 1List of ethnomedicinal plants used for skin diseases from Eastern Ghats, India
Family name Botanical name/local name/voucher number Part used Diseases Mode of administration/preparation
Wd Dandruff Wood made into paste, used in head bath
Verbenaceae Gmelina asiatica L. Gummudu/20699 Fr Eczema Fruit pulp as an external applicationVerbenaceae Premna latifolia Roxb. Nelli chettu/23836 Sb Ringworm Stem bark ground with pepper and infusion
given orally in the early morningsVerbenaceae Vitex altissima L.f. Nemaliadugu/20811 Sb Wounds Stem bark ground, made into paste, applied
externallyZingiberaceae Globba marantina L. Kalingadumpa/20655 Rt Leucoderma Fresh rihzomes crushed, mixed with
Skin diseases such as eczema, itches, scabies, etc., areprevalent in most of the tribal enclaves in the area. Basicamenities such as drinking water and sanitation are not avail-able and the hygienic conditions are therefore appalling.The investigations on ethnobotanical information for curingvarious skin ailments resulted in selection of a number ofspecies used by different aboriginals of Eastern Ghats. Theinteresting thing is that the same plant is being used by dif-ferent tribal societies with varied parts of the plant, however
the illness is being caused by the same class of pathogens.The biological properties ofAndrographis paniculata and
Plumbago zeylanica against the pathogenic microorganismswere hitherto not reported (Perumal Samy et al., 1999).However, the extracts of both species have exhibited maxi-mum inhibition onPseudomonas aeruginosa andStaphylo-cocus aureus at lower concentrations.
Among the 23 plants screened, highest inhibitory zoneswere observed in the extracts ofPentanema indicum (32 mm)againstMicrococcus luteus, Plumbago zeylanica (30 mm)againstMicrococcus roseus, Sphaeranthus indicus (24 mm)
356 A. Jeevan Ram et al. / Journal of Ethnopharmacology 90 (2004) 353–357
Table 2In vitro antimicrobial studies of selected medicinal plants from EasternGhats, India
againstMicrococcus luteus and Micrococcus roseus andAcanthospermum hispidum (18 mm) againstPseudomonasaeruginosa and Candida albicans. The folk-claims on theuse of these crude drugs against infectious diseases weresubstantiated by the present observations.
The results were encouraging, as all the selected plants ap-peared to contain antimicrobial substances. Further attemptsare being made to elucidate the molecular basis for the in-hibitory properties.
A. Jeevan Ram et al. / Journal of Ethnopharmacology 90 (2004) 353–357 357
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Journal of Ethnopharmacology 90 (2004) 359–365
Toxicity of crude rhizome extract ofKaempferia galanga L. (Proh Hom)
D. Kanjanapothia,∗, A. Panthonga, N. Lertprasertsukeb, T. Taesotikula, C. Rujjanawated,D. Kaewpinitd, R. Sudthayakornd, W. Choochotec, U. Chaithongc, A. Jitpakdic, B. Pitasawatc
a Department of Pharmacology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailandb Department of Pathology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand
c Department of Parasitology, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailandd Chulabhorn Research Institute, Chiang Mai 50200, Thailand
Received 25 July 2001; received in revised form 8 October 2003; accepted 20 October 2003
Keywords: Kaempferia galanga; The Hippocratic screening test; Acute and subacute toxicities in rats; Dermal irritation test in rabbits
1. Introduction
Kaempferia galanga (“Proh hom” in Thai; Zingiber-aceae) is an acaulescent perennial that grows in SouthernChina, Indochina, Malaysia and India. The rhizomes ofthe plant, which contains essential oils, have been usedin a decoction or powder for indigestion, cold, pectoraland abdominal pains, headache and toothache. Its alco-holic maceration has also been applied as liniment forrheumatism (Keys, 1976; Lieu, 1990). In Chinese medicine,Kaempferia galanga rhizomes have been used as an aro-matic stomachic, and also as incense. The constituentsof this rhizome, hitherto reported, have included cineol,borneol, 3-carene, camphene, kaempferol, kaempferide,
∗ Corresponding author.
cinnamaldehyde,p-methoxycinnaamic acid, ethyl cinna-mate, and ethylp-methoxycinnamate (Nakao and Shibu,1924). Ethyl p-methoxycinnamate was reported to inhibitmonoamine oxidase (Noro et al., 1983). The methanolic ex-tract of Kaempferia galanga, which identified as ethyl cin-namate, ethylp-methoxycinnamate andp-methoxycinnamicacid, showed larvicidal activity against the second stagelarva of dog roundworm,Toxocara canis (Kiuchi et al.,1988). Evaluation for amebicidal activity in vitro againstthree species ofAcanthamoeba: Acanthamoeba culbertsoni,Acanthamoeba castellanii, and Acanthamoeba polyphaga;the causative agents of granulomatous amebic encephalitisand amebic keratitis, found that theKaempferia galangaextract possessed an effective amebicidal for all threespecies (Chu et al., 1998). Vimala et al. (1999)foundthat the rhizome extract ofKaempferia galanga exhibitedEpstein-Barr virus (EBV) activiation inhibitory activity
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when screened for anti-tumour promoter activity usingthe short-term assay of inhibition of 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced EBV early antigen in Rajicells.
Recently,Pitasawat et al. (1998)demonstrated signifi-cant larvicidal activity againstCulex quinquefasciatus inthree of ten plant extracts, including those fromKaempferiagalanga, Illicium vernum and Spilanthes acmella, whichhad LC50values of 50.54, 54.11 and 61.43 ppm, respec-tively. Subsequently, larvicidal and repellent activities ofKaempferia galanga fractions, i.e. the hexane fraction,dichloromethane fraction 1, dichloromethane fraction 2, andmethanolic fraction have been affirmed byChoochote et al.(1999). They declared that the hexane fraction possessedlarvicidal potency againstCulex quinquefasciatus (LC50 =42.33 ppm), repelledAedes aegypti (ED50 = 30.73 ug/cm2),and provided biting protection for 3 h in the laboratory. Itcould also protect in the field againstArmigeres suball-batus, Anopheles barbirostris, Anopheles aconitus, Man-sonia uniformis, Culex quinquefascitus, Culex gelidus,Culex tritaeniorhynchus and Aedes aegypti. Additionally,it did not cause dermal irritation when applied on humanskin. Before applying the effective formula ofKaempferiagalanga for mosquito control, it is important to evaluatethe toxic effects of the extract, which is essential when asafe dose has to be selected. Therefore, the present studywas initiated for analysing the acute and subacute tox-icites and dermal irritation of the extract in laboratoryanimals.
2. Materials and methods
2.1. Laboratory animals
Adult Sprague–Dawley rats of either sex, aged 6–8 weekswith a weight of 250–300 g were purchased from the Na-tional Laboratory Animal Center, Salaya Mahidol Univer-sity, Nakorn Pathom, Thailand. Adult albino rabbits of ei-ther sex, weighing between 4 and 5 kg were obtained fromthe Animal Unit of the Faculty of Medicine, Chiang MaiUniversity, Thailand. The animals were kept in an animalroom where the temperature was maintained at 22± 3◦Cunder a 12 h light–dark cycle. They were provided with foodand water ad libitum for 1 week to acclimatize them beforestarting the experiment.
2.2. Preparation of plant extract
The rhizomes ofKaempferia galanga were purchasedin Chiang Mai province, Thailand. Voucher specimen(PHCO-CM-012) was deposited at the Department of Phar-macology, Faculty of Medicine, Chiang Mai University,Thailand. The extraction was performed by macerating1.5 kg of dried and powderedKaempferia galanga with5 l of hexane or 95% ethanol at room temperature for 2
days. After suction filtering through a Buchner funnel, thehexane or ethanolic filtrates were evaporated by a rotaryevaporator at 40–60◦C, and then lyophilized to yield hex-ane or ethanolic extracts. Extracts isolated fromKaempferiagalanga were kept at−20◦C until testing.
2.3. Hippocratic screening test
The effect of the ethanolic extract ofKaempferia galangaon the general behavior of conscious animals was evaluatedin rats, as described byMalone and Robichaud (1962). In-traperitoneal administration of five dose levels of test sub-stance suspended in 5% Tween 80 were given to groups ofnonfasted rats. A control group received an equal volume ofthis vehicle. Two females and two males from a total of 24animals were used for each dosage level. Signs and symp-toms induced by the test substance were recorded prior toadministration. Then again at 5, 15, and 30 min, and 1, 2,and 24 h after administration, and once daily thereafter for7 days. The rats that died within 24 h were autopsied andrecorded for pathological changes. On the seventh day aftertreatment, the live animals were sacrificed for the examina-tion of their internal organs (heart, lungs, liver, spleen, kid-neys, adrenals, testes, ovaries, uterus, thymus, brain, eyes,stomach, intestines, etc.) for abnormal signs. Any changesin their vital organs compared with those of the control an-imals were recorded.
2.4. Acute toxicity studies
Ethanolic extract ofKaempferia galanga suspended in5% Tween 80 was administered to the groups of rats in asingle oral dose by gavage using a feeding needle (at leastthree doses). The control group received an equal volumeof the 5% Tween 80 vehicle. Ten females and ten maleswere used for each dosage level. They were deprived offood, but not water 16–18 h prior to the administration ofthe test suspension. Observations of toxic symptoms weremade and recorded systematically at one, two, four andsix hours after administration. Finally, the number of sur-vivors was noted after 24 h and these animals were thenmaintained for a further 13 days with observations madedaily. At the conclusion of the experiment, all survivinganimals were sacrificed with an injection of pentobarbitaland their organs such as liver, lungs, heart, spleen, adrenals,kidneys, testes and ovaries were excised and weighed. Thepathological observations of these tissues were performedon gross and microscopic bases. Histological examinationswere performed on the preserved tissues with particu-lar emphasis on those which showed gross pathologicalchanges.
The toxicological effect was assessed on the basis of mor-tality, which was expressed as LD50. If a test at one dose levelof at least 5 g/kg body weight produced no compound-relatedmortality, then a full study using three dose levels might notbe necessary.
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2.5. Subacute toxicity
The animals were divided into five groups of eight fe-males and eight males totaling 80 rats. Ethanolic extract ofKaempferia galanga suspended in 5% Tween 80 was admin-istered orally by gavaging at three dose levels of the extractdaily to three groups of rats for a period of 28 days. Thecontrol group received an equal volume of the 5% Tween80 vehicle. In order to assess reversibility, an alcoholic ex-tract ofKaempferia galanga was administered to a group ofrats at 100 mg/kg daily for 28 days, with no treatment forthe following 14 days. All rats were weighed and observeddaily for physiological and behavioral responses. Any ratsthat died during the test period were tested pathologically,and all animals were examined at the end of the test period.
2.6. Parameters
2.6.1. Blood analysisAll surviving animals fasted overnight and were anes-
thetized afterwards for blood collection from a commoncarotid artery. Blood samples were collected into heparinizedand dry non-heparinized centrifuge tubes. A blood analysis(both hematology and chemistry) was carried out. The hep-arinized blood was used for a hematological study whichincluded WBC and differential leukocyte counts, platelet,hematocrit and hemoglobin estimation. The non-heparinizedblood was allowed to coagulate before being centrifugedand the serum was separated. The serum was assayed forglucose, creatinine, blood urea nitrogen (BUN), aspartatetransaminase (AST), alanine transaminase (ALT), alkalinephosphatases (Alk-P), total protein and albumin.
2.6.2. Tissue analysisImmediately after the blood collection, the animals were
sacrificed for tissue studies. The organs such as liver, lungs,heart, spleen, adrenals, kidneys, testes and ovaries were re-moved, blotted free of blood and weighed immediately ona Precisa electronic balance for subsequent analysis. Eyes,brain, thymus, intestines, uterus, epididymis, seminal vesi-cles, prostate glands, and thoracic spine and muscle withthe sciatic nerve were also observed. Histological examina-tions were performed on the preserved tissues with particu-lar emphasis placed on those that showed gross pathologicalchanges.
2.7. Dermal irritation test
In the present study, determination of the effect of thehexane fraction ofKaempferia galanga on the skin wasconducted to examine the potential for producing extensivetissue damage or corrosions. The method was performedaccording to the slightly modified procedure of OECD TestGuidelines (OECD, 1981). Six adult albino rabbits (threemales, three females) with healthy, intact skin were used.Approximately 24 h before the test, their fur was shaved off
the dorsal area of their trunk. Care had to be taken to avoidabrading the skin.
A 0.5 ml dose of 250 mg/ml hexane fraction ofKaempfe-ria galanga dissolved in absolute ethanol was applied to asmall area (2 cm×3 cm) of the test site, which was then cov-ered with a gauze patch. The patch was held in place withnon-irritating tape. The animal accessability to the patchwith resulting ingestion/inhalation of the test substance hadto be prevented. A symmetrical test site area for each an-imal served as a control and was applied in an equal vol-ume of absolute ethanol. Croton oil was used as a positivecontrol. Exposure duration was 4 h. At the end of the expo-sure period, the residual test substance was removed, wherepracticable, using absolute ethanol or an appropriate sol-vent without altering the existing response or the integrity ofthe epidermis. The animals were examined for signs of ery-thema, edema and the responses were scored at 30–60 min,and then at 24, 48 and 72 h after patch removal. Furtherobservations might be needed, as necessary, to establishreversibility.
2.8. Statistical analysis
Either the analysis of variance (ANOVA) or Student’st-test (SPSS/PC computer program) was employed to an-alyze the results statistically. A statistical comparison wascarried out using the Duncan Multiple Range Test. All val-ues were expressed as back transformed mean± S.D. Dif-ferences below the probability level of 0.05 were consideredstatistically significant.
3. Results
3.1. Hippocratic screening test
The general behavioral changes of the rats were observedfollowing intraperitoneal injections of the ethanolic extractof Kaempferia galanga at 25, 100, 250, 800 and 2000 mg/kgdoses, which were graded through time. Doses of 25, 100 and250 mg/kg did not cause any detectable changes, whereas,a dose of 2000 mg/kg seemed to be lethal and caused twoout of four rat deaths within 24 h. Signs and symptoms,which occurred in response toKaempferia galanga extract,decreased in motor activity and respiration. A loss of screengrip was observed when high doses (800 and 2000 mg/kg)were given. A 2000 mg/kg dose ofKaempferia galanga ex-tract caused analgesia. The intensity of responses grew withincreasing doses, and the effects ofKaempferia galanga ex-tract persisted more for than 2 h. The rats that died froma high dose (2000 mg/kg) ofKaempferia galanga extractshowed signs of respiratory failure (decreased respiratoryrate and irregular breathing) before death. The internal or-gans of both controlled and treated groups did not show anyunusual signs and were found to be normal in both size andcolor.
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Table 1Body and organ weights (g) of rats treated with ethanolic extract ofKaempferia galanga in an acute toxicity
Data are expressed as mean± S.D., n = 10. No statistical differencebetween control andKaempferia group (P > 0.05).
3.2. Acute toxicity
No death was recorded during the treatment period in ei-ther the control or treated groups given 5 g/kg of ethano-lic extract of Kaempferia galanga orally. The animals didnot show any changes in general behavior or other physio-logical activities. There were no significant differences be-tween the control and treated groups in the body and organweights of male and female rats (Table 1). Moreover, therewas no significant difference in the testicular weight of malerats and, similarly, there was no significant difference in theovarian weight of female rats. Pathological examinations ofthe tissues on a gross and microscopic basis indicated thatthere were no detectable abnormalities. No pathological al-terations were grossly detected. The organs of both controland treated groups were unremarkable and comparable toeach sex. No further evidence of histopathological changeswere seen. The architectures of the internal organs exam-ined and their cellular appearances were comparatively un-remarkable in both groups and sexes.
3.3. Subacute toxicity
No death was recorded during the treatment period ineither the control or treated groups. The animals did not
show any changes in general behavior or other physiolog-ical activities. The body and organ weights of the maleand female rats, which were administered an alcoholicextract of Kaempferia galanga at 25, 50 or 100 mg/kgdoses daily for 28 days, are given inTable 2. There wereno significant differences in the body and organ weightsbetween control and treated animals of both sexes. Sim-ilarly, a withdrawal from Kaempferia galanga treatmentproduced no significant changes in both male and femalerats.
3.4. Hematological and biochemical observations
The hematological analysis (Table 3) showed no signif-icant differences in any of the parameters examined in ei-ther the control or treated groups of both sexes. However,the differential leukocyte counts (Table 4) showed a slightbut significant decrease of lymphocyte count in the 50 and100 mg/kg male rat groups with no significant differencesin neutrophils, eosinophils and monocytes in all male ratgroups. In contrast, there were no significant changes in anyof the differential leukocyte counts in female rats. Bloodchemistry analysis (Table 5) revealed no significant changesin any of the parameters examined in either the control ortreated groups of both sexes.
In the separated groups, the ethanolic extract ofKaempfe-ria galanga was administered at 100 mg/kg daily for 28days followed by a treatment-free period of 14 days, whena slight but significant decrease in hemoglobin (HGB)was observed in male rats (Table 3). In accordance withthe previous 28 days treatment, the decrease of lympho-cyte count was still significant in the male rats that hadbeen withdrawn from treatment. Moreover, a significantincrease in neutrophil count, with no marked changes inthe other parameters, was observed. However, there wasno significant change in any of the leukocyte counts infemale rats (Table 4). Surprisingly, the rats that had beenwithdrawn from treatment displayed a significant decreasein BUN, protein, and Alk-P in males and a significantfall of AST in females (Table 5). However, no gross ab-normalities and no further evidence of histopathologicalchanges were seen in all control and treated rats of bothsexes.
3.5. Dermal irritation test
Dermal application of 250 mg/ml hexane fraction ofKaempferia galanga dissolved in absolute ethanol producedno irritation in both male and female rabbits. No signs oferythema, eschar and edema were observed. No other reac-tions were seen during the test. In the case of the positivecontrols that had been treated with croton oil, well-definederythema and moderate edema were observed in both maleand female rabbits from 24 to 72 h after application. Nodifferences in the degree of skin irritation were seen whenmale and female rabbits were compared.
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Table 2Body and organ weights (g) of rats treated with ethanolic extract ofKaempferia galanga in a subacute toxicity
Data are expressed as mean± S.D., n = 8. No statistical difference between the control andKaempferia group (P > 0.05).a A separate group was administered at 100 mg/kg daily for 28 days followed by no treatment for 14 days.
Table 3Hematological values of rats treated with ethanolic extract ofKaempferia galanga in a subacute toxicity
Data are expressed as mean± S.D., n = 8.a A separate group was administered at 100 mg/kg daily for 28 days followed by no treatment for 14 days.∗ Significantly different from the control (P < 0.05).
4. Discussion
In toxicity studies, including the Hippocratic screeningtest, acute, subacute and dermal toxicities were elucidated insmall laboratory animals. Ethanolic extract ofKaempferiagalanga was used in most of these tests with the exceptionof the dermal irritation test, since this form was convenientto prepare, could be easily applied, and its larvicidal potency
(LD50 = 50.54 ppm) was comparable to that of the hexanefraction (LD50 = 42.33 ppm) (Choochote et al., 1999). Inthe dermal irritation test, however, it was necessary to use thesame form (hexane fraction) as that used for skin applicationin both laboratory and field repellent tests (Choochote et al.,1999).
The Hippocratic screening test is commonly used in thepreliminary screening of medicinal plants to detect inter-
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Table 4Differential white blood cell count of rats treated with ethanolic extract ofKaempferia galanga in a subacute toxicity
Data are expressed as mean± S.D., n = 8.a A separate group was administered at 100 mg/kg daily for 28 days followed by no treatment for 14 days.∗ Significantly different from the control (P < 0.05).
esting pharmacological activities (Malone and Robichaud,1962). The test has revealed some pharmacological ac-tivities of Kaempferia galanga, which are observed fromdrug-induced signs and symptoms. According to the screen-ing study, the ethanolic extract ofKaempferia galanga hasbeen found to cause a dose-related decrease in motor activ-ity and respiration, and a loss of screen grip and analgesia.These effects, therefore, suggest a CNS depressant activity.However, a loss of the righting reflex has not been observed.This indicates that the extract possesses some selectiveCNS depressant or sedative activity. The loss of screen gripcan be taken as an indication for skeletal muscle relaxantactivity, of which, the site of action can be peripheral (atneuromuscular junction) or central. The extract tested shows
Table 5Blood chemistry values of rats treated with ethanolic extract ofKaempferia galanga in a subacute toxicity
Data are expressed as mean± S.D., n = 8.a A separate group was administered at 100 mg/kg daily for 28 days followed by no treatment for 14 days.∗ Significantly different from the control (P < 0.05).
an analgesic activity at the highest dose (2000 mg/kg). It ispossible that the CNS depression and paralysis of skeletalmuscle, which are caused by the extract, tend to modify theresponse to pain stimulation.
Following a dose of 2000 mg/kg of ethanolic extract, givenintraperitoneally, two of four rats died within 24 h. Markedrespiratory depression (decrease respiratory rate and irreg-ular breathing) was found to occur before death. This res-piratory failure could be due to central or peripheral action.Centrally, the failure could be due to a CNS depression,whereas, peripherally the failure could possibly be due toan inhibitory action at the neuromuscular junction.
Based on the results in an acute toxicity study, it wasconcluded that a dose of 5 g/kg of ethanolic extract of
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Kaempferia galanga, given orally, appeared to be prefer-ably non-toxic. This is in accordance with the study ofMokkhasmit et al. (1971), who administered 50% ethanolicextract ofKaempferia galanga to groups of Yenken DenkenTokyo mice. They reported that 10 g/kg ofKaempferiagalanga extract, which was administered by oral and subcu-taneous routes, did not produce any toxic symptoms. Theremust be a point, however, at which it can be concluded thata test substance is practically non-toxic or non-lethal afteran acute exposure. This test limit for acute oral toxicityis generally considered to be 5.0 g/kg body weight. If nomortality is observed at this dose level, a higher dosage isgenerally not necessary (Hayes, 1989).
In a subacute toxicity study, it appeared that the ethano-lic extract ofKaempferia galanga at a dose of 25 mg/kg didnot produce any marked changes in both male and femalerats, as evidenced by the parameter examined. A dose of 50and 100 mg/kg, which was administered for 28 days, causeda decrease in the lymphocyte count. Although the decreasewas statistically significant, it might not have had clinicalrelevance. The decrease in lymphocyte count was still ev-ident after 14 days in males that were maintained withouttreatment, thus suggesting a lack of reversibility in this pa-rameter. It is difficult to explain why, upon the removal ofK galanga extract administration from rats, the neutrophilcount was increased in the male ones only. However, bac-terial infection may have occurred during the experimentalperiod. In the same way, the significant changes in some ofthe blood chemistry parameters in both male (BUN, proteinand Alk-P) and female rats (AST), which had been with-drawn from treatment for 14 days, were very doubtful. Eventhough the changes noted were slight, they were statisticallydifferent from the control. It is important to stress that thesignificant changes seen were mild in nature, but it shouldbe borne in mind that these changes did occur. However, atpresent the clinical relevance of the findings noted upon theremoval ofKaempferia galanga extract are not known andwarrants a more extensive study.
The dermal irritation test must be performed for furtherhuman safety in case of substance exposure. Animals havebeen used to assess dermal irritation by the observation ofvisible changes ranging from erythema and edema to corro-sion and ulceration. Information that derives from tests fordermal irritation serves to identify the possible risk to thepopulation who use and are exposed to substances such asmosquito repellents. In the present study, the treatment ofhexane fraction produced no signs of irritation. It also pro-duced no irritation in human volunteers in both laboratoryand field repellent studies (Choochote et al., 1999). Thisfinding indicates that hexane fraction ofKaempferia galangacan be categorized as a ‘non-irritant’.
It can be concluded thatKaempferia galanga demon-strates less toxicity, but it is considered as an effectivebotanical insecticide with high larvicidal activity and a pro-tective effect against mosquitoes (Choochote et al., 1999).Chu et al. (1998)found thatKaempferia galanga extract ex-
hibited amebicidal activity in vitro against three species ofAcanthomoeba: Acanthamoeba culbertsoni, Acanthamoebacastellanii, and Acanthamoeba polyphaga, that were notlytic for normal macrophage cultures. Similary, the rhizomeextract ofKaempferia galanga exhibited Epstein-Barr virusactiviation inhibitory activity that had no cytotoxicity effectin Raji cells (Vimala et al., 1999). However, a subchronictoxicity test should have been conducted to establish the ad-verse effects of a repeated response toKaempferia galangaextract. Moreover, it is conceivable that humans and ani-mals may receive or be exposed to this substance throughapplied use or by chance. More research in the productionand use of this substance should be encouraged in orderto provide an additional weapon in the overall strategy ofvector and disease control.
Acknowledgements
The authors are thankful to Dean of Faculty of Medicinefor providing necessary research facilities. Acknowledgmentis extended to the Chulabhorn Research Institute for financialsupport, and the Faculty of Medicine Endowment Fund forResearch Publication for helping to defray the publicationcost.
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Journal of Ethnopharmacology 90 (2004) 367–374
Inhibition of sustained repetitive firing in cultured hippocampal neuronsby an aqueous fraction isolated fromDelphinium denudatum
a Department of Neurology, Medical College of Virginia, Virginia Commonwealth University,P.O. Box 980599, Richmond, VA 23298-0599, USA
b Department of Biochemistry and Molecular Biophysics, Medical College of Virginia, Virginia Commonwealth University,P.O. Box 980599 Richmond, VA 23298-0599, USA
c Department of Pharmacology and Toxicology, Medical College of Virginia, Virginia Commonwealth University,P.O. Box 980599, Richmond, VA 23298-0599, USA
d H.E.J. Research Institute of Chemistry, Third World Centre for Research on Chemical Sciences,University of Karachi, Karachi 75270, Pakistan
Received 3 May 2002; received in revised form 28 September 2002; accepted 20 October 2003
Abstract
In this report we investigated the effects of the aqueous fraction (AF) isolated fromDelphinium denudatumon sustained repetitive firing incultured neonatal rat hippocampal pyramidal neurons. Blockade of SRF is one of the basic mechanisms of antiepileptic drugs (AED) at thecellular level.
The effects of aqueous fraction (0.2–0.6 mg/ml) were compared with the prototype antiepileptic drug, phenytoin (PHT). Using the whole cellcurrent-clamp technique, sustained repetitive firing was elicited in neurons by a depolarizing pulse of 500 ms duration, 0.3 Hz and 0.1–0.6 nAcurrent strength. Similar to phenytoin, aqueous fraction reduced the number of action potentials (AP) per pulse in a concentration-dependentmanner until no action potentials were elicited for the remainder of the pulse. There was a corresponding use-dependent reduction in amplitudeandVmax (velocity of upstroke) of action potentials. TheVmax and amplitude of the first action potential was not affected by phenytoin,while aqueous fraction exhibited concentration-dependent reduction. At 0.6 mg/ml aqueous fraction reducedVmax to 58–63% and amplitudeto 16–20% of the control values. The blockade of sustained repetitive firing by aqueous fraction was reversed with hyperpolarization ofmembrane potential (−65 to−75 mV) while depolarization of membrane potential (−53 to−48 mV) potentiated the block.
Effective and safer therapy of epilepsy with currentlyavailable antiepileptic drugs (AEDs) is an unfulfilled chal-lenge (Heinemann et al., 1994). Conventional antiepilepticdrugs such as phenytoin (PHT), carbamazepine (CBZ), val-proic acid (VPA), phenobarbital, and benzodiazepines andnewer antiepileptic drugs such as gabapentin, lamotrigine,oxcarbazepine, remacemide, tiagabine, topiramate and viga-
batrin are all synthetic compounds. Antiepileptic drug ther-apy is associated with dose-related and chronic toxicity,adverse effects on cognition and behavior and teratogeniceffects (Smith and Bleck, 1991; Holmes, 1993; Devinsky,1995; Mattson, 1995; Samren et al., 1997). Natural prod-ucts have contributed significantly in the modern drug dis-covery process and are a rich source of novel compounds tosearch for new drugs with better efficacy and a safer profile(Farnsworth, 1994; Cragg et al., 1997).
Sustained repetitive firing of action potentials in responseto a depolarizing pulse is a non-synaptic voltage sensitiveproperty of central nervous system neurons (Macdonald
368 M. Raza et al. / Journal of Ethnopharmacology 90 (2004) 367–374
et al., 1985). Inhibition of sustained repetitive firing ofneurons in culture is one of the basic mechanisms of ac-tions of currently available antiepileptic drugs at the cel-lular level (Macdonald and Kelly, 1995; Rho and Sanker,1999). Antiepileptic drugs effective in the therapy of gen-eralised tonic-clonic and partial seizures such as pheny-toin, carbamazepine, valproic acid and status epilepticussuch as diazepam (DZP) limit sustained repetitive firing inuse-dependent and voltage-dependent manner (DeLorenzo,1988; Macdonald, 1989; White, 1999).
Among newer antiepileptic drugs, felbamate (White et al.,1992), gabapentin (Wamil and McLean, 1994), lamotrig-ine (Cheung et al., 1992), oxcarbazepine (Wamil et al.,1994), remacemide (Wamil et al., 1996) and topiramate(DeLorenzo et al., 2000) also limit sustained repetitive firingin use-dependent manner.
Phenytoin, carbamazepine, and valproic acid limit sus-tained repetitive firing of action potentials at the clinicallyrelevant therapeutic concentrations (Macdonald, 1989). DZPand other benzodiazepines (Skerrit et al., 1984a,b; McLeanand Macdonald, 1988b) and barbiturates (McLean andMacdonald, 1984) limit sustained repetitive firing at thetherapeutic concentrations attained during the treatment ofstatus epilepticus. Blockade of sustained repetitive firing ina use-dependent manner provides an electrophysiologicalbasis for preventing seizure discharge from an epileptogenicfocus in the brain by antiepileptic drugs that do so withoutinterrupting normal communication between neurons.
Delphinium denudatumis a traditional herb used forthe treatment of epilepsy in the sub-continent, especiallyPakistan (Said, 1970). We found anticonvulsant activity inaqueous fraction (AF) isolated from this medicinal herbon the maximal electroshock test (MEST), subcutaneouspentylenetetrazole and subcutaneous bicuculline tests andinhibitory effects on sustained repetitive firing were re-ported in preliminary form previously (Raza et al., 2001).In this report we investigated the effects of aqueous frac-tion on sustained repetitive firing in hippocampal pyramidalneurons in culture and compared the results of the effectsof aqueous fraction on sustained repetitive firing with theprototype antiepileptic drug, phenytoin.
2. Material and methods
2.1. Preparation of hippocampal cell cultures
Neuronal cultures were prepared from hippocampal tissueisolated from 2-day-old neonatal Sprague–Dawley rat pups(Harlan Sprague–Dawley) by a modification of the methodof Banker and Cowan (1977)as described bySombati andDeLorenzo (1995). Briefly, hippocampal cells were pre-pared from 2-day-old rat pups and grown on a confluent hip-pocampal astroglial feeder layer. Astrocytes were preparedfrom 2 day old pups by the method ofAbney et al. (1981).The glial cultures were maintained for two weeks in 35 mm
culture dishes (Costar), coated with 10�g/ml poly l-lysine(MW 30,000–70,000) and fed twice weekly with MEM(Minimal Essential Medium), 10% fetal bovine serum,2 mM l-glutamine and 10 mM glucose. After confluence(usually 4 days), the glial cultures were exposed to 5�Mcytosine arabinoside to inhibit further proliferation. Oneday prior to neuronal plating, the glial feed was replacedby N3-supplemented neuronal feed which contained 25 mMHEPES (N-2-hydroxyethylpiperazine-N-2-ethansulfonicacid) buffer (pH 7.4), 2 mM glutamine, 5�g/ml insulin,100�g/ml transferrin, 100�M putrescine, 30�M sodiumselenite, 20�M progesterone, 1 mM sodium pyruvate,0.1% ovalbumin, 20�g/ml T3 and 40�g/ml corticosterone.Hippocampal cells were plated at a density of 7.5 × 105
cells/dish onto the confluent glial bed. Cultures were main-tained at 37◦C under 5% CO2 and 95% air and fed twofeedings/week (two half media change) with neuronal feed.The glutamine, MEM and HEPES buffer were obtainedfrom GIBCO (Gaithersburg, MD).
2.2. Experimental apparatus and electrophysiologicalstudies
Neuronal cultures between 12 and 17 days old were usedfor electrophysiological studies. Whole cell current-clamprecordings (Hamill et al., 1981) were carried out with thinborosilicate glass (WPI, Sarasota, FL) micropipette elec-trodes. Prior to recording, the culture medium was replacedwith extracellular recording solution. Glass micropipetteelectrodes (4–8 M� resistance) were pulled in two stageson Flaming-Brown P-80 C electrode puller (Sutter Instru-ment Co.) and fire polished with microfurge (Narishige,Japan) and filled with the pipette solution.
Cultures were transferred to the moveable stage of an in-verted microscope (Swift Instruments, USA) equipped withphase contrast optics and micro manipulators (Narishige,Japan) to patch clamp the cells with microelectrode underdirect visual control. Recordings were amplified using anAxopatch 200 A amplifier (Axon Instruments, Burlingame,CA) and filtered with a four-pole bessel filter before digi-tization. All data were simultaneously visualised on a Stor-age Oscilloscope V-134 (Hitachi) and stored on a VCR afterdigitization (at 44 kHz) with a VR-10 Digital Data Recorder(Instrutech Corp.). Data were subsequently offline analysedby Strathclyde Electrophysiology Software WCP Version1.2 (Dagan Corporation, Minneapolis, MN) and played backon a chart recorder (Astro-Med Dash IV, Warwick, RI).
Changes in control and testing solutions were accom-plished through modified 6 or 8 barrel sewer pipe per-fusion technique (Gibbs et al., 1996) in which recordingsolution or different concentrations of aqueous fraction(0.2–0.6 mg/ml), or phenytoin (20–40�M) flowed out ofparallel teflon tubes (0.2 mm internal diameter) at a constantrate (0.5 ml/h) in a laminar pattern. Aqueous fraction orphenytoin were dissolved in DMSO at a final concentrationof 0.05% in recording solution. The recording solution was
M. Raza et al. / Journal of Ethnopharmacology 90 (2004) 367–374 369
placed in conical tubes attached with teflon tubing imme-diately before the experiments. Higher concentrations wereused first and upon detection of activity, the concentrationwas reduced gradually to study concentration-effect rela-tionships. Rapid (∼=500 ms) and complete solution changeswere conducted by moving the tube assembly laterally inrelation to the neuron under study at a distance of approxi-mately 50–75�m. This technique allowed perfusion with-out any cross contamination or flow artifact. The recordingsolution was continuously aspirated from chamber at a rateof 0.5 ml/min. Hippocampal pyramidal cells with restingmembrane potential greater than−50 mV were selectedfor study. Pipette capacitance and access resistance werecompensated about 60%.
2.3. Experimental solutions
Extracellular (recording) solution (in mM): 145 NaCl, 2.5KCl, 10 HEPES, 1 MgCl2, 2 CaCl2 and 10d-glucose. Toblock excitatory and inhibitory synaptic transmission, 0.5–1kynurenic acid and 50�M bicuculline were added. The pHwas adjusted to 7.25 with 1 M NaOH and the osmolarityto 325 mOsm with sucrose. Pipette (intracellular) solution(in mM): 140 K+ gluconate, 1 MgCl2, 0.5 CaCl2 and 10HEPES. Cellular ATP reconstitution system consisting of2 mM Mg-ATP, 20 mM phosphocreatinine and 50 U/ml cre-atinine phosphokinase were also added. The pipette solutionwas filtered with 0.2�M pore size filters (Nalgene). Thissolution’s pH was adjusted to 7.25 with KOH and osmolar-ity to 325 mOsm with sucrose.
2.4. Studies on sustained repetitive firing
Sustained repetitive firing was induced by S-95 Tri-levelStimulator (MSC, Greenvale, NY) by depolarizing pulses of500 ms duration, 0.3 Hz and increasing current strength from0.1 to 0.6 nA through the micropipette. The duration andfrequency of depolarizing pulses were kept constant for allexperiments. Current strength was gradually increased untilthe cell under study exhibited a suitable number of actionpotentials throughout the duration of the depolarizing pulse.All data were analyzed as described earlier. The results wereexpressed as changes inVmax (velocity of upstroke of theAP) and amplitude of each action potential. Values ofVmaxwere taken as an index of inward sodium current duringgeneration of action potentials (McLean and Macdonald,1986a).
Hippocampal pyramidal neurons exhibited different pat-terns of sustained repetitive firing with varied numbers of ac-tion potentials per 500 ms depolarizing pulse, as the cultureswere prepared from the entire hippocampus. Neurons fromdifferent subfields of the hippocampus vary somewhat intheir response to a depolarizing pulse (Johnston and Amaral,1998). Cells eliciting uniform patterns of action potentialsthroughout the 500 ms pulse (less then 20% of the total cellsrecorded) were selected for studies. In all the experiments
with sustained repetitive firing, controlVmax (the maximalrate of rise of action potentials that is the index of inwardsodium current) values were found to be less than 160 V/s.
2.5. Chemicals
All the chemicals were purchased from Sigma ChemicalCo. (St. Louis, MI) unless otherwise specified.
2.6. Collection of plant and preparation of aqueous fraction
Delphinium denudatumwas collected from its local habi-tat (Swat) and identified by Prof. Mustafa Shameel (BotanyDepartment, University of Karachi). A voucher herbariumspecimen (AN-96) was deposited in the herbarium of theBotany Department. The aqueous fraction (AF) was pre-pared as described previously (Raza et al., 2001). Briefly,the roots (8 kg) were dried in air, crushed to coarse powderand percolated in ethanol (95%) for 6 days. The solventwas evaporated under reduced pressure and the resultantethanolic extract was dissolved in distilled water and de-fatted with hexane. The aqueous layer was then furtherextracted with chloroform at pH 1.0, 7.0 and 12.0. Theremaining water-soluble aqueous fraction (450 g) was usedfor the experiments.
2.7. Statistics
Statistical significance of results was evaluated by theStudent’st-test with sigma plot version 5. AP value of lessthan 0.002 was considered significant.
3. Results
3.1. Effect of phenytoin on sustained repetitive firing
Phenytoin blocked sustained repetitive firing in a use-dependent and voltage-dependent manner. Representativecurrent clamp recordings from a hippocampal neuron il-lustrating the concentration-dependent effect of phenytoin(20–40�M) are shown inFig. 1A. The effects of phenytoin(20–40�M) on the amplitude and number of action poten-tials of the same neuron are shown inFig. 2A. In all thecells (n = 11) phenytoin had no significant effect on theamplitude of the first action potential. Phenytoin exhibiteduse-dependent reduction in amplitudes of the subsequentaction potentials that also corresponded with reduction inVmax. Fig. 3A illustrates the effect of phenytoin (20–40�M)on the Vmax of the action potentials from the same neu-ron shown inFig. 2A. Phenytoin did not exhibit significantconcentration-dependent reduction inVmax and amplitude ofthe first action potentials (AP #1).
The voltage-dependent block of sustained repetitive fir-ing by phenytoin was seen in 10 out of 11 cells. When thecells were hyperpolarized from−65 to −75 mV, the lim-
370 M. Raza et al. / Journal of Ethnopharmacology 90 (2004) 367–374
Fig. 1. (A) Concentration-dependent limitation of sustained repetitive firing of a hippocampal neuron by phenytoin. Current-clamp recordings froma singleneuron at different concentrations of phenytoin. The action potentials (APs) were evoked at a depolarizing current strength of 0.4 nA. The membranepotential (Em) of the neuron was−58 mV. Reduction in number of action potentials as well as amplitude is evident as the concentration of phenytoin wasincreased and corresponds to progressive reduction inVmax (Fig. 3) in the same neuron. Washout period= 1 s. (B) Concentration-dependent limitationof sustained repetitive firing in a hippocampal neuron by aqueous fraction. The Current-clamp recordings from a single hippocampal neuron are shownto illustrate the limitation of sustained repetitive firing at different concentrations of aqueous fraction. sustained repetitive firing was elicited by 500 msdepolarizing pulses of 0.5 nA. TheEm of above neuron was held at−61 mV throughout the recording.
itation of sustained repetitive firing was reduced and re-versed, causing the number of action potentials per pulseto increase. Depolarization of the membrane potential from−53 to −48 mV reduced the number of action potentialsper pulse and slowed the recovery of sustained repetitivefiring effect to the resting values of the membrane poten-tials. Phenytoin (50�M) had no significant effect on restingmembrane potential (−56.3±4.2 mV versus a control valueof −60.4 ± 5.6 mV (n = 8).
Fig. 3. (A) Concentration-dependent effect of phenytoin onVmax during sustained repetitive firing. The data present the mean± S.E.M. obtained from 50identical records of action potentials from the same neuron shown inFigs. 1A and 2A. The use-dependent limitation ofVmax by phenytoin was paralleledby a progressive reduction ofVmax until action potential generation ceased. The reduction in the amplitude of action potentials (Fig. 2A) correspondedto the reduction inVmax. Phenytoin had an insignificant effect on theVmax of action potential #1. In control solutions (pre-PHT and wash) the rapidreduction ofVmax after action potential #1 was due to depolarization. (B) Concentration-dependent effects of aqueous fraction onVmax during sustainedrepetitive firing. The data present the mean± S.E.M. obtained from 53 records of action potentials from the same hippocampal neuron in culture shownin Figs. 1B and 2B. In the control solution the depolarizing pulse of 500 ms duration at 0.5 nA current strength evoked 13 action potentials with areduction inVmax due to depolarization. Aqueous fraction (0.2–0.6 mg/ml) progressively decreased theVmax until no action potentials were elicited.
3.2. Effect of aqueous fraction on sustained repetitive firing
Results of the studies on the effects of aqueous fraction(0.2–0.6 mg/ml) on sustained repetitive firing in hippocam-pal pyramidal neurons are shown inFigs. 1B, 2B, 3B and 4.
Aqueous fraction at a concentration of 0.6 mg/mlhad no significant effect on resting membrane potential(−58.2 ± 4.1 mV versus control−60.8 ± 3.9 mV) ofpyramidal neurons (n = 11). Current clamp recordings
M. Raza et al. / Journal of Ethnopharmacology 90 (2004) 367–374 371
Fig. 2. (A) Effect of phenytoin (PHT) on the amplitude of the action potentials during sustained repetitive firing. The data present the means± S.E.M.
obtained from 50 identical records of action potentials at each concentration of phenytoin (20, 30 and 40�M) and control (pre-PHT and wash) of thesame neuron shown inFig. 1. Phenytoin had no effect on the amplitude of AP#1. (B) Effect of aqueous fraction (AF) on the amplitude of action potentialsduring sustained repetitive firing. The data present the mean± S.E.M.s obtained from 70 identical records of action potentials at each concentration ofaqueous fraction and control (pre-AF and wash) of the same neuron shown inFig. 1B. There was a progressive reduction in the amplitude of actionpotentials until no action potential was fired for the remainder of the depolarizing pulse. Aqueous fraction also reduced the amplitude of action potential#1 in a concentration dependent manner.
372 M. Raza et al. / Journal of Ethnopharmacology 90 (2004) 367–374
Fig. 4. Inhibitory effects of aqueous fraction (0.6 mg/ml) on sustainedrepetitive firing in a hippocampal pyramidal cell at increasing currentstrength of the depolarizing pulse. The number of action potentials in-creased initially with sequentially larger depolarizing pulses (0.2 and0.3 nA) and ceased near the end of the 500 ms pulse at 0.4 nA. Aque-ous fraction (0.6 mg/ml) exerted a potent blockade of sustained repetitivefiring at all the higher current strengths (Em = −57 mV).
showed a concentration-dependent effect of aqueous frac-tion (0.2–0.6 mg/ml) on the number and amplitude of actionpotentials fired during the pulse (Fig. 1B). At the concentra-tion of 0.2 mg/ml the amplitudes of action potentials beganto reduce progressively. This effect was also evident at in-creasing strengths of depolarizing current pulses from 0.2to 0.4 nA, as shown from current clamp recordings from an-other hippocampal pyramidal cell (Fig. 4).
The amplitude of the action potential #1 was slightly lowerthan the control values at the concentration of 0.2 mg/ml andshowed a concentration-dependent reduction (Fig. 2B). Inall the cells studied, aqueous fraction at the concentration of0.6 mg/ml reduced the amplitude of action potential #1 to16–20% of the control values. In addition to the reduction ofamplitude, at the concentration of 0.4–0.6 mg/ml, the num-ber of action potentials gradually reduced to approximately60% of the pre-aqueous fraction values. At the concentrationof 0.6 mg/ml a rapid decline in the number and amplitudeof successive action potentials was seen until firing ceasedfor the remainder of the pulse. In all the cells studied theamplitude returned to pre-aqueous fraction values as soonas the cells were superfused with recording solution.
The Vmax values of the action potential #1 followed aprogressive concentration-dependent reduction correspond-ing to the amplitude of action potentials (Fig. 3B). TheVmax of action potential #2 decreased abruptly due to de-polarization and subsequent spikes were fired at a steadystate lower level ofVmax beginning from the concentra-tion of 0.2–0.4 mg/ml. At the concentration of 0.6 mg/ml,the Vmax of the first spike was significantly lower thanthe control value. Due to the sharp decline ofVmax, onlytwo to four spikes were elicited and the generation ofaction potentials failed for the remainder of the depolariz-ing pulse (seeFig. 3B). In all the cells studied, theVmaxof the action potential #1 was observed to be between78 and 85% of the control values at the concentration of0.2 mg/ml, which showed a concentration-dependent re-duction. TheVmax of action potential #1 was reduced to67–74% at the concentration of 0.4 mg/ml and 58–63% atthe concentration of 0.6 mg/ml of aqueous fraction. It wasobserved that cells continued to fire almost similar num-bers of action potentials at each concentration of aqueousfraction.
In all the cells studied for sustained repetitive firing, re-duction in the number of action potentials per pulse by AFwas found to be dose dependent and significantly reducedat the concentration of 0.6 mg/ml. This is comparable to theaction of 40�M phenytoin (Fig. 5). The voltage-dependentlimitation of sustained repetitive firing by aqueous fractionat the concentration of 0.6 mg/ml was observed in 11 out of13 cells. Hyperpolarization of cells at membrane potentialsof −75 to−85 mV resulted in restoration of sustained repet-itive firing and the number of action potentials per pulse in-creased. Depolarization to−48 mV potentiated the block ofsustained repetitive firing by aqueous fraction as evidencedby a decrease in the number of action potentials per pulseand by slowed the recovery of sustained repetitive firingblocked by aqueous fraction.
4. Discussion
The results of the present study demonstrate that aqueousfraction is effective in producing a use-dependent or voltage-dependent block of sustained repetitive firing in hippocam-pal neurons in culture. This activity on sustained repetitivefiring was similar to the well-characterized effects of pheny-toin on sustained repetitive firing. Thus, aqueous fractiondemonstrated anticonvulsant activity identical to phenytoinat the cellular level in blocking sustained repetitive firing.Since a use-dependent and voltage-dependent block of sus-tained repetitive firing mediates the anticonvulsant proper-ties of several well-characterized anticonvulsant agents, theresults indicate that the aqueous fraction has the similarmechanism of action.
The use-dependent block of sustained repetitive firingsuggests that drugs such as local anaesthetics, phenytoinand carbamazepine, preferably bind to the inactive state of
M. Raza et al. / Journal of Ethnopharmacology 90 (2004) 367–374 373
Fig. 5. Effect of phenytoin (40�M) and aqueous fraction (0.6 mg/ml) on the number of action potentials per 500 ms of a depolarizing pulse duringsustained repetitive firing. Data provide the mean± S.E.M. and represent values obtained from all the neurons recorded for phenytoin (n = 11) andaqueous fraction (n = 19). ∗Significant (P < 0.002) reduction in number of action potentials by aqueous fraction and phenytoin.
sodium channels (Courtney, 1975). The modulator receptorhypothesisHille (1992)suggests that the compounds bind todifferent states (resting, active and inactive) of the sodiumchannels with different affinities. Antiepileptic drugspreferably bind to the inactivated state of sodium channel(Catterall, 1999) and thus gradually reduceVmax and theamplitude of subsequent action potentials by slowing therate of recovery from inactivation till no action potentialsare elicited for the remaining duration of the depolariz-ing pulse (Courtney and Etter, 1983). In use-dependentblock, the initial action potential (AP #1) is unaffected byantiepileptic drugs such as phenytoin, carbamazepine &valproic acid (Macdonald, 1989; McLean and Macdonald,1983, 1986a,b), since the sodium channels are in the restingstate at the negative resting membrane potential. Our resultsshowed thatVmax values of the first action potential werereduced to 58–63% of the control values at 0.6 mg/ml ofaqueous fraction, suggesting the presence of compounds inaqueous fraction that in addition to interacting with the in-active state of the sodium channel, are also interacting withthe resting state. Thus, the effect of aqueous fraction on theamplitude of the initial action potential was different fromphenytoin, which had insignificant effect on amplitude andVmax of action potential #1.
The effects of aqueous fraction in producing a voltage-dependent block of sustained repetitive firing are consistentwith the effects of currently available antiepileptic drugsphenytoin, carbamazepine, valproic acid (McLean andMacdonald, 1986a,b; Macdonald and McLean, 1986), in-dicating that aqueous fraction preferentially interacts withvoltage-gated sodium channels that change from open to in-active state during depolarization. Presumably, compounds
present in aqueous fraction appear to potentiate the block ofsustained repetitive firing at depolarized values of neuronalmembrane potential (−48 mV). Thus, similar to phenytoin,aqueous fraction can reduce neuronal excitability by in-creasing the inhibition of the sodium channel in depolarizedtissue of the epileptic focus.
Antiepileptic drugs that inhibit sustained repetitive fir-ing also exhibit strong suppression of tonic hind limb ex-tension in MEST (Meldrum, 1996). This is related to abil-ity of antiepileptic drug to prevent the spread of seizuredischarges during epileptic seizures. Aqueous fraction hasshown strong anticonvulsant activity in sustained repetitivefiring and we have recently found very strong suppressionof hind limb tonic extension in MEST model by a purifiedfraction of aqueous fraction (Raza et al., 2001), suggestingthat compounds present in aqueous fraction are potent an-ticonvulsants and may lead to discovery of a new class ofantiepileptic drugs.
Acknowledgements
M.R. was a recipient of research fellowship from theFriends Education and Medical Trust. This research was sup-ported by National Institute of Neurological Disorders andStroke grants RO1 NS23350 and PO1 NS25630 to R.J. De-Lorenzo, the Nathan and Sophie Gumenick NeuroscienceResearch Fund, and the Milton L. Markel Alzheimer’s Dis-ease Research Fund. The experiments were performed ac-cording to guidelines of Institutional Animal Care and UseCommittee of the Medical College of Virginia, VirginiaCommonwealth University.
374 M. Raza et al. / Journal of Ethnopharmacology 90 (2004) 367–374
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Songphol Phadungpata, Banchong Chaoraia, Raywadee Butrapornba Department of Medical Sciences, Medicinal Plant Research Institute, 88/7 Soi Bamrasnaradura, Tivanond Road, Nonthaburi 11000, Thailand
b Department of Medical Sciences, National Institute of Health, 88/7 Soi Bamrasnaradura, Tivanond Road, Nonthaburi 11000, Thailand
Received 23 October 2002; received in revised form 19 September 2003; accepted 20 October 2003
Portulaca grandiflora Hook. (Portulacaceae) (Backerand Bakhuizen Van Den Brink, 1963; Liu and Chen,1976) is a succulent plant. Its stems, mostly in branches,are approximately 10–30 cm high. Leaves, with sizes ofabout 12–35 mm in length and about 1–4 mm in width,are linear-subulate, thick, fleshy and spirally arranged. Thepetiole is mostly very short with axillary hairs. Its colorfulflowers are 2–3 cm across with conspicous stamens, andarranged in a terminal capitulum which is surrounded bywhorls of leaves. Two sepals are about 6 mm long with avery small apical keel. Its petals are broadly obovate, ratherdeeply notched, and they are in red, pink, or purple. Fruitsare subglobose of 4–6 mm in diameter. In the pericarp, acircumscissile suture is formed, along with articulation andthe fruit bursts. Seeds are very small and shining. The plantgrows throughout Thailand and is usually cultivated as anornamental annual herb.
According to oriental traditional medicine,Portulacagrandiflora is used for the relief of sore throat and skin rash.
It is also used for detoxification.Portulaca grandiflora wasreported for its effect on hepatitis B surface antigen (Zhengand Zhang, 1990). In addition, antimutagenic effect on themutation induced by alfatoxin B1 and cyclophosphamide inmice was demonstrated (Liu et al., 1990).
Owing to its pharmacological effects as well as increas-ing trend in using herbs as alternative remedies in severaldeveloping countries including Thailand, the present studywas to perform a 6-month toxicity ofPortulaca grandiflorawater extract in rats in order to obtain scientific informationon its safety for consumption.
2. Materials and methods
2.1. Preparation of Portulaca grandiflora
The aerial part ofPortulaca grandiflora Hook. (Portula-caceae) were collected from the central part of Thailand.The botanical identification were determined using thedescription of Backer (1963) and Liu (1976), and com-pared with the authentic specimens at two herbaria, i.e., theBangkok Herbarium (BK) of the Department of Agricultureand the Forest Herbarium (BKF) of the Royal Forest Depart-
376 P. Chavalittumrong et al. / Journal of Ethnopharmacology 90 (2004) 375–380
ment, Ministry of Agriculture and Cooperative, Thailand.A voucher specimen ofPortulaca grandiflora (Bansiddhi43-12) were deposited at the Botanical Section, MedicinalPlant Research Institute, Department of Medical Sciences,Thailand. The plants were washed, then dried in an oven at40◦C and grounded.
Fifteen grams of groundedPortulaca grandiflora wereextracted twice with 30 ml of distilled water using a refluxmethod for 2 h. The filtrate was then evaporated at low pres-sure to obtain dried extract of 3.14 g. The dried extract wasdissolved in distilled water to concentrations needed for thestudy.
2.2. Treatment of the animals
Sixty male Wistar rats weighing 200–220 g and 60 femalerats weighing 160–180 g from the National Laboratory An-imal Center, Mahidol University, Nakornpathom province,were used in the study. All animals were housed in the an-imal facility of the Department of Medical Sciences. Thetemperature in an animal room was kept at 25± 1◦C with60% relative humidity. The animals were allowed to havefree access to food and clean water. The study was approvedby an Institutional Animal Care and Use Committee, De-partment of Medical Sciences.
2.3. Chronic toxicity study
The Wistar rats of each sex were randomly divided intofive groups containing 12 animals per sex. Group 1 (watercontrol) received water 5 ml/kg per day and the groups 2–5were orally given the water suspension ofPortulaca grandi-flora extract at doses of 10, 100, 1000 and 1000 mg/kg perday, respectively. Body weights and food intake were mea-sured weekly and the animals were observed for signs ofabnormalities throughout the study. At the end of 6 months,the one to four groups of rats were fasted for 18 h, thenanesthetized with ether and sacrificed by drawing bloodsamples from the inferior vena cava. Blood was collectedfor hematological and biochemical examinations. The fifthgroup of rats (1000R) was further cared without givingthe extract for another 14 days before sacrificing and theirblood was also collected for hematological and biochemicaltests.
Hematological analysis was performed using an automatichematological analyser (Cell dyne 3500, Abbott). Hemato-logical parameters measured were white blood cell (WBC),neutrophil (%), lymphocyte (%), monocyte (%), eosinophil(%), basophil (%), red blood cell (RBC), hemoglobin, hema-tocrit (Hct), mean cell volume (MCV), mean corpuscularhemoglobin (MCH), mean corpuscular hemoglobin concen-tration (MCHC), red cell distribution width (RDW), platelet,mean platelet volume (MPV), plateletcrit (PCT) and plateletdistribution width (PDW).
Biochemical analysis of serum samples was performedusing an automatic chemistry analyser (Hitachi model 912,
The positions, shapes, sizes and colors of internal or-gans, namely, brain, heart, both kidneys and lungs, stomach,liver, intestine, spleen, urinary bladder, left and right adrenalglands, testis in male rats or ovary, uterus and mammarygland in female rats were visually observed for any signsof gross lesions. These organs were collected, weighed todetermine relative organ weights. The relative organ weightwas calculated using the following formula: relative organweight (kg) = (organ weight(g)/body weight(g)) × 1000.The organs were then preserved in 10% buffered formalinsolution. The organs were processed for histopathologicalstudies. Tissue slides were stained with hematoxylin andeosin and were examined by a pathologist.
2.4. Statistical analysis
Data were analyzed by an SPSS program version 9.0.The data were firstly tested for homogeneity of variance byLevene test. Bonferroni test was used for analysis of meanswhen the variance was homogenous, whereas Tamhane testwas used for comparisons of the means when there was nohomogeneity of the variance. Histopathological results wereanalyzed by Fisher’s Exact test. Statistical significance wasset atP < 0.05. Clinical signs and gross pathology were notstatistically analyzed.
3. Results
3.1. Effects on food intake and body weight
The food intake of all tested male groups was significantlylower than their control group for several weeks (data notshown). Among the female groups, significant difference inthe food intake was also demonstrated (data not shown).
We measured the body weights of all animals every weekthroughout the study. The final body weights were reportedin Tables 1 and 2. It was found that the body weights ofmale and female animals given the water extract ofPortu-laca grandiflora at any doses tested were not significantlydifferent from their controls.
3.2. Effects on actual organ weight and relative organweight
The actual and relative organ weights were weighed afterbeing sacrificed. No significant changes in the actual organweights were seen in all male and female rats given thewater extract ofPortulaca grandiflora as compared with thecontrol groups (data not shown).
P. Chavalittumrong et al. / Journal of Ethnopharmacology 90 (2004) 375–380 377
Table 1Relative organ weight of male rats orally givenPortulaca grandiflora extract for 6 monthsa
Parameter Dose ofPortulaca grandiflora extract (mg/kg body weight per day)
a Values are mean± S.D.∗ P < 0.05 as compared with the control group.
No significant differences in the relative weights of thebrain, the heart, the kidneys (left and right), the urinarybladder, the liver, the spleen, the lung, the adrenal glands (leftand right) and the testis of male rats treated withPortulacagrandiflora extract at the doses of 10, 100 and 1000 mg/kgper day were shown (Table 1). In female rats, there wereno significant differences in relative weights of any organscollected in the study as shown inTable 2.
3.3. Effects on hematological parameters
Significant changes in the number of white blood cell,neutrophil (%), lymphocyte (%), monocyte (%), basophil(%), eosinophil (%), the number of red blood cells,hemoglobin, Hct, MCV, MCH, MCHC, RDW, platelet,MPV, PCT and PDW were not observed inPortulaca
Table 2Relative organ weight of female rats orally givenPortulaca grandiflora extract for 6 monthsa
Parameter Dose ofPortulaca grandiflora extract (mg/kg body weight per day)
grandiflora extract-treated male rats at any doses testedas compared to the water control group (Table 3). At thedose of 100 mg/kg per day, the male group showed sig-nificantly decreased in basophil (%) as compared with thecorresponding control group.
The female rats givenPortulaca grandiflora at any dosesof the extract exhibited no significant changes in any hema-tological tests (Table 4).
3.4. Effects on blood chemistry
It was demonstrated that there was no significant al-teration in all biochemistry parameters tested in the malerats given 10 mg/kg per day of thePortulaca grandifloraextract (Table 5). The levels of bilirubin were significantlydecreased in the male rats givenPortulaca grandiflora
378 P. Chavalittumrong et al. / Journal of Ethnopharmacology 90 (2004) 375–380
Table 3Hematological results of male rats orally givenPortulaca grandiflora extract for 6 monthsa
Parameter Dose ofPortulaca grandiflora extract (mg/kg body weight per day)
at the doses of 100 and 1000 mg/kg per day. The levelof uric acid in the male group receiving 1000 mg/kg perday of the extract was significantly lower than the controlgroup.
The female rats givenPortulaca grandiflora at the doses of10, 100 and 1000 mg/kg per day had no significant changesin any biochemistry parameters tested (Table 6).
3.5. Effects on histopathology of internal organs
No remarkable changes in the internal organs of both maleand female rats receiving the extract at all doses tested werenoticed by the gross examinations. Among the male animals
receivingPortulaca grandiflora extract, fatty changes in theliver were found in 1, 2 and 4 of the rats receiving 10, 100and 1000R mg/kg per day ofPortulaca grandiflora extract,respectively (Table 7).
Histopathological examinations did not reveal anychanges in the internal organs of the female rats given thewater extract ofPortulaca grandiflora (Table 8).
4. Discussion
We studied toxicity ofPortulaca grandiflora in the Wistarrats in order to demonstrate whetherPortulaca grandiflora
P. Chavalittumrong et al. / Journal of Ethnopharmacology 90 (2004) 375–380 379
Table 5Results of blood chemistry of male rats orally givenPortulaca grandiflora extract for 6 monthsa
Parameter Dose ofPortulaca grandiflora extract (mg/kg body weight per day)
Each value represents numbers of rats with pathological finding/total numbers of rats examined.
was safe for a long-term consumption. We observed signsof abnormalities during the study and measured laboratoryparameters that might relate to alteration in functions of vitalorgans, for instance, liver and kidney. The Wistar rats wereorally given the water extract ofPortulaca grandiflora at thedoses of 10, 100 and 1000 mg/kg per day for 6 months. Toevaluate whether any significant changes were indeed dueto the toxicity ofPortulaca grandiflora, the 1000R groupsof male and female animals were added as recovery groupsin our study.
The percentage of basophil of the male rats receivingthe extract at the dose of 100 mg/kg per day was signifi-cantly lower than the control group. It was, however, withinits normal range, i.e., 0–1.5% (Smith, 1995) and was notdose-dependent. Therefore, it may indicate that the waterextract ofPortulaca grandiflora did not affect the hemato-logical parameters.
The amount of bilirubin measured in the male groupsorally givenPortulaca grandiflora extract at the doses of 100and 1000 mg/kg per day was significantly decreased but itwas within the normal level which ranges from 0–0.55 mg/dl(Gad, 1992). In addition, it was shown that uric acid, ofwhich its normal range is 1.2–7.5 mg/dl (Gad, 1992), wassignificantly lower in male rats receiving 1000 mg/kg per dayof the water extract, however, no significant change in thisparameter in the male recovery (1000R) group was demon-strated. It may suggest that there were no deleterious effectsof Portulaca grandiflora on the functions of any organs.
Histopathological findings showed fatty changes in thelivers of some male animals. The numbers of the animalswith this phenomenon were not increased as the dosesof the extract were increasing. In addition, no changes inhistopathology of other organs were observed. Therefore,our study suggests thatPortulaca grandiflora extract didnot produce any toxicity to internal organs examined.
In summary, there was no relevance of serious signsand significant changes in hematological, biochemicaland histopathological parameters that resulted from thelong-term consumption ofPortulaca grandiflora water ex-tract. It is, therefore, concluded thatPortulaca grandifloraat the doses given in our study did not have any remarkabletoxic effects in the rats.
Acknowledgements
The authors are grateful to Assistant Professor Dr. Som-nuek Jesdapatarakul of the Department of Pathology, Facultyof Medicine, Srinakharinwirot University, for histopatho-logical examinations of tissue slides. We acknowledgeDr. Busarawan Sriwanthana for her assistance with themanuscript preparation. We would also like to thank thestaffs of the Animal Facility of the Department of MedicalSciences for taking care of the animals throughout the study.
References
Backer, C.A., Bakhuizen Van Den Brink, R.C., 1963. Flora of Java, vol.1. Gronigen, N.V.P. Noordhoff, pp. 216–218.
Gad, S.C., 1992. The rat: pathology. In: Gad, S.C., Chengellis, C.P. (Eds.),Animal Models in Toxicology. Marcel Dekker, New York.
Liu, D., Yin, X., Wang, H., Zhou, Y., Zhang, Y., 1990. Antimutagenicityscreening of water extracts from 102 kinds of Chinese medicinal herbs.Zhongguo Zhong Yao Za Zhi, vol. 15. pp. 617–622, 640 (in Chinese).
Liu, T.S., Chen, C.H., 1976. Flora of Taiwan, Cutting. vol. 2. Taiwan,Epoch Publishing Co., Ltd., pp. 314–318.
Smith, J.E., 1995. Comparative hematology. In: Beutler, E., Lichtman,M.A., Coller, B.S., Kipps, T.J. (Eds.), Williams Hematology, 5th edi-tion, McGraw-Hill, New York, pp.77–85.
Zheng, M.S., Zhang, Y.Z., 1990. Anti-HBsAg herbs employing ELISAtechnique. Zhong Xi Yi Jie He Za Zhi 10, pp. 560–562, 518 (inChinese).
Journal of Ethnopharmacology 90 (2004) 381–387
Hepatotoxic effect of (+)usnic acid fromUsnea siamensis Wainio in rats,isolated rat hepatocytes and isolated rat liver mitochondria
(+)Usnic acid is the normal component of lichen cellsand is one of the most common and abundant lichen metabo-lites. This natural monobasic acid (Fig. 1) exists as (+) and(−) enantiomers which found to be more effective than thesynthetic derivatives. Usnic acid is well known as antibioticand endowed with several biological and physiological ac-tivities including antiparasitic, antimitotic, antiproliferative,anti-inflammatory, analgesic and antipyretic (Campanellaset al., 2002; Cocchietto et al., 2002; Vijayakumar et al.,2000; Kumar and Muller, 1999; Cardarelli et al., 1997;Lauterwein et al., 1995; Okuyama et al., 1995; Al-Bekairiet al., 1991). The particularly clinical relevance is its effectagainst a large variety of Gram-positive bacteria, irrespec-tive of their resistant phenotypes. Usnic acid inhibits thegrowth of multi-resistant strain ofStaphylococcus aureus,enterococci and mycobacteria without the side effect on
∗ Corresponding author.
oral saprophyte flora (Cocchietto et al., 2002). The (+)enantiomer is more active than (−) form, and exerts ratherselective activity againstStaphylococcus mutants (Ghioneet al., 1988). This natural compound has shown a greatrelevance in pharmacology and clinics. To be further de-veloped, more research is needed especially the details ofmechanism of its different actions and its toxicity. In thepresent investigation, hepatotoxic effect of (+)usnic acidwas studied in rats, isolated rat hepatocytes and isolatedrat liver mitochondria using carbon tetrachloride as the ref-erence hepatotoxin. (+)Usnic acid is derived fromUsneasiamensis Wainio, known in Thai as Foi-lom.
2. Materials and methods
2.1. Materials
(+)Usnic acid was kindly provided by Nijsiri Ruan-grungsi, Associate Professor in the Department of Pharma-cognosy, Chulalongkorn University.
382 P. Pramyothin et al. / Journal of Ethnopharmacology 90 (2004) 381–387
Fig. 1. Structure of (+)usnic acid (C18H16O7).
2.2. Test animals
Male Wistar albino rats weighing 200–250 g were ob-tained from National Laboratory Animal Center, MahidolUniversity. They were allowed free access of standard ro-dent food and tap water throughout the investigation.
2.3. Hepatotoxic study in rats
Rats were divided into four groups of eight animals each.The control groups were given saline (0.1 ml per day, i.p.)or DMSO (0.1 ml per day, i.p.) for 5 days. The test groupswere treated with (+)usnic acid (dissolved in DMSO) at thedoses of 50 or 200 mg/kg per day, intraperitoneally for 5days. At the end of 5 days treatment, blood sample weretaken for the measurement of serum transaminase activity(serum AST, ALT), livers were exposed for histopathologicalexamination.
2.3.1. MicroscopyElectron micrographs were performed using transmission
electron microscope (TEM).Liver slices (in 1 mm3) were fixed with 1–2% OsO4 in
0.1 M phosphate buffer, pH 7.3 for 2 h. After washing withphosphate buffer, liver cubes were dehydrated with ethanol(from 35 to 100%) then infiltrated with propylene oxide andplastic mixture for 2–3 days. The embedded liver cubes weresectioned with ultramicrotome and dyed with uranyl acetateand lead citrate prior to photomicrography.
2.4. Hepatotoxic study in isolated rat hepatocytes
2.4.1. Preparation of isolated rat hepatocytesIsolation of rat hepatocytes was routinely prepared at
9.00 a.m. using the method ofBerry and Friend (1969)asmodified by Stacey and Priestly (1978)and Pramyothin(1986). Under ether anesthesia, liver was perfused withCa2+-free physiological solution (96 mM NaCl, 1.4 mMKCl, 0.7 mM MgSO4, 2.5 mM KH2PO4, 30 mM NaHCO3and 21.7 mM sodium gluconate, equilibrated with 95%O2/5% CO2 at pH 7.4) via the portal vein. When the per-fusion of all hepatic lobes was rapid and complete, 100 mlof 0.5% collagenase in the same Ca2+-free physiologicalsolution was added and allowed to digest under the recir-culating condition. Flow was maintained at 30–35 ml/min
with the pressure head of 20 cm water, and temperature wasmaintained at 37◦C throughout the procedure. After perfu-sion with collagenase (10–15 min), liver was dispersed withblunt spatula in 50 ml of 0.5% fresh collagenase buffer, andincubated at 37◦C in a shaker water bath for 10 min. Bovineserum albumin (BSA) was added to give a final concentra-tion of 12 mg/ml and cells were harvested through nylonmesh (250–61�m). Hepatocytes were separated from othercells and cellular debris by differential centrifugation (50 g,1 min). The cell pellet was washed twice with this freshphysiological medium containing 12 mg/ml BSA and oncewith the incubation medium. Hepatocytes were finally sus-pended in Eagle’s basal medium containing 12 mg/ml BSAat the concentration of approximately 40 mg wet weightcells per milliliter or approximately 4–5× 106 cells permilliliter. Trypan blue exclusion index was routinely per-formed. Preparation with the trypan blue exclusion indexlesser than 90% was never used.
2.4.2. Wet weight calibrationDuplicate aliquots of 1 ml cell suspension were put into
preweighted tubes, and centrifuged at 5000 rpm for 5 min.Supernatant was discarded, the upside down tubes were al-lowed to stand in room temperature for 2 h. Then tubes wereweighed and cell pellet was calculated as milligram of wetweight cells per milliliter of cell suspension.
2.4.3. Incubation of isolated rat hepatocyte suspensionIn every experiment, the aliquot of 3 ml cell suspension
was incubated in 25 ml Erlenmeyer flask in the metabolicshaker bath, under the atmosphere of carbogen (95% O2/5%CO2) at 37◦C for 1 h.
2.4.4. Cytotoxicity studyCell suspension isolated from four control rats was used.
For each cell preparation, triplicate samples were used forcontrol and treated groups including DMSO, (+)usnic acid(0.01, 0.1, 1 mM), carbon tetrachloride (10�l), and (+)usnicacid plus carbon tetrachloride. After 1 h incubation, aliquotof 0.5, 1.5 and 0.5 ml of each sample were used for the deter-mination of transaminase activity, malondialdehyde (MDA)formation and glutathione content, respectively.
2.4.5. Determination of transaminase activity (AST, ALT)Aliquot of 0.5 ml cell suspension was centrifuged at
1000 rpm for 1 min, the supernatant was measured fortransaminase activity using the method ofReitman andFrankel (1957).
2.4.6. Lipid peroxidation and glutathione (GSH)determination
Aliquot of 1.5 ml cell suspension was used in the thiobar-bituric acid assay for the formation of malondialdehyde bythe method ofBuege and Aust (1978). Glutathione was mea-sured using 0.5 ml cell suspension by the method ofEllman(1959)as modified byJollow et al. (1974).
P. Pramyothin et al. / Journal of Ethnopharmacology 90 (2004) 381–387 383
Fig. 2. Electron micrographs: (A) control (13,600×); (B) DMSO, same as control (13,600×); (C) (+)usnic acid 50 mg/kg per day, i.p., 5 days, slightlyswelling of mitochondria (13,600×); (D) (+)usnic acid 200 mg/kg per day, i.p., 5 days, markedly swelling of mitochondria and endoplasmic reticulum(13,600×); enlargement from (D) of mitochondria in (E) (68,000×) and endoplasmic reticulum in (F) (68,000×). N = nucleus, M= mitochondria, E= endoplasmic reticulum.
384 P. Pramyothin et al. / Journal of Ethnopharmacology 90 (2004) 381–387
2.4.7. Determination of aniline hydroxylase activityUsing cell suspension isolated from rats preadministered
with isonazid in drinking water (0.1%, w/v, pH 7.4) for 10days. After 15 min incubation of 3 ml cell suspension with0.1 ml 5 mM aniline and (+)usnic acid (1 and 10 mM), allsamples were used for the determination of aniline hydrox-ylase activity by the method ofGibson and Skett (1986).
2.5. Hepatotoxic study in isolated rat liver mitochondria
2.5.1. Preparation of rat liver mitochondriaIntact rat liver mitochondria was prepared by the method
of Hogeboom (1955). The mitochondrial protein was deter-mined by the method ofLowry et al. (1951)as modified byMiller (1959) using bovine serum albumin as standard.
2.5.2. Oxygen consumption measurementsThe oxygen uptake by intact mitochondria was measured
polarographically by Clarke-type oxygen electrode con-nected to an oxygen monitor (YSI model 53) and recordedon a strip chart recorder (Gilson model N2).
2.5.3. ATPase activityThe mitochondrial ATPase activity was measured by de-
termining the amount of inorganic phosphate liberated at theend of incubation period. The reaction was terminated byadding 1 ml aliquot of reaction mixture to 1 ml of ice-coldtrichloroacetic acid. After centrifugation, the amount of in-
Table 1Effects of 5 days treatment of (+)usnic acid on serum AST and ALT in rats
Group Dose per day, 5 days Serum ASTa (S.F. units per milliliter) Serum ALTa (S.F. units per milliliter)
S.F. units per milliliter× 0.48= IU/l.a Values are mean± S.E.∗ Significantly different from control group,p < 0.05.∗∗ Significantly different from DMSO group,p < 0.05.∗∗∗ Significantly different from CCl4 group,p < 0.05.
organic phosphate in the supernatant was determined by themethod ofFiske and Subbarow (1925).
2.5.4. Experimental conditionsAll experiments were performed at 37◦C. The composi-
tion of reaction mixtures and other experimental conditionsare described in the table legends. The mitochondria usedin this study must have the RCI (respiratory control in-dex) value not less than five with glutamate plus malateas substrate. The experimental results reported here werereproducible with at least four separate mitochondrialpreparations.
2.6. Statistical analysis
Statistical analysis of the results were performed withStudent’st-test.
3. Results
After 5 days treatment of (+)usnic acid in rats, therewas no significant change in serum transaminase activity(serum AST, ALT) (Table 1) while the electron micrographsillustrated the signs of liver cell damage as shown by themarkedly swelling of mitochondria and endoplasmic retic-ulum, especially at the 200 mg/kg dose of (+)usnic acid(Fig. 2).
P. Pramyothin et al. / Journal of Ethnopharmacology 90 (2004) 381–387 385
Table 3Effect of (+)usnic acid on aniline hydroxylase activity in isolated rathepatocytes
a Values are mean± S.E.∗ Significantly different from control group,p < 0.05.∗∗ Significantly different from control (DMSO) group,p < 0.05.
The study of cytotoxic effect of (+)usnic acid in isolatedrat hepatocytes demonstrated the dose related pattern in therelease of cellular transaminase (AST, ALT) and malondi-aldehyde formation (index of lipid peroxidation). DMSO, atthe volume used (10�l) to dissolve (+)usnic acid had no ef-fect on these criteria. When increasing the dose of (+)usnicacid, transaminase activity (AST, ALT) and lipid peroxida-tion were increased, the glutathione content was decreased.CCl4 (10�l) was used as the reference hepatotoxin, similarresults were obtained as shown in the high dose (+) usnicacid group (1 mM). Coadministration of (+)usnic acid andCCl4 exerted the additive effects on the release of cellu-lar transaminases, the formation of MDA and GSH content(Table 2). The aniline hydroxylase activity was also foundto be increased with high doses of (+)usnic acid (1, 10 mM)(Table 3).
(+)Usnic acid stimulated respiration in isolated rat livermitochondria using glutamate plus malate and succinate as
Table 4Effect of (+)usnic acid on rate of oxygen consumption in isolated ratliver mitochondria
Composition of reaction system: 37.5 mM HEPES buffer pH 7.2, 3.68 mMMgCl2, 86.25 mM KCl, 13.02 mM sucrose, 5.21 mM succinate or 5.21 mMpotassium glutamate plus 5.21 mM potassium malate, mitochondrial frac-tion (1.21 mg protein per milliliter) and (+)usnic acid as indicated. Totalvolume was 1.92 ml. The mitochondria were preincubated with (+)usnicacid for 1 min before substrates were added. The rates of oxygen up-take denoted the difference between the rates after and before substratesaddition.
a Values are mean± S.E.∗ Significantly different from control (DMSO) group,p < 0.05.
Table 5Effect of (+)usnic acid on ATPase activity in isolated rat liver mitochon-dria
Composition of reaction system: 35.9 mM HEPES buffer pH 7.2, 3.51 mMMgCl2, 82.58 mM KCl, 17.06 mM sucrose, 5 mM ATP, mitochondrialfraction (2.18 mg protein per milliliter) and (+)usnic acid as indicated.Total volume was 2.93 ml. The mitochondria were preincubated with(+)usnic acid for 1 min before ATP was added. The reaction mixtureswere further incubated for 10 min after ATP addition.
a Values are mean+ S.E.∗ Significantly different from control (DMSO) group,p < 0.05.
substrates. The rate of oxygen consumption in state 4 respi-ration increased when increased doses of (+)usnic acid start-ing from 0.15, 4.5 and 6�M doses showed the highest rateof mitochondrial respiration, respiring with succinate andglutamate plus malate, respectively. After the highest rate,there was a slow down in respiration even when increasedthe doses of (+)usnic acid (Table 4). Mitochondrial ATPaseactivity was also stimulated by (+)usnic acid, with the dosestarting from 0.3�M. At 2 �M dose of (+)usnic acid showedthe maximal stimulation. Doses higher than 2�M illustratedthe slower rate of ATPase stimulation (Table 5).
4. Discussion
In the present investigation, the hepatotoxicity inducedby (+)usnic acid in rats requires both high dose and timeof exposure using serum AST, ALT and histopathologicalchanges as the criteria. (+)Usnic acid seems to be a lesserhepatotoxin due to its lesser effect on the transaminase ac-tivity and liver cell injury. Membrane like structures suchas mitochondria and endoplasmic reticulum are affected byhigh dose of (+)usnic acid as seen clearly in the electronmicrographs.
In order to search further for hepatotoxic mechanisms of(+)usnic acid, isolated rat hepatocytes were used, togetherwith carbon tetrachloride (CCl4) as the reference heptotoxin.CCl4 is well known for its hepatotoxicity. It is metabolizedin rats by the cytochrome P450 system, especially CYP 2E1to a highly reactive metabolite, trichloromethyl-free radical(CCl3•). This free radical may react with oxygen and gen-erates the trichloromethylperoxy radicals (OOCCl3
•). Bothradicals may attack lipids on cell membrane and membranelike structures such as mitochondria and endoplasmic retic-
386 P. Pramyothin et al. / Journal of Ethnopharmacology 90 (2004) 381–387
ulum, stimulating lipid peroxidation, causing disturbance ofCa2+ homeostasis, and resulting in cell death. The dam-age to cell membrane integrity causes the release of cellu-lar hepatospecific enzymes, mainly the transaminase (AST,ALT). Lipid peroxidation chain reaction generates the prin-cipal products called malondialdehyde. Glutathione is thestrong nucleophilic molecules found in most cells with itsfunction as an antioxidant. Free radicals are usually de-stroyed by GSH. CCl4 induced cell death may be determinedby increasing the release of cellular transaminases, increas-ing the production of MDA and decreasing cellular level ofGSH (Recknagel, 1983; Recknagel et al., 1989; Lawrenceand Dean, 1996). In the present investigation, (+)usnic acidand CCl4 demonstrated similar cytotoxic effects and addi-tive results are obtained with the combination of (+)usnicacid and CCl4. Therefore, (+)usnic acid appears to havethe same hepatotoxic mechanisms as presented by CCl4.Furthermore, aniline hydroxylase, the CYP 2E1 in the cy-tochrome P450 system, which metabolizes both aniline andCCl4 in rats, is increased in the presence of (+)usnic acid.This indicates that (+)usnic acid may be metabolized byCYP 2E1. Rat CYP2E1 protein expression is known to bestimulated by many xenobiotics. These xenobiotics appar-ently act by stabilizing the enzyme (binding as substrate) orincreasing translational efficiency of the CYP 2E1 mRNA(Henderson and Wolf, 1992). This observation is clear inthe present investigation, in which the aniline hydroxylaseactivity is increased when increased doses of (+)usnic acid.
Isolated rat liver mitochondria was used to demonstratethe direct effect of (+)usnic acid on mitochondrial functions.(+)Usnic acid caused maximal stimulation of both state 4respiration (three- to seven-folds, depending on substratesused) and ATPase activity (seven-fold). This uncoupling ef-fect of oxidative phosphorylation is dose-dependent and issimilar to results reported in mouse liver mitochondria byAbo-Khatwa et al. (1996). Loss of respiratory control ap-pears after the maximal stimulation.
In conclusion, hepatotoxic effect of (+)usnic acid maymediated by itself or by its reactive metabolite(s) causingloss of membrane integrity and destruction of mitochondrialfunctions.
Acknowledgements
This study was supported in part by the Graduate ResearchFund and Grant to support High Potential Research Unit,Ratchadaphise-Ksomphot Endowment Fund, ChulalongkornUniversity.
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Berry, M.N., Friend, D.S., 1969. High yield preparation of isolated ratliver parenchymal cells; a biochemical and fine structure study. Journalof Cell Biology 43, 506–520.
Buege, J.A., Aust, S.D., 1978. Microsomal lipid peroxidation. In: Colow-ick, S.P., Kaplan, N.O. (Eds.), Methods in Enzymology. AcademicPress, New York.
Campanella, L., Delfini, M., Ercole, P., Iacoangeli, A., Risuleo, G., 2002.Molecular characterization and action of usnic acid: a drug that inhibitsproliferation of mouse polyomavirus in vitro and whose main targetis RNA transcription. Biochimie 84, 329–334.
Cardarelli, M., Serino, G., Campanelli, L., Ercole, P., De Cicco Nardone,F., Alesiani, O., Rossiello, F., 1997. Antimitotic effects of usnic acidon different biological system. Cell Molecular Life Sciences 53, 667–672.
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Keywords:Ethnobotany; Medicinal plants; Red-headed Yao people; Yunnan
1. Introduction
The second half of the twentieth century has seen a grad-ual loss in the value of plants in therapeutics and, as a con-sequence, the interest in the use of plants in therapy has alsodecreased. Nevertheless, research in this field has increased.What is evident is that popular knowledge on the medicinaluses of plants has not disappeared. It exits, but each day itbecomes weaker and we must make an effort to inventorywhatSchultes (1991)recently called the prolific and promis-ing treasure-trove of the ethnopharmacological knowledge,before it is too late. In industrialized countries, the erosionof popular information on plants is much faster than in de-veloping ones (Bonet et al., 1992).
The Yao people are an ethnic group with a population ofover seven million. They live in China, Vietnam, Laos, Thai-land, France and the United States. The Yao people are oneof the oldest nationalities in the south of China. They live inthe mountains and the forests of Guangxi, Hunan, Guang-dong, Yunnan and Guizhou (Luo and Qing, 1994; Dai andLi, 1995; Dai and Qiu, 1997). During the long history of
production, the Yao people have gradually grown to under-stand the nature and function of plants and animals. Theyhave been using these plants and animals to prevent harmfrom poisonous insects, violent animals, diseases and poorliving conditions for thousands of years. They have accumu-lated rich living experiences and created their own brillianttraditional medicine culture, which is a main part of theirtraditional knowledge (Bai, 1990). According to our field in-vestigation and literature studies, the Yao people in Jinpingof Yunnan Province have developed advanced technology ofusing medicinal plants and animals to treat diseases.
The Yao people have different branches. The Red-headedYao is a main branch in Jinping. The Red-headed Yaomainly inhabit the mountains in Jinping county, HongheHani Autonomous Prefecture, Southeast Yunnan Province ofSouthwest China. It is located between 103◦13′–103◦23′Eand 22◦46′–22◦54′N. It borders with Vietnam in the south.In this area, the majority of the population resides in remotevillages where modern facilities are lacking. The elevationvaries from 1020 to 2597 m. The climate here is sub-tropical,and the annual average temperature is 10.6◦C. Likewisethe annual average rainfall is 3000 mm (Xu and Xie,1989).
Unlike the Dai, Tibetan, Uigur and Mongolian in China,the Red-headed Yao people in Jinping do not have theirown medicine theory yet. Their medicine is a form of folk
390 C.-l. Long, R. Li / Journal of Ethnopharmacology 90 (2004) 389–395
medicinal practice. It is still in the stage of accumulatingand improving medicinal knowledge. However, there arespecial features in diagnosing and curing diseases, as wellas, administration in the Red-headed Yao people’s medicine.It is interesting that there are some similarities between theRed-headed Yao people and other branches of Yao peoplein Guangxi and Hunan provinces although they are far fromeach other with a distance of over 1000 km. Major researchresults on medicinal plants from the Red-headed Yao societyare concluded in the present paper.
2. Materials and methods
In 1996, we started to collect literature related to theRed-headed Yao people and traditional medicine. The liter-ature was studied extensively (Mo, 1991; Waller, 1993; Luoand Qing, 1994; Dai and Li, 1995; Chen, 1998). A surveyin seven districts of Jinping was performed six times dur-ing different seasons in a period of 3 years. We adoptedanthropological methods, ethnobotanical methods and par-ticipatory rural appraisal methods in the field (Etkin, 1993;Lipp, 1989; Hedberg, 1993; Pei and Long, 1998). In eachvisit, plants were collected from different parts of the re-gions. Following methodology byCroom (1983), detailedfield record was taken on the medicinal uses of the plants.
Information was obtained though general conversationwith elderly villagers, local healers and herbalists at thetime of each visit. Whenever possible, the conversationswere recorded without the knowledge of the informant(Longuefosse and Nossin, 1996). Voucher specimens of 66plants with medicinal uses were collected by the authorsfrom the field (Martin, 1995). The parts of the plants usedto treat diseases, types of diseases to be cured, doses, timeand methods of administration were carefully recorded inthe field (Alba and Brito, 1996).
All plants collected from the fields were identified inten-sively. The voucher specimens are deposited in the VoucherHerbarium at the Department of Ethnobotany, Kunming In-stitute of Botany, Chinese Academy of Sciences.
3. Results
As a result of rich flora in this region, a rich folk medicineis expected (Li, 1994). In general, the Red-headed Yao peo-ple use the most common and accessible plants. A largenumber of medicinal plants are cultivated spontaneously orsubspontaneously in open areas, along lanes, or in bushland.Sixty-six medicinal plant species were collected and iden-tified during the field investigation and herbarium studies.In Table 1, the medicinal plants are alphabetically arrangedand the data are presented in the following sequences: num-ber of collection of each plant, scientific name, family name,local name, part of plant used, method of preparation, theailments for which medicine is given.
Sixty-six medicinal plant species belong to 43 fami-lies, and 61 genera. The family with the most species wasCompositae (including seven medicinal plants), the secondbiggest family was Rubiaceae (including five species), andthe third largest families were Rosaceae and Labiatae (eachfamily has three medicinal plant species). The members ofthe four families altogether represent more than 27% of thetotal species of medicinal plants.
The medicinal functions of 27 species (41%) have neverbeen recorded in the literature on medicinal plants of Jinping(Zeng, 1984; Luo, 1987; Huang, 1992; Zhu, 1995; Dai andQiu, 1997). Those species includeMicrosorium superficiale(mainly for tummy bug and stomach convulsion),Phyma-todes lucida(for stomachache and hepato-cirrhosis),Illig-era celebica(for numbness of limbs),Clematis chrysocoma(mainly for dysmenorrhea),Piper macropodum(for rheuma-toid arthritis), Sarcandra hainanensis(for osteoarthritisand arthritis), Begonia cathayana(mainly for bronchi-tis), Begonia truncatiloba(for ulcers),Melastoma normale(mainly for rheumatoid arthritis and cystitis),Hypericumacmosepalum(for quadriplegia),Fragaria nilgerrensis(fordysmenorrhea),Ficus chapaensis(for adynamia),Sabiafasciculata(for fracture and hysteria),Helwingia japonica(for diarrhoea),Jasminum fuchsiaefolium(for rheumatoidarthritis), Luculia intermedia (mainly for tuberculosis),Aeschynanthus bracteatus(for rheumatoid arthritis),Cary-opteris paniculata(for inflammation),Elsholtzia fruticosa(for diarrhoea),Commelina diffusa(for defervesce anddetoxification),Asparagus lycopodineus(for cough),Parisvietnamensis(for themorrhoids),Smilax mairei (mainlyfor venereal disease),Rhaphidophora decursiva(mainlyfor fracture),Iris decora (for hemorrhoids and diarrhoea),Dendrobium williamsonii(for spermatorrhea) andCarexbaccans(mainly for measles) (seeTable 1).
The medicinal value of 23 species (35%) are noted inprevious works but they are different in treatment of dis-eases and the parts used from these reported previously(Zeng, 1984; Luo, 1987; Huang, 1992; Zhu, 1995; Daiand Qiu, 1997). They areAstilbe rivularis, Urena lobata,Hydrangea macrophylla, Potentilla kleiniana, Crotalariaferrnginea, Euodia lepta, Apium graveolens, Gaultherialeucocarpa, Embelia oblongifolia, Maesa indica, Uncariasessilifructus, Valeriana jatamansi, Adenostemma lavenia,Gnaphalium affine, Senecio scandens, Lysimachia christi-nae, Plantago asiatica, Glechoma longituba, Leucas ciliata,Amomum tsaoko, Reineckia carnea, Acorus tatarinowiiandCymbidium hookerianum.
The healing properties of some plants such asPiperthomsonii, Rubus ellipticusvar.obcordatus, Rubia mangith,Mussaenda laxiflora, Ligularia duciformis and Myriactiswallichii are similar to those reported from other parts ofthe country (Luo, 1987; Huang, 1992; Zhu, 1995; Dai andQiu, 1997).
There were several plant species with special uses. TheyarePiper macropodum, Amaranthus lividus, Urena lobata,Rubus ellipticusvar. obcordatus, Uncaria sessilifructus,
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391Table 1List of medicinal plants used by the Red-headed Yao people in Jinping of Yunnan Province, China
Number ofcollection
Scientific name Family name Local name Part used Preparation of administration Popular uses or diseases treated
Teched, bellyache, tummy bug, numbness of limbs,hemorrhoids, diarrhea, gall, injuries from falls,dysmenorrhea, invigorant
99033 Adenostemma lavenia(L.)Kuntze
Compositae fa ha mi Leaf, whole plant Poultice Flu, toothache, injuries from falls, hepatitis, pneumonia,quinsy, enteritis, lymphadenitis, tummy bug, tummy calculus,vesical calculus
99007 Aeschynanthus bracteatusWall.
Gesneriaceae di yang heng Whole plant Medicine bath, broth Rheumatoid arthritis, constitutional, invigornt, healthprotection
99078 Amaranthus lividusL. Amaranthaceae gan jiang mi Leaf, seed, wholeplant
Myrsinaceae ze hu pei Fruit, whole plant Decoction, medicinebath
Dispel wind and dampness, numbness of limbs, injuries fromfalls
99023 Euodia lepta(Spreng.)Merr.
Rutaceae bei la gong diang Root, leaf Medicine bath, poultice Rheumatoid arthritis, sciatica, quadriplegia, flu, pneumonia,injuries from falls, gall, numbness of limbs
99069 Ficus chapaensisGagnep. Moraceae deng di xiu (dixiu ang)
Root, stem, leaf Medicine bath, broth Invigorant, adynamia, giddily
Scientific name Family name Local name Part used Preparation of administration Popular uses or diseases treated
99035 Gaultheria leucocarpaBl.var. crenulata (Kurz) T. Z.Hsu
Ericaceae jian cai za Whole plant Decoction, medicinebath
Rheumatoid arthritis, injuries from falls, numbness of limbs,tummy bug, flu, toothache, cough, constitutional,
99004 Glechoma longituba(Nakai) Kupr.
Labiatae di pou (di bi) Stem, leaf, whole plant Decoction, medicinebath
Vesical calculus, calculus, injuries from falls, fracture, gall,cough, flu, diarrhea, dysmenorrhea, hysteritis, leucorrhea,bronchitis, scare, pneumonia, pulmonary tuberculosis
99041 Gnaphalium affineD. Don Compositae dou mei Whole plant Decoction Cough, flu, numbness of limbs, quadriplegia, neurasthenic,sterility and dystocia
99060 Helwingia japonica(Thunb.) Dietr.
Cornaceae ye shan hua Leaf, fruit, whole plant Decoction, poultice Diarrhea, tummy bug, ulcer, viper bite, injuries from falls,fracture
99056 HemiphragmaheterophyllumWall.
Scrophulariaceae di bi Whole plant Decoction, poultice Dysmenorrhea, pulmonary tuberculosis, quinsy, injuries fromfalls, lumbago, numbness of limbs, measles, ulcer, toothache
99057 Hydrangea macrophyllaDC. f. hortensiaWils.
Hydrangeaceae fang han mi Root, stem, leaf,flower
Decoction Fret, diarrhea, scare
99034 Hypericum acmosepalumN. Robson
Hypericaceae xiang ge da Root, stem Decoction Numbness of limbs, injuries from limbs, quadriplegia,arthritis, cough
Numbness of limbs, injuries from falls, fracture, hysteritis,invigorant
99054 Sarcandra hainanensis(P’ei) Swamy et Bailey
Chloranthaceae jiu jie feng Whole plant Broth Numbness of limbs, osteoarthritis, arthritis, teched
99027 Saussurea deltoidea(DC.)C. B. Clarke
Compositae deng duo bu ai Root, leaf, whole plant Decoction, medicinebath
Dyspepsia, windy, little milk, fracture, adynamia, tummybug, numbness of limbs
99036 Senecio scandensBuch.-Ham ex D. Don
Compositae qian li guan Whole plant Medicine bath Bellyache, gall, measles, ulcer, hemorrhoids
99055 Smilax maireiLevl. Smilacaceae deng ji yang gong Tuber Decoction Rheumatoid arthritis, tummy bug, dysmenorrhea,hemorrhoids, gall, venereal disease
99053 Uncaria sessilifructusRoxb.
Rubiaceae ying diu Root, leaf, whole plant Decoction Scare, teched, high blood pressure, giddily, bellyache,hysteritis, rheumatoid arthritis, arthritis, hemiplegia, sciatica,injuries from falls, ulcer
Windy, dyspepsia, scour, diarrhea, numbness of limbs,quadriplegia, injuries from falls, gall, fracture, ulcer
394 C.-l. Long, R. Li / Journal of Ethnopharmacology 90 (2004) 389–395
Lysimachia christinae, Plantago asiatica, Glechoma longi-tuba, Smilax mairei, Acorus tatarinowiiandCarex baccans.They are mixed up together and boiled for two to threehours. Then the patients can be bathed in this medicine wa-ter when it cools down. In the past, the local people tookmedicine bath every day. Today, following improvement ofliving conditions and medicinal technology, especially short-age of medicinal plant resources, the people have to reducethe number of medicine baths they take. At present, onlyfor important traditional festivals of the Yao people suchas the Dragon Boat Festival, Chong Yang Festival and PanWang Festival, do the local people take medicine baths. Bythis way, some gynaecology and obstetrics diseases can betreated effectively. It will be necessary to carry out phyto-chemical and pharmacological studies of these plants in or-der to verify the validity of these uses.
4. Discussions and conclusions
Knowledge of using plants as remedies is apparently theresult of transmission from the old to the new generation. Inspite of the socio-economic welfare, well-developed roadsand medical facilities, the tradition of using plants for thetreatment of some diseases still continues in the region. TheRed-headed Yao medicinal herb doctors are already veryold. The younger generations, however, have learned verylittle from the old healers. To prevent traditional medici-nal knowledge from being lost, it is an extremely urgenttask to collect and arrange that traditional knowledge andspread it throughout the Red-headed Yao societies (Rao,1996).
On the protection and utilization of medicinal plant, theRed-Yao people have a rich experience. First, according todifferent seasons and climate conditions, the local peoplepick different parts of the medicinal plant to treat diseases.Second, when the local people pick medicinal plants, theydo not pick the root to treat diseases. If only the root isneeded for the cure, they invariably pick old roots and leavethe new root so the plant can persist. Third, the Red-headedYao medicinal herb doctors think it is important to protectbig trees. By protecting the forest and the whole ecologicalenvironment they are providing an excellent natural envi-ronment for the growth of medicinal plants.
The Red-headed Yao women play an important role inthe spread and utility of medicinal plants. The women candiscriminate the medicinal plants and remember their func-tions and methods of disease treatment. The Red-headed Yaomedicine can also treat some gynaecology and obstetrics ef-fectively, it is convenient to the Red-headed Yao women tomaster the medicine and use it to treat diseases.
There are some plants such asPhymatodes lucida, Piperthomsonii, Amaranthus lividus, Fragaria nilgerrensis, Po-tentilla kleiniana, Apium graveolens, Ligusticum brachy-lobum, Valeriana jatamansi, Kalimeris indica, Plantagoasiatica and Amomum tsaoko, which are not only used as
medicine to treat diseases, but also as food to supplementnutrition.
The Red-headed Yao’s medicine is an experienced medic-inal at practice. In the course of development, it was influ-enced deeply by religious culture and feudal superstition. Itis an urgent problem to solve how to take effective measuresto get rid of the binding of religious culture and feudal su-perstition to traditional knowledge and to progress towardsregularization and science.
Under the pressure of poor living conditions, and a con-stant fight with the poor natural environment, poisonous in-sects, violent animals and diseases, they summed up a se-ries of effective traditional medical knowledge. We shouldrespect the traditional knowledge and protect their intellec-tual property right when we collect and arrange the folk cul-ture knowledge (Posey, 1990). If it is possible, the nationshould provide some preferential policy to encourage theRed-headed Yao doctors to spread their medicinal knowl-edge from generation to generation (Vagelos, 1991).
Acknowledgements
This research was supported by the Chinese Academyof Sciences (KSCX2-SW-117), Ministry of Science andTechnology of China (2001DEA10009), National ScienceFoundation of China (30170102), and Yunnan Province(2001C0058M & 2001PY017). Tony Cunningham, GaryMartin, Darrell Posey, Thomas Carlson, and Jennifer Sow-erwine gave fruitful comments and suggestions duringthe writing of this paper. The authors wish to thank allthe Red-headed Yao people in Jinping county, who havehelped to make this research possible, particularly the olderRed-headed Yao medicinal herb doctors who kindly pro-vided us with hospitality in their villages. Special thanksgo to Mr. Yu Zhi-Yong who helped to arrange interviews.Thanks are also expressed to Professor Li Heng, a botanistat Kunming Institute of Botany, who helped in the iden-tification of the plants collected from the fields. Sincereappreciation is expressed to Ms. Diana Aljets for her criticalreading of the original manuscript.
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Journal of Ethnopharmacology 90 (2004) 397–401
Ligusticum wallichi-induced vasorelaxation mediated bymitogen-activated protein kinase in rat aortic smooth muscle
Bokyung Kima,∗, Junghwan Kima, Aeran Kima, Yoon-Sun Kima, Youn Ri Leea,Young Min Baea, SungIl Choa, Mee-Ra Rhyub
a Department of Physiology, College of Medicine, Konkuk University, Danwol-dong 322, Chungju, Choong-Buk 380-701, South Koreab Food Chemistry & Biotechnology Division, Korea Food Research Institute, Kyonggi-Do 463-420, South Korea
Received 16 May 2003; received in revised form 30 October 2003; accepted 3 November 2003
Keywords: Aorta; Ligusticum wallichi; Mitogen-activated protein kinases; Vasorelaxation
1. Introduction
Ligusticum wallichi, a popular Chinese herbal medicine,has been used orally with other herbs for “heart disease” forthousands of years. Current research in nutrition science pro-vides a better understanding of the possible link between theplant and heart disease. A component ofLigusticum wallichiincreases myocardial contractility and coronary circulation(Chiou et al., 1991; Hwang, 1993). In contrast, this plant caninhibit the muscle contractions induced by vasoconstrictorsand low systemic blood pressure (Hwang, 1993). However,little is known about the effects ofLigusticum wallichi onthe contraction of smooth muscle and the mechanisms un-derlying the contractile system.
It is well established that smooth muscle contraction isregulated by intracellular Ca2+ ([Ca2+]i ) and the phos-phorylation of myosin light chain (MLC) (Karaki, 1989;Somlyo and Himpens, 1989). However, various kindsof vasoconstrictors induce a further contraction at agiven [Ca2+]i , and elicit a sustained contraction under
Ca2+-depleted conditions, referred to as “Ca2+-independentcontraction,” in intact and membrane-permeabilized smoothmuscle (Hori et al., 1992; Kim et al., 2003a). In previous re-ports, several molecules, including protein kinase C (PKC)and MAPKs, have been suggested as candidate regulatorsof Ca2+-independent contraction (Nixon et al., 1995; Leeet al., 1999; Kim et al., 2003b).
MAPKs constitute a family of serine/threonine-specificprotein kinases which play a central role in intracellularsignal transduction initiated by extracellular stimuli, in-cluding growth factors, neurotransmitters, and hormones(Kosako et al., 1994). The three MAPK isoforms, extra-cellular signal-regulated kinase (ERK1/2), p38 MAPK andstress-activated protein kinase (SAPK)/c-Jun N-terminalkinase (JNK), are central elements in transducing the mes-sages in mammalian cells (Miyata and Nishida, 1999). Invascular smooth muscle, MAPKs are activated by recep-tor agonists, including angiotensin II, phenylephrine, andendothelin-1 (Dessy et al., 1998; Touyz et al., 1999). Thereis accumulating evidence that the MAPK pathway is closelylinked with the increase in smooth muscle contraction un-der Ca2+-dependent and -independent conditions (Khalilet al., 1995; Dessy et al., 1998; Kwon et al., 2003). Further-
398 B. Kim et al. / Journal of Ethnopharmacology 90 (2004) 397–401
more, recent studies have shown that MAPKs contributeto the intracellular signal transduction initiated by herbalmedicines (Cheung et al., 2000; Kim et al., 2000), and it isassumed that MAPKs can be involved in the regulation ofcontractions mediated byLigusticum wallichi. However, ithas not been demonstrated that MAPKs contribute to thevascular activity mediated byLigusticum wallichi.
In this study, we investigated the effects ofLigusticumwallichi on vascular reactivity and the involvement of theMAPK pathway inLigusticum wallichi-induced vasoactiv-ity in rat aortic smooth muscle contraction, using a pharma-cological approach, with parallel experiments based on theisolated-tissue bath and western blotting analyses.
2. Materials and methods
2.1. Preparation of extract
Dried Ligusticum wallichi, obtained from the local mar-ket, was cut into small pieces and ground with a commer-cial mixer. The powder was homogenized with 10 volumesof chloroform and the homogenates were centrifuged at10,000× g for 20 min, and then filtered. The filtrates wereevaporated and lyophilized at 37◦C and the solids were dis-solved in 100% ethanol at concentration of 2 g/ml to producea stock for bioassay. A voucher specimen (No. LW-04) hasbeen deposited in the Korea Food Research Institute (KFRI),Kyonggi-Do, Korea.
2.2. Animals and measurement of isometric contraction
Male Sprague–Dawley rats (200–250 g) were stunned andbled. The thoracic aorta was isolated and cut into strips(2–3 mm wide and 7–8 mm in length). The endothelium wasremoved by gently rubbing the inner surface of the vesselwith cotton thread moistened with physiological salt solu-tion (PSS). Each strip was attached to a holder under a rest-ing contraction of 10 mN. After equilibration for 20 min ina 5 ml muscle bath, each strip was repeatedly exposed to a70 mM K+ solution until responses became stable. PSS con-tained (mM): NaCl 136.9, KCl 5.4, CaCl2 1.5, MgCl2 1.0,NaHCO3 23.8, glucose 5.5, and ethylenediaminetetraaceticacid (EDTA) 0.01. The high-K+ solution was prepared byreplacing NaCl with equimolar K+. These solutions weresaturated with a 95% O2 and 5% CO2 mixture at 37◦C andpH 7.4. Muscle contraction was recorded isometrically witha force-displacement transducer (FT03, Grass, RI, USA)connected to a polygraph system (7WC, Grass).
2.3. Measurement of MAPK activity
Aortic strips were isolated in the way described forcontraction measurement experiments, and snap-frozen inliquid N2 after treatment with various stimulants and atdifferent times. Samples were then homogenized in sam-ple buffer containing 50 mM Tris–HCl (pH 7.4), 5 mM
EGTA, 20 mM �-glycerophosphate, 1 mM NaF, 2 mMNa3VO4, 5�g/ml aprotinin, 5�M leupeptin, 1% triton-X100, 0.3 mM phenylmethylsulfonyl fluoride, 5 mM dithio-threitol, 10% glycerol, and 150 mM NaCl. The homogenatewas centrifuged at 14,000×g for 10 min at 4◦C, and the su-pernatant was collected. Protein concentrations were deter-mined using a Bio-Rad protein assay kit (CA, USA), whichis a colorimetric assay for protein based on the Bradforddye-binding procedure. Protein homogenates were diluted1:1 (vol:vol) with sodium dodecyl sulphate (SDS) samplebuffer containing 40 mM Tris–HCl (pH 6.8), 8 mM EGTA,4% 2-mercaptoethanol, 40% glycerol, 0.01% bromophenolblue, and 4% SDS, and then boiled for 5 min. Equal amounts(30–50�g per lane) of proteins were separated in each laneof a 10% SDS–polyacrylamide gel. Electrophoretically sepa-rated proteins were transferred to a nitrocellulose membrane(Amersham Pharmacia, USA). Membranes were incubatedwith phosphate-buffered saline, 0.1% Tween 20 (PBST)containing 5% non-fat dried milk for 30 min, and then incu-bated with individual polyclonal anti-phosphorylated MAPKantibodies diluted 1:1000–5000 for 5 h at room temperatureor overnight at 4◦C. Following incubation with horseradishperoxide-conjugated anti-rabbit IgG (1:1000) for 60 min,the blots were developed using the enhanced chemilumi-nescence (ECL) detection system (Amersham Pharmacia,USA). Quantitative analysis of antibody-specific bands wasperformed with an image analyser (Bio-Profil, VL, France).
2.4. Materials
Polyclonal anti-phosphorylated ERK1/2 antibody waspurchased from Promega (Amersham Pharmacia, USA).Polyclonal anti-phosphorylated p38 MAPK antibodywas purchased from Upstate Biotech (NY, USA). Nore-pinephrine, �-glycerophosphate, NaF, Na3VO4, apro-tinin, leupeptin, and phenylmethylsulfonyl fluoride werepurchased from Sigma (MO, USA). 12-Deoxyphorbol13-isobutyrate was purchased from Funakoshi (Japan).Triton-X 100 and dithiothreitol were purchased from Amer-sham Pharmacia (NJ, USA). PD98059 and SB203580 werepurchased from Tocris Cookson (Bristol, UK).
2.5. Statistical analysis
The results of experiments are expressed as means±S.E.M. Unpaired Student’st-test was used to compare thedata, andP < 0.05 was considered to be significantly dif-ferent.
3. Results
3.1. Effects of Ch1LW on norepinephrine-inducedcontraction
Isolated rat thoracic aorta was contracted with nore-pinephrine (NE) and then exposed to Ch1LW, a chloro-
B. Kim et al. / Journal of Ethnopharmacology 90 (2004) 397–401 399
Fig. 1. Effects of Ch1LW on the vasoconstrictor-induced contraction in rataortic smooth muscle. After the responses to 10�M norepinephrine (NE)was established, 1 and 10 mg/ml Ch1LW was added cumulatively (A).In Panel B, strips were pre-incubated with Ca2+-free medium containing1 mM EGTA for 30 min to remove external Ca2+, and then 1�M DPBwas added to the muscle strips. After establishing DPB-induced sustainedcontraction, 1 and 10 mg/ml Ch1LW was added sequentially.
form extract of Ligusticum wallichi. Ch1LW (1 mg/ml)significantly inhibited contraction induced by 10�M NE(52± 9.4% of NE-response), and the contraction was com-pletely abolished with 10 mg/ml Ch1LW (Fig. 1A). Todetermine the effects of Ch1LW on Ca2+-independent con-traction, the extract was tested on phorbol ester-inducedcontractions in Ca2+-free medium. After incubation of mus-cle strips in Ca2+-free medium containing 1 mM EGTA for30 min to remove external Ca2+, 1�M 12-deoxyphorbol13-isobutyrate (DPB) was added to the medium. Whenthe DPB-elevated contraction reached a steady-state level,Ch1LW (1 mg/ml) was added, resulting in an inhibi-tion of the contraction in Ca2+-free medium (31± 7.1%of DPB-response) (Fig. 1B). Complete inhibition ofDPB-induced contraction to the resting level was achievedwith 10 mg/ml Ch1LW. In the quiescent preparation, Ch1LW(1–10 mg/ml) did not evoke any changes of contraction.Together, these results show the existence of additionalmechanism involved in Ch1LW-induced vasorelaxation thatinhibits both Ca2+-dependent and -independent pathways.Neither NE- nor DPB-mediated contractions were affectedby the equivalent concentration of the vehicle ethanol (datanot shown).
3.2. Effects of Ch1LW on the activity of MAPK
To determine whether MAPKs influenceLigusticumwallichi-induced vasorelaxation, the activity of MAPKswas measured using phosphorylated MAPK antibodies inrat aortic smooth muscle. To determine whether the activityof MAPKs measured with the antibodies reflected the real
Fig. 2. The activities of MAPKs during NE-stimulation in rat aortic smoothmuscle. The time course of changes in phosphorylated ERK1/2 (A) andp38 MAPK (B) induced by NE. Strips were stimulated with 10�M NEfor 0, 5, 15 and 30 min, respectively, and then western blotting analysiswas carried out as described inSection 2. Results are presented as percentof phosphorylation relative to resting state. Values are means± S.E.M.
from three independent experiments. The insets indicate a representativeresult of western blotting with MAPK antibodies.
activation of the kinases, the selective inhibitors, PD98059and SB203580, were used (Dessy et al., 1998). Fig. 2 illus-trates the effects of NE on the activities of ERK1/2 and p38MAPK in rat aortic smooth muscle strips. Treatment withNE (10�M) elicited a sustained increase in the activity ofERK1/2 in a time-dependent manner (Fig. 2A). The max-imal response to NE was observed at 15 min. In addition,NE (10�M) also increased the activity of p38 MAPK witha pattern similar to that observed for ERK1/2 (Fig. 2B).PD98059 (10�M), an inhibitor of ERK1/2, was added tothe medium at the beginning of incubation for 30 min, andinhibited NE-stimulated ERK1/2 activity (data not shown).SB203580 (10�M), an inhibitor of p38 MAPK, signifi-cantly diminished the activity of the kinase elevated by NE(10�M) (data not shown). The effects of Ch1LW on theactivity of MAPKs induced by 10�M NE are illustratedin Figs. 3 and 4. Application of Ch1LW (10 mg/ml) to theresting muscle did not cause any change in the activity of
400 B. Kim et al. / Journal of Ethnopharmacology 90 (2004) 397–401
Fig. 3. Effects of Ch1LW on the increase in the activity of ERK1/2evoked by NE in rat aortic smooth muscle. After aortic strips pre-treatedwith 10�M NE or saline for 15 min, Ch1LW (10 mg/ml) was treatedfor 15 min sequentially. Western blotting analysis was carried out asdescribed inSection 2. Panel B shows the statistical results for changesof ERK1/2 activity, which are presented as percent of phosphorylationrelative to non-stimulating resting state. Values are means± S.E.M. fromthree independent experiments.∗∗P < 0.01 vs. control.
Fig. 4. Effects of Ch1LW on the increase in the activity of p38 MAPKevoked by NE in rat aortic smooth muscle. After aortic strips pre-treatedwith 10�M NE or saline for 15 min, 10 mg/ml Ch1LW was treated for15 min. Panel B shows the statistical results for the changes of p38MAPK activity, which are presented as percent of phosphorylation relativeto non-stimulating resting state. Values are means± S.E.M. from threeindependent experiments.
ERK1/2 in rat aortic smooth muscle. However, the sameconcentration of Ch1LW significantly abolished the activityof ERK1/2 elevated by 10�M NE (Fig. 3). Furthermore,10 mg/ml Ch1LW alone slightly increased p38 MAPK ac-tivity in resting muscle. However, in NE-stimulated muscle,Ch1LW did not reduce the activity of p38 MAPK, butcaused it to increase further (Fig. 4).
4. Discussion
In the present study, we have demonstrated that a chloro-form extract ofLigusticum wallichi inhibited the contractioninduced by a vasoconstrictor in rat aortic smooth muscle.This result is consistent with an earlier study in which thisplant inhibited the contractions induced by a receptor ago-nist and by membrane depolarization (Wu et al., 1989). It hasbeen reported that tetramethylpyrazine (TMP), a componentof Ligusticum wallichi, directly affects Ca2+-influx throughthe cellular membrane and Ca2+-release from the sarcoplas-mic reticulum, which results in a decrease in [Ca2+]i in thevascular system (Dai and Bache, 1985; Pang et al., 1996).These results suggest thatLigusticum wallichi has a potentvasorelaxation effect and this effect may afford protectionto the cardiovascular system. Furthermore, in the presentstudy, an extract of this plant inhibited phorbol ester-inducedCa2+-independent contractions in medium where externalCa2+ was chelated by EGTA. These results imply thatLigus-ticum wallichi causes vasorelaxation mediated by the inhi-bition of both Ca2+-dependent and -independent pathways.Furthermore, these results suggest that TMP is not an essen-tial component in theLigusticum wallichi-induced inhibitionof Ca2+-independent contraction, and there may exist anunidentified substance(s) that inhibits phorbol ester-inducedCa2+-independent contraction.
MAPKs constitute a family of kinases believed to play im-portant roles in the stimulus-induced contraction of smoothmuscle (Kosako et al., 1994; Dessy et al., 1998; Kwon et al.,2003). In vascular smooth muscle, MAPKs have been im-plicated as components of the signal transduction that re-sults in the activation of contractile proteins (Gerthofferet al., 1997). Adam et al. (1995)established conclusively thatMAPKs are modulators for Ca2+-independent contractionin vascular smooth muscle. It has been suggested that ac-tivation of MAPKs initiates phosphorylation of caldesmon,an actin-regulating protein, and this might contribute to thesensitization of contractile proteins to intracellular Ca2+(Gerthoffer et al., 1997). NE evokes activation of MAPKsin vascular smooth muscle (Yu et al., 1996). In the presentstudy, to determine whether MAPKs influenceLigusticumwallichi-induced inhibition, the activity of MAPKs was mea-sured using specific antibodies and inhibited with selectiveinhibitors in rat aortic smooth muscle (Dessy et al., 1998;Kwon et al., 2003). Although both kinases, ERK1/2 and p38MAPK, were activated by NE with similar time courses, theydisplayed different patterns of inhibition byLigusticum wal-
B. Kim et al. / Journal of Ethnopharmacology 90 (2004) 397–401 401
lichi. The extract ofLigusticum wallichi completely blockedthe activity of ERK1/2 elevated by the agonist. The activ-ity of ERK1/2 induced by NE was also blocked by a se-lective inhibitor of the kinase. However, the extract did notinhibit the activity of p38 MAPK, which was reduced by aselective blocker of the kinase. In the present study,Ligus-ticum wallichi extract increased the activity level of p38MAPK in both resting and NE-stimulated muscle. These re-sults indicate that inhibition of ERK1/2, but not p38 MAPK,contributes to Ch1LW-induced vasorelaxation in rat aorticsmooth muscle.
In conclusion, Ch1LW, an extract ofLigusticum wallichi,strongly inhibited NE- and phorbol ester-mediated contrac-tions, and the extract significantly attenuated the NE-inducedactivity of ERK1/2, but not p38 MAPK. These results sug-gest thatLigusticum wallichi extract-induced vasorelaxationis mediated by inhibition of ERK1/2 in rat aortic smoothmuscle.
Acknowledgements
This work was supported by a Grant-in-Aid for ScientificResearch from Ministry of Agriculture and Forestry, Korea.
References
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a Laboratory of Immunomodulator, Korea Research Institute of Bioscience and Biotechnology, Daejeon 305-600, South Koreab Laboratory of Pathology, College of Oriental Medicine, Deajeon University, Daejeon 300-176, South Korea
c College of Pharmacy, Chungnam National University, Daejeon 305-764, South Korea
Received 20 July 2003; received in revised form 27 October 2003; accepted 3 November 2003
Dendropanax morbifera Leveille (Araliaceae) is an en-demic species in Korea and distributes in the southern partof Korea (Han et al., 1998). The roots and stems of thisplant are used in folk medicine for the treatment of mi-graine headache, dysmenorrheal, and remove winddamp-ness (Bae, 2000). Recently, polyacetylene compounds havebeen isolated from the genusDendropanax and showed tohave cytotoxic activity (Setzer et al., 1995; Bernart et al.,1996), antiseptic effects (Kawazu et al., 1973), and ma-jor allergen (Oka et al., 1999). (9Z,16R)-16-Hydroxy-9,17-octadecadiene-12,14-diynoic acid (dendrotrifidic acid) wasisolated from the leaves ofDendropanax trifidus Makino(Kobayashi et al., 1977), and this tree was reported to havean antifungal activity (Kawazu et al., 1973). A triterpene ox-ide, dendropanoxide (2), was also known as epoxyglutinaneand campanulin (Tori et al., 1977), and glutinol (3) showedin vitro cytotoxic activity against P-388 and KB cells (Gilet al., 1994).
The complement system plays a significant role in thehost defense. The complement can be activated by a cascademechanism of the classical pathway (CP), alternative path-way (AP), or the MBL/MASP (mannan binding lectin/MBL-associated serine protease) pathway (Kirschfink, 1997). The30 odd complement fragments that make up the system in-clude proteolytic pro-enzymes, non-enzymatic componentsthat form functional complexes, co-factors, regulators, andreceptors (Ember and Hugli, 1997). The proteolytic cascadeallows for a considerable amplification, since each pro-teinase molecule activated at one step can generate multiplecopies of an activated enzyme later in the cascade, whichin turn cleaves non-enzymatic components, such as C3, C4,and C5. The larger fragments derived from C3, C4, andC5 (i.e. C3b, C4b, and C5b) are involved in biologic effec-tors functions, such as in opsonization, phagocytosis, andimmunomodulation. However, the smaller molecules, C3a,C4a, and C5a, called anaphylatoxins, induce the releaseof mediators from the mast cells and lymphocytes, whichcause a variety of inflammatory diseases and can be fatalif occurring after organ transplantation (Abbas et al., 1997;Min et al., 2001). Therefore, modulation of complementactivity should be useful in the therapy of inflammatorydiseases.
404 B.-Y. Park et al. / Journal of Ethnopharmacology 90 (2004) 403–408
As a part of our continuing research to find novel anticom-plement active compounds from natural plants, we studiedthe MeOH extract of leaves ofDendropanax morbifera. Thispaper describes the isolation and structure determination, aswell as the anticomplement activity against the CP of thecomplement system of a new polyacetylene compound (1)including to six known compounds (2–7). The anticomple-ment effect on the CP was reported for the first time fromDendropanax morbifera.
2. Materials and method
2.1. Plant material
The leaves ofDendropanax morbifera Leveille were col-lected during May 2000 at Cheju-island (Korea) and iden-tified by Dr. Tae-Jin Kim, Korea Research Institute of Bio-science and Biotechnology (KRIBB), Korea. The voucherspecimen (PB3965.3) was deposited at the herbarium of theKRIBB. Plant material was dried at room temperature.
2.2. General instrumental equipment
Optical rotations were measured with a JASCO DIP-370automatic polarimeter in CHCl3. IR spectrum was obtainedon a Jasco Report-100 spectrometer. FAB-MS was mea-sured on a JMS-AX 110/110A.1H- and13C-NMR spectrawere recorded on a Bruker DMX 300 spectrophotometer,the chemical shifts being represented as pounds per minutewith tetramethylsilane as an internal standard. Column chro-matography was performed on Silica gel 60 (70–230 and230–400 mesh, Merck) and YMC-GEL ODS-A (S-75�m,YMC). In TLC and preparative TLC pre-coated silica gel 60F254 and RP-18 F254Splates (Merck) were used. Spots weredetected under UV light or after spraying with 10% H2SO4reagent, followed by heating.
2.3. Isolation and identification of compounds
The powdered leaves ofDendropanax morbifera (1.1 kg)were extracted with MeOH (3 days, three times). The MeOHextract (110 g) was subjected to column chromatography(CC) on silica gel, which was using a stepwise gradient ofhexane–acetone to give five fractions. The Fr. 2 (11.47 g)was dissolved in acetone kept overnight at room temper-ature to yield compound2 (1.38 g) as a colorless needle.The rest portion of Fr. 2 (8.0 g) was subjected to CC on sil-ica gel, eluted with hexane–acetone (100:0→ 2:1) to fur-nish nine sub-fractions (Fr.−2A, 3.0 g; Fr.−2B, 0.16 g;Fr.−2C, 3.7 g; Fr.−2D, 0.20 g; Fr.−2E, 0.28 g; Fr.−2F,1.8 g; Fr.−2G, 0.93 g; Fr.−2H, 30.33 g; Fr.−2I, 0.43 g). Fr.−2D was applied to preparative TLC (silica gel), which wasthen eluted with hexane–acetone (9:1) to give compound3(49.0 mg). Fr.−2F was subjected to CC on RP-18 silica gel(97%, MeOH in H2O) gave compounds4 (150.0 mg) and
Table 11H- and 13C-NMR data of compound1 (300 MHz, in CDCl3)
δ values in pounds per minute and coupling constants (in parentheses) inhertz.
5 (30.0 mg), while RP-18 silica gel (75→ 100% MeOHin H2O) CC and preparative TLC (RP-18 silica gel, 100%MeOH) of Fr. −2G afforded compounds6 (22.0 mg) and7 (25.6 mg). Column chromatography of Fr.−5 on RP-18silica gel (80% MeOH in H2O) and Sephadex LH-20 (Phar-macia, MeOH) yielded compound1 (1.1 g).
2.4. Anticomplement assay through the classical pathway
A diluted solution of normal human serum (comple-ment serum, 80�l) was mixed with a gelatin vernal buffer(GVB2+, 80�l) with or without sample. Each sample wasdissolved in DMSO, and it was used as negative control.The mixture was pre-incubated at 37◦ for 30 min, afterwhich sensitized erythrocytes (sheep red blood cells, 40�l)were added. After incubation under the same conditions,the mixture was centrifuged (4◦C, 1500 rpm) and the op-tical density of the supernatant (100�l) was measured at450 nm (Yamada et al., 1985). Anti-complement activitywas determined as a mean of triplicate measurements andexpressed as the IC50 values from complement-dependenthemolysis of the control (Oh et al., 1996).
2.5. MTPA (Mosher) esters of compound 1
2.5.1. (R)-MTPA ester of 1 (1a)A solution of1 (5.3 mg) in CH2Cl2 (3 ml) was treated with
(R)-MTPA (10 mg) in the presence of EDC-HCl (20 mg)and 4-DMAP (10 mg). The mixture was refluxed for 6 h andpoured into ice water, and then the whole was extracted withan AcOEt. The AcOEt extract was successively washed with5% aqueous HCl, saturated aqueous NaHCO3 and NaCl,then dried over MgSO4 powder and filtered (Yoshikawaet al., 1999). Removal of the solvent from the filtrate un-der reduced pressure furnished a residue, which was purifiedby preparative TLC (silica gel, CHCl3/MeOH 10:1) to give(R)-MTPA ester (1a). 1H NMR; δ 3.03 (1H, d, H-11), 5.16(1H, br d, J = 9.9 Hz, H-18b), 5.34 (1H, m, H-10), 5.42(1H, m, H-18�), 5.50 (1H, m, H-9), 5.89 (1H, ddd, H-17).
2.5.2. (S)-MTPA ester of 1 (1b)(S)-MTPA (10.2 mg), EDC-HCl (20 mg), and 4-DMAP
(10.3 mg) were added to a mixture of1 (5.7 mg) in CH2Cl2(3 ml). Work up as mentioned above gave the (S)-MTPAester (1b). 1HNMR; δ 3.04 (1H, d, H-11), 5.15 (1H, br d,J = 9.9 Hz, H-18b), 5.35 (1H, m, H-10), 5.40 (1H, m,H-18a), 5.51 (1H, m, H-9), 5.88 (1H, ddd, H-17).
3. Results and discussion
Repeated column chromatography of a MeOH extract ofDendropanax morbifera (leaves) on silica gel followed by
reverse phase C-18, Sephadex LH-20, and preparative TLClet to the isolation of seven compounds1–7. The known com-pounds were identified as dendropanoxide (2) (Tori et al.,1977), �-glutinol (3) (Tanaka et al., 1996), �-amyrin (4)(Kang, 1987), �-amyrin (5) (Kang, 1987), trans-phytol (6)(Brown, 1994), and�-sitosterol (7) (Chang et al., 1981) bycomparing data with those previously reported (Fig. 1).
Compound1 obtained as a viscous liquid with a nega-tive optical rotation ([α]D −25◦). The molecular formulaof C18H24O3 was estimated from the HR-FABMS with amolecular ion peak atm/z 311.1623 [M+ Na]+. The IRspectrum of1 showed the presence of a conjugated triplebond (2230 cm−1), a conjugated double bond (1700 cm−1),and hydroxyl groups (3400 cm−1). The1H-NMR spectrumshowed signals for five olefinic protons atδ 5.92 (ddd,J =17.1, 10.2, 5.4 Hz), 5.50 (m), 5.40 (m), 5.35 (m) and 5.23(br dt), and a methine atδ 4.91 (d,J = 5.4 Hz) (Table 1).The13C-NMR spectrum, aided by DEPT and HMQC spec-tra, revealed the presence of a carbonyl carbon atδ 179.4,four olefinic carbons atδ 136.2, 133.0, 122.0 and 117.0, fourquaternary carbons atδ 64.1, 71.2, 74.3 and 80.1, and oneoxygen-bearing carbon atδ 63.5 (Table 1).
Analysis of the1H–1H COSY, HMQC, and HMBC ofcompound1 allowed its structural fragments to be deter-mined. In the COSY spectrum, a terminal spin system be-gan with an olefinic methylene protons atδ 5.23 and 5.40,which were coupled with another olefinic proton atδ 5.92that were further linked to an oxymethine proton atδ 4.91.The COSY spectrum also showed a separate spin system thatbegan with a methylene proton atδ 3.02 (d,J = 6.9 Hz).This proton was coupled to an olefinic proton atδ 5.35,which, in turn, coupled to its vicinal partner (δ 5.50) thatwas linked overlapped methylenes (H2-3–H2-8). This lattersignal also correlated with a methylene (δ 2.03). In addi-tion, partial structures were linked using the HMBC spec-trum. Long-range correlations betweenδH 3.02 (H-11) andδC 80.1 (C-12)/δC 71.2 (C-13), andδH 4.91 (H-16) andδC 64.1 (C-14)/δC 74.3 (C-15) showed that the conjugateddiynes were linked to C-11 and C-16, respectively. A car-bonyl carbon (δC 179) correlated withδH 2.30 (H-2) andδH 1.30 (overlapped methylenes) in the HMBC spectrum,indicating the end of the spin system. This was further sup-ported by the presence of the prominent ion peak atm/z 144[C8H16O2]+ in the FABMS. This was further confirmed theseven continuous methylenes.
The stereochemistry of C-9 double bond was determinedto be in theZ configuration based on irradiation of H-8 andH-11 peaks in the1H-NMR spectrum. The coupling constant(J = 10.5) of H-9 (δ 5.50) and H-10 (δ 5.35) was observedon irradiation atδ 2.03 (H-8) andδ 3.02 (H-11), respectively.In order to determine the absolute stereochemistry at C-16,compound1 was tested by the modified Mosher’s method(Dale and Mosher, 1973; Ohtami et al., 1991). Under thereaction conditions with (R)-MTPA and (S)-MTPA in thepresence of 4-DMAP and EDC-HCl,1 gave 16-(R)-MTPAester (1a) and 16-(S)-MTPA ester (1b). The proton signals
406 B.-Y. Park et al. / Journal of Ethnopharmacology 90 (2004) 403–408
Fig. 1. Structures of compounds (1–7) isolated fromDendropanax morbifera leaves.
assigned for H-9 and H-11 in the (S)-MTPA ester (1b) wereobserved at lower field compared to those of the (R)-MTPAester (1a), while the proton signal due to H-17 and H-18in the former ester was observed at a higher field com-pared to those in the latter ester (Fig. 2). Consequently, theabsolute stereochemistry at C-16 was determined to beS
Fig. 2. Chemical shift difference for the (S)-MTPA ester (1b) and (R)-MTPA ester (1a) in pounds per minute.
(Min et al., 2000). Finally, the structure of1 was determinedas (9Z,16S)-16-hydroxy-9,17-octadecadiene-12,14-diynoicacid.
Compounds1–7 were tested for their anti-complementactivity in the complement system. The results (IC50 values)are summarized inTable 2. Of these, compound1 showed
B.-Y. Park et al. / Journal of Ethnopharmacology 90 (2004) 403–408 407
Table 2Inhibitory effects of compounds isolated from the leaves ofDendropanaxmorbifera on the classical pathway of the complement system in vitro
a Data are expressed as mean± S.E.M. of three experiments.b This compounds were used as positive control.
significant effect on the CP of the complement system withIC50 value of 56.98�M, compared to tiliroside (IC50 =70.09�M), which were used as positive controls (Jung et al.,1998). On the other hand, compounds2–7 were completelyincapable of inhibiting complement activity.
The family Araliaceae is a rich source of C17 linear poly-acetylene almost isolated fromPanax ginseng (Hirakuraet al., 1991; Kwon et al., 1997). Some reports have ap-peared of this type of compound in genusDendropanax,such as falcarinol fromDendropanax trifidus (Kawazuet al., 1973), and dehydrofalcarinol, falcarindiol, dehydro-falcarindiol, dendroarboreols A and B fromDendropanaxarboreus (Bernart et al., 1996). The biological evaluation ofpolyacetylenes from this family has been variously reportedas a skin sensitizer and irritant, toxic trace constituent ofcarrot, probable phytoalexin, thromboxane inhibitor, andcytotoxic activity (Bernart et al., 1996). Furthermore, linearpolyacetylenes have become a major element in the searchfor bioactive substances from marine sponges. These com-pounds have been also reported cytotoxic, antimicrobial, an-tiviral, RNA cleaving, and enzyme-inhibitory activities, aswell as brine-shrimp lethality (Jung et al., 2002). Although,to our knowledge this is the first report of anticomplementactivity against the CP of the complement system of poly-acetylene isolated fromDendropanax morbifera. Based onthe biological activity of compound1 against complementactivity, more detailed study will be need to clarify the ac-tion against anticomplement activity of polyacetylene fromDendropanax morbifera.
Acknowledgements
This research was supported by a grant (PF0300401-00)from the Plant Diversity Research Center of the 21stCentury Frontier Research Program funded by theMinistry of Science and Technology of the Koreangovernment. We are grateful to Korea Basic ScienceInstitute, Daejeon, Korea, for NMR and mass spectralmeasurements.
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Chang, I.M., Yun, H.S., Yamasaki, K., 1981.�-Sitosterol and�-sitosteryl-3-O-�-d-glucopyranoside isolated fromPlantago asiatica seed. KoreanJournal of Pharmacognosy 12, 12–24.
Dale, J.A., Mosher, H.S., 1973. Nuclear magnetic resonance enantiomerreagents. Configurational correlationsvia nuclear magnetic resonancechemical shifts of diastereomeric mandelate,O-methylmandelate, and�-methoxy-�-trifluoromethylphenylacetate (MTPA) esters. Journal ofthe American Chemical Society 95, 512–519.
Ember, J.A., Hugli, T.E., 1997. Complement factors and their receptors.Immunopharmacology 38, 3–15.
Gil, R.R., Ulubelen, A., Chai, H.B., Pezzuto, J.M., Cordell, G.A., 1994.Biologically active compounds from the Euphorbiaceae 2. Two triter-penoids ofEuphobia cyparissias. Planta Medica 60, 594–596.
Han, S.H., Jung, Y.H., Oh, M.H., Ko, M.H., Oh, Y.S., Koh, S.C., Kim,M.H., Oh, M.Y., 1998. Phytogenetic relationships of theDendropanaxmorbifera and D. trifidus based on PCR-RAPD. Korean Journal ofGenetics 20, 173–181.
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Jung, K.Y., Oh, S.R., Park, S.H., Lee, I.S., Ahn, K.S., Lee, J.J., Lee,H.K., 1998. Anticomplementary activity of tiliroside from the flowerbuds ofMagnolia fargesii. Biological and Pharmaceutical Bulletin 21,1077–1078.
Jung, H.J., Min, B.S., Park, J.Y., Kim, Y.H., Lee, H.K., Bae, K.H., 2002.Gymnasterkoreaynes A–F, cytotoxic polyacetylenes fromGymnasterkoraiensis. Journal of Natural Products 65, 897–901.
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Journal of Ethnopharmacology 90 (2004) 409–414
Anti-inflammatory activity ofSedum kamtschaticum
Dong Wook Kima, Kun Ho Sonb, Hyeun Wook Changc,KiHwan Baed, Sam Sik Kange, Hyun Pyo Kima,∗
a College of Pharmacy, Kangwon National University, Chunchon 200-701, South Koreab Department of Food& Nutrition, Andong National University, Andong 760-749, South Korea
c College of Pharmacy, Yeungnam University, Gyongsan 712-749, South Koread College of Pharmacy, Chungnam National University, Chungnam, South Korea
e Natural Products Institute, Seoul National University, Seoul 110-460, South Korea
Received 8 March 2003; received in revised form 1 November 2003; accepted 6 November 2003
Eicosanoids including prostaglandins (PG) and leukotrie-nes (LT) are inflammatory mediators. They are biosynthe-sized by cyclooxygenases (COX) and lipoxygenases (LOX)in many cell types and deeply associated with inflamma-tory disorders, acute as well as chronic inflammation. Aninhibition of eicosanoid production is one of the impor-tant therapeutic strategies for various inflammatory diseases.Among the eicosanoid generating enzymes, an inducibleisoform of cyclooxygenase, COX-2, was found to be piv-otal to produce prostanoids in the inflammatory site (Seibertet al., 1994). Several COX-2 inhibitors have been developedand clinically prescribed showing less side effects. In accor-dance with this respect, development of COX-2 inhibitorsor modulators of COX-2 expression may be a significantsubject to find new anti-inflammatory agents.
Sedum kamtschaticumFischer (S. k.), Crassulaceae, isperennial and common in Korea, China, and Japan. Thewhole part of this plant has been used in the form of water
extract as a folk medicine for improving blood circulation,anti-anxiety and anti-inflammation (Bae, 1999). AlthoughS. k. is widely distributed in North-East Asia, the chem-istry and biological activity are rarely known. Especially,its anti-inflammatory activity has not been described despiteof frequent use for anti-inflammatory crude drug. Duringour program to search for new anti-inflammatory agents, themethanol extract from the whole part of S. k. was found tostrongly inhibit PGE2 production from lipopolysaccharide(LPS)-induced RAW 264.7 cells, a mouse macrophage cellline, in the preliminary experiment. This finding suggestsan anti-inflammatory potential of this plant material. There-fore, the present investigation was carried out to establishCOX-2 modulating potential and in vivo anti-inflammatoryactivity of S. k.
2. Materials and methods
2.1. Chemicals
N - [2- (Cyclohexyl) - 4-nitrophenyl]-methanesulfonamide(NS-398) was purchased from Biomol (Plymouth Meeting,
410 D.W. Kim et al. / Journal of Ethnopharmacology 90 (2004) 409–414
PA). Prednisolone was obtained from Upjohn Co. (Kalama-zoo, MI). Arachidonic acid (AA, 99%), lipopolysaccharide(LPS, Escherichia coli0127:B8), 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT), croton oil,�-carrageenan (CGN), carboxymethyl cellulose (CMC),and 12-O-tetradecanoylphorbol 13-acetate (TPA) were pur-chased from Sigma-Aldrich. DMEM and other cell culturereagents including FBS were obtained from Gibco BRL(Grand Island, NY). Heat-killedMycobacterim butyricumwas a product of Difco Lab. (Detroit, MI). EIA kit for PGE2
Fig. 1. Inhibition of PGE2 production from LPS-treated RAW 264.7 cells bySedum kamtschaticum. (A) Effect of pretreatment of S. k. on PGE2
production. S. k. and LPS were simultaneously added to RAW cells. After 24 h, PGE2 concentration was measured using EIA kit as described inSection2. LPS treatment produced 2.53± 0.02 nM of PGE2 for 24 h from the basal level of 0.35± 0.08 nM of PGE2. Hundred percent PGE2 production in thisfigure represented the difference of these two values. (B) Effect of S. k. on PGE2 production by pre-induced COX-2. To induce COX-2, LPS was treatedon RAW cells for 24 h. After washing thoroughly, S. k. and arachidonic acid were added and incubated for another 30 min. PGE2 concentration in themedia was measured using EIA kit. Hundred percent production represented 2.19± 0.02 nM of PGE2, n = 3. ∗P < 0.005, significantly different fromLPS-treated control. (C) Effect of S. k. on COX-2 expression (western blotting). S. k. and LPS were simultaneously added to RAW cells. After 20 h,cells were harvested and homogenized. The same amount of protein was used for electrophoresis (10�g per lane).
and anti-COX-2 antibody (No. 160116) were purchasedfrom Cayman Chem. (Ann Arbor, MI).
2.2. Plant material
The whole plant ofSedum kamtschaticumFischer (S. k.)was collected on August 15, 2000 at Taejon, Korea. Thisplant material was authenticated by Dr. K. Bae and a voucherspecimen has been deposited in the Herbarium of College ofPharmacy, Chungnam National University under the regis-
D.W. Kim et al. / Journal of Ethnopharmacology 90 (2004) 409–414 411
tration number CNU 869. The dried whole plant (1 kg) wasextracted in methanol (5 l) at room temperature for 7 days.The residue was evaporated to dryness under vacuo (212 g)and used throughout this study.
2.3. Animals
Male ICR mice (16–20 g) and Spraque–Dawley (SD)rats (150–180 g) were obtained from Korea ExperimentalAnimal Co. (Seoul) and fed with laboratory chow (PurinaKorea) and water ad libitum. Animals were acclimatizedin a specific pathogen-free animal facility under the condi-tions of 20–22◦C, 40–60% relative humidity, and 12 h/12 h(light/dark) cycle at least for 7 days.
2.4. RAW 264.7 cell culture, measurement of PGE2concentration, and western blotting of COX-2
RAW 264.7 cells obtained from American Type CultureCollection were cultured in DMEM supplemented with 10%FBS and 1% antibiotics under 5% CO2 at 37◦C and acti-vated with LPS. All procedures including western blottingtechnique were based on the previously described proce-dures (Chi et al., 2001). Briefly, cells were cultured in 96-well plates (2×105 cells per well). After 2 h pre-incubation,LPS (1�g/ml) and various concentrations of S. k. or NS-398were added and incubated for 24 h, unless otherwise speci-fied. S. k. and NS-398 were dissolved in DMSO and dilutedwith serum-free DMEM into appropriate concentrations.Cell viability was accessed by MTT bioassay (Mossman,1983). PGE2 concentration in the media was measured us-ing EIA kit following the manufacturer’s recommendation.To determine the direct inhibitory activity of COX-2 byS. k., cells were incubated with LPS (1�g/ml) for 24 h inorder to allow COX-2 induction, and washed thoroughlythree times with serum-free DMEM. S. k. and AA (10�M)were added and incubated for another 30 min. PGE2 con-centration in the media was measured using EIA kit as sameas described above. For a western blotting of COX-2, cellswere cultured in 6-well plates (5× 106 cells per well) in thepresence or absence of LPS (1�g/ml) with/without S. k. for20 h. After preparing cell homogenate, proteins were sep-arated on 4–15% Tris-glycine gel (Novex Laboratory) byelectrophoresis and bands were blotted to PVDF membrane.COX-2 band was visualized using anti-COX-2 antibodyand horseradish peroxide-conjugated secondary antibody.
2.5. In vivo anti-inflammatory activity
In order to evaluate the inhibitory activity ofSedumkamtschaticumagainst animal models of acute inflamma-tion, mouse croton oil-induced ear edema and rat CGN-induced paw edema were employed according to thepreviously described procedures (Kim et al., 1993; Winteret al., 1962). Briefly, 2.5% croton oil in acetone was top-ically applied to ears of mice (20�l per ear). Five hours
later, the ear thickness was measured using a dial thicknessgauge (Lux Scientific Instrument, USA). For provoking pawedema, 1% CGN dissolved in pyrogen free saline (0.05 ml)was injected to right hind paw of rats. After 5 h, a swellingof the treated paw was measured using plethysmometer(Ugo Basile, Italy). S. k. and prednisolone dissolved in 0.5%CMC were administered orally 1 h prior to the treatmentof inflammagen. To measure anti-inflammatory activity inan animal model of subchronic inflammation, TPA-inducedear edema assay (multiple treatment with TPA) was carriedout using a slightly modified procedure ofStanley et al.(1991). TPA dissolved in acetone (3�g/10�l) was topicallyapplied to ears of mice once a day for 3 days. S. k. andprednisolone dissolved in 0.5% CMC were orally admin-istered once a day for 3 consecutive days 2 h after TPAtreatment. At 12-h interval, the ear thickness was measuredusing a dial thickness gauge. For evaluating inhibitory ac-tivity against chronic inflammation, rat adjuvant-inducedarthritis assay (AIA) was used according to the previouslyreported (Kim et al., 1999). An arthritic inflammation wasprovoked by injection ofMycobacterium butyricum(0.6 mgper rat) dissolved in mineral oil to right hind paw of rats.S. k. and prednisolone were orally administered everyday.The swelling of the treated and the untreated paws wasmeasured using plethysmometer for 20 days.
2.6. Analgesic activity
For measuring analgesic activity, standard acetic acid-induced writhing test was employed according to the previ-ously described procedure (Bentley et al., 1983). S. k. andaspirin were orally administered to mice. One hour later,100�l of acetic acid (0.7%) was administered intraperi-toneally and numbers of writhing were counted for 10 minstarting 10 min after administration of acetic acid solution.
Table 1Inhibition of Sedum kamtschaticumagainst ear edema and paw edema
Values in parenthesis represent percent inhibition of edematic response.NT; not tested;n = 5.
a Mouse croton oil-induced ear edema.b Rat CGN-induced paw edema.∗ P < 0.05, significantly different from the inflammagen-treated group
in each column.
412 D.W. Kim et al. / Journal of Ethnopharmacology 90 (2004) 409–414
2.7. Statistical analysis
All values were represented as arithmetic mean± S.D.One-way ANOVA was used to determine the statistical sig-nificance.
3. Results
A treatment of LPS (1�g/ml) in RAW 264.7 cells in-duced COX-2 expression producing a large amount ofPGE2 and the peak concentration of COX-2 was previ-ously determined to be 10–20 h after a treatment of LPS(Chi et al., 2001). When added simultaneously with LPS,S. k. (5–25�g/ml) clearly inhibited PGE2 production from
Table 2Inhibition of Sedum kamtschaticumagainst ear edema induced by the multiple treatment of TPA
Group/dose (mg/kg per day) Ear thickness increased (mm)
Values in parenthesis represent percent inhibition of ear edematic response,n = 5.a Hours after first treatment of TPA.b TPA was topically applied to ears of mice once a day for 3 days.∗ P < 0.05, significantly different from the TPA-treated group in each column.
Days after adjuvant injection
0 2 4 6 8 10 12 14 16 18 20
Incr
ease
d ed
ema
volu
me
of t
he t
reat
ed p
aw(m
l)
0.0
0.5
1.0
1.5
2.0
2.5
3.0
**
* * ** * * **
Fig. 2. Effect ofSedum kamtschaticumon rat adjuvant-induced arthritis.Mycobacterium butyricumwas subplantarly injected to right hind paw of rats. S.k. (1, 5, 10, 50, 200 mg/kg per day) was orally administered everyday. The edema volume increased of the treated paw from the vehicle-treated controlwithout adjuvant was represented here. Adjuvant-treated without test compound (�), S. k. (50 mg/kg per day,�), prednisolone (5 mg/kg per day,�).Note: All doses of S. k. did not show a significant inhibition,n = 6. ∗P < 0.05, significantly different from adjuvant-treated group.
LPS-induced RAW 264.7 cells concentration-dependentlyfor 24-incubation period (IC50 = 6.7�g/ml) as shownin Fig. 1A. NS-398 (COX-2 inhibitor) potently inhibitedPGE2 production as expected (95% inhibition at 10�M).In contrast, S. k. at the same concentration range, whenadded after COX-2 was fully induced, did not significantlyinhibited PGE2 production, whereas NS-398 still inhibitedPGE2 production (Fig. 1B). This finding indicated that S.k. did not inhibit COX-2 enzyme activity directly. Instead,S. k. suppressed COX-2 induction at 5–25�M, revealed bywestern blotting experiment (Fig. 1C). S. k. and NS-398at the tested concentration did not show any cytotoxic-ity judged by MTT assay, indicating that the inhibitionof PGE2 production by S. k. was not associated with itscytotoxicity.
D.W. Kim et al. / Journal of Ethnopharmacology 90 (2004) 409–414 413N
umbe
r of
Wri
thin
g
0
5
10
15
20
50 200 10 50 200 400Aceticacid Aspirin S. k.
mg/kg
*
*
*
Fig. 3. Analgesic activity ofSedum kamtschaticumin mice. The standardacetic acid-induced writhing in mice was employed. Acetic acid-treatedgroup showed 16.3 ± 4.8 writhings for 10 min, n = 6. ∗P < 0.05,significantly different from acetic acid-treated group.
Table 1demonstrated that S. k. showed dose-dependentanti-inflammatory activity by oral administration againstanimal models of acute inflammation (47% inhibition at200 mg/kg in croton oil-induced ear edema and 30% inhibi-tion at 800 mg/kg in CGN-induced paw edema). A referencecompound, prednisolone, showed 54 and 36% inhibition at10 mg/kg against ear edema and paw edema, respectively. Ina subchronic model of skin inflammation provoked by mul-tiple treatment of TPA (Table 2), S. k. significantly inhibitedear edema up to 60 h (23% inhibition at 200 mg/kg per day,60 h). Prednisolone (2 mg/kg per day) also showed similarinhibitory activity against same animal model (25%, 60 h).Against an animal model of chronic inflammation, however,S. k. did not show the significant inhibitory activity in ratAIA-induced arthritic inflammation at doses of 5–200 mg/kgper day for 20 days, while prednisolone (5 mg/kg per day)showed potent inhibition as shown inFig. 2.
S. k. possessed strong analgesic activity on acetic acid-induced writhing at doses of 10–400 mg/kg with an IC50value of 125 mg/kg (Fig. 3). Aspirin also showed analgesicactivity, 43 and 56% inhibition at 50 and 100 mg/kg, respec-tively.
In order to evaluate the acute toxicity, S. k. was orally ad-ministered to mice up to 3.2 g/kg (two-fold dilution). During14 days after administration, no apparent toxicity includ-ing dead animals and the change of major organ weights(lung, heart, liver, spleen, and kidney) was observed (datanot shown).
4. Discussion and conclusions
The present investigation clearly demonstrated that themethanol extract of S. k. inhibited PGE2 production frommacrophage cell line in vitro, at least partly, by down-regulation of COX-2 expression. And it possessed anti-
inflammatory activity in vivo against several animal modelsof inflammation, although the potency of anti-inflammatoryactivity by S. k. was less than that of prednisolone.
S. k. reasonably inhibited acute as well as subchronicinflammation dose-dependently. S. k., however, did not sig-nificantly inhibit chronic inflammation in rat AIA test. Theprecise reason for this result is not understood at present.But, it is speculated that S. k. may not affect lymphocyteactivity, since T-lymphocytes play a major role in AIAmodel (Otterness and Bliven, 1985). This speculation maybe also supported by the evidence that S. k. (5–100 mg/kgper day) only weakly inhibited picryl chloride-induceddermatitis (less than 25% inhibition, data not shown), inwhich activated T-lymphocytes participate to provoke de-layed hypersensitivity reaction. No significant toxicity ofthis plant material by oral administration may indicate thatit is practically nontoxic and may be used safely in humanat moderate doses.
The genusSedumis classified as eleven species and threevarieties in Korea (Lee, 1997). Some species of the genusSedumhave been used as a folk medicine for inflamma-tory diseases (Bae, 1999). Although S. k. is the most com-mon among them, a few reports have been published. Oneof the related species,Sedum sarmentosum, was previouslydescribed to contain alkaloids and terpens as major con-stituents, and its anti-hepatoxic property was also demon-strated (Aimin et al., 1998; Kang et al., 2000).
All results from the present investigation suggest that S.k. may be used as an anti-inflammatory agent with analgesicactivity. The inhibition of prostanoid production by COX-2down-regulation may be, at least in part, one of the cellularaction mechanism(s) of anti-inflammation by S. k. As far asour best knowledge, this is the first report of in vivo anti-inflammatory activity of S. k. and provides a scientific basisof medicinal use of this plant material against inflammatorydisorders.
Acknowledgements
This research was financially supported by a grant(PF0320302-00) from Plant Diversity Research Center of21st century Frontier Research Program funded by Ministryof Science and Technology of Korean government.
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Journal of Ethnopharmacology 90 (2004) 415–419
Protective effect of Moutan Cortex extract onacetaminophen-induced hepatotoxicity in mice
Yun-Hee Shon, Kyung-Soo Nam∗Department of Pharmacology, College of Medicine and Intractable Disease Research Center,
Dongguk University, Sukjang-Dong 707, Kyongju 780-714, South Korea
Received 23 May 2003; received in revised form 27 October 2003; accepted 6 November 2003
Acetaminophen (N-acety1-p-aminophenol, paracetamol,AAP) is a clinically important over-the-counter drug com-monly used for its analgesic and antipyretic properties.At the therapeutic doses, AAP is considered a safe drug.However, it can cause hepatic necrosis, nephrotoxicity,extrahepatic lesions, and even death in humans and experi-mental animals when taken in overdoses (Ray et al., 1996;Webster et al., 1996). AAP is metabolized by a cytochromeP450-dependent pathway to an electrophilic metabolite,N-acetyl-p-benzoquinone imine (NAPQI) (Lee et al., 1996).Therapeutic doses of this drug are safely biotransformedand eliminated as non-toxic conjugates of sulfate and glu-curonic acid (Nelson, 1990), and only a small fraction isconverted to NAPQI which is detoxified by glutathione(GSH) and eventually eliminated in the urine or bile (Coleset al., 1988). Alternatively quinone compound, NAPQI canbe reduced by a direct two-electron reduction pathway tonon-toxic hydroquinones by quinone reductase (QR). The
direct two-electron reduction occurs without any productionof the toxic semiquinone and may provide protection againstthe toxicity caused by NAPQI (Hajos and Winston, 1992).However, during overdose of AAP, the glucuronidationand sulfation routes become saturated and more extensivebioactivation of AAP occurs, leading to rapid depletion ofhepatic GSH levels. Thus, unconjugated NAPQI can exertits cytotoxic effect by covalent binding to cellular macro-molecules, membrane lipid peroxidation, and alteration ofcalcium homeostasis (Cohen and Khairallah, 1997). Re-cently, another pathway of AAP toxicity was proposedthe potential of AAP to influence the integrity of genomicDNA, and it may lead to cell death in the liver (Ray et al.,1996).
Moutan Cortex, the root cortex ofPaeonia suffruticosaAndr., is an important Chinese herbal medicine used as anal-gesic, antispasmodic, and anti-inflammatory agent. The drughas long been used in remedies for female diseases.
The current authors previously demonstrated the protec-tive effect of Moutan Cortex against AAP-induced cytotox-icity in vitro (Shon and Nam, 2002). In the present study,we examined the effect of Moutan Cortex on AAP-inducedhepatotoxicity in mice and elucidated the mechanisms un-derlying this effect.
416 Y.-H. Shon, K.-S. Nam / Journal of Ethnopharmacology 90 (2004) 415–419
2. Materials and methods
2.1. Plant material and extraction
Moutan Cortex was purchased from Dongguk UniversityOriental Medical Center in Kyongju, Korea. A voucherspecimen (no. 00M-18) has been deposited in the herbar-ium of the Intractable Disease Research Center, DonggukUniversity, Kyongju, Korea. Dried Moutan Cortex (60 g)was extracted with distilled water (240 ml) for 3 h atroom temperature. The extract was filtered, and the filtrate(100 ml) was concentrated in vacuo and lyophilized. Thelyophilized extract (3.4 g) of Moutan Cortex was dissolvedin phosphate-buffered saline (PBS).
2.2. Animals and treatments
Adult male ICR mice weighing 25–35 g were obtainedfrom the Dae-Han Laboratory Animal Research Center(Eumsung, Korea) and given access to lab chow and topwater ad libitum. Mice were acclimated for 1 week be-fore experiments. There were primarily four experimentalgroups: control (vehicle), Moutan Cortex alone, AAP alone,and Moutan Cortex plus AAP. Moutan Cortex was dis-solved in PBS and administered (200 or 400 mg/kg, oralgavage) for 14 days. AAP dissolved in PBS was admin-istered (400 mg/kg, i.p.) on day 14 after Moutan Cortexexposure. Food and water were readministered to the ani-mal 2 h after the AAP treatment. Animals were sacrificed24 h after AAP administration and tissues (blood and liver)were obtained for biochemical analyses. Portions of freshlivers were collected in liquid N2 and preserved at−70◦Cuntil further analysis. The remaining portions of the liverswere homogenized in 0.25 M sucrose. Microsomes and thecytosol fractions were prepared from liver homogenatesby differential centrifugation. The protein concentrations(homogenate, microsome, and cytosol) were determined byusing a bicinchoninic protein assay kit (Sigma, St. Louis,MO) with bovine serum albumin as the standard.
Hepatotoxicity was determined by measuring serum ALT(EC 2.6.1.2) activity. ALT activity was determined with aSigma kit (no. 59-UV) based on the method ofBergmeyeret al. (1978)in which enzyme activity is calculated from thedecrease in NADH absorbance atλ 340 nm.
2.4. Determination of glutathione (GSH) levels
The GSH content of liver homogenates was determined byan enzymatic recycling procedure (Griffith, 1980). The ex-tent of 2-nitro-5-thiobenzoic acid formation was monitoredat 405 nm. The GSH content was calculated in comparisonwith a standard GSH curve. The GSH levels were expressedas nmol/mg protein.
2.5. Quinone reductase (QR) activity
The QR specific activity of liver cytosol fractionwas measured by the addition of 200�l of a mixture(0.5 M Tris–HCl (pH 7.4), bovine serum albumin, 1.5%Tween-20, 7.5 mM FAD, 150 mM glucose-6-phosphate,50 mM NADP, yeast glucose-6-phosphate dehydrogenase,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-mide (MTT), and 50 mM menadione in distilled water) to50�l of a suitable dilution of the tissue cytosol fraction ac-cording to the method ofProchaska and Santamaria (1988).The induction of QR activity was calculated from the ratioof the specific enzyme activity of the sample-treated mouseliver in comparison with the solvent control.
2.6. Microsomal enzyme assays
The activity of microsomal aniline hydroxylase was mea-sured as the rate ofp-aminophenol formation from aniline,according to the method ofImai et al. (1966). Hydroxyla-tion of p-nitrophenol to nitrocatechol was determined by themethod ofKoop (1986).
2.7. Quantitative evaluation of DNA fragmentation
To measure hepatic DNA fragmentation by spectropho-tometry, a portion of the liver frozen in liquid N2 was ho-mogenized in chilled lysis buffer (10 mM Tris–HCl, 20 mMEDTA, 0.5% Triton X-100, pH 8.0). Homogenates were thencentrifuged at 27,000×g for 20 min to separate intact chro-matin in the pellet from DNA cleavage product in the su-pernatant. Pellets were resuspended in 0.5N perchloric acid,and concentrated perchloric acid was added to supernatantsamples to reach a final concentration of 0.5N. Resuspendedpellets and supernatant were heated at 90◦C for 15 min andcentrifuged at 1500× g for 10 min to remove protein. Re-sulting supernatants were then reacted with diphenylaminefor 16–20 h at room temperature. Absorbance at 600 nm wasmeasured using a Gilford spectrophotometer (Gilford, Ober-lin, OH). The amount of DNA fragmentation in control sam-ple (frag. DNA/(frag. DNA+ intact DNA)) is expressed asthe percentage of total DNA that appears in the supernatantfraction. Treatment effects on DNA fragmentation are re-ported as the percentage of control fragmentation.
2.8. Statistical analysis
The data were analyzed for statistical significance usingStudent’st test.P values of less than 0.05 were consideredto be significant.
3. Results
AAP-induced hepatotoxicity and its prevention byMoutan Cortex pre-exposure are shown inTable 1. AAP at
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Table 1Effect of Moutan Cortex administration on AAP-induced increase in serumalanine aminotransferase (ALT) levels
a Each value represents the mean± S.D. of 10 mice.∗ P < 0.05; control vs. AAP.∗∗ P < 0.05; AAP vs. Moutan Cortex+ AAP.
400 mg/kg dose caused severe hepatoxicity as indicated bysharp increase of serum ALT activity. Exposure of 200 or400 mg/kg Moutan Cortex alone did not induce any toxic-ity, whereas Moutan Cortex followed by AAP afforded ahepatoprotection (Table 1).
GSH concentrations in the liver were determined to estab-lish whether Moutan Cortex-induced reduction in the hep-atotoxicity is associated with hepatic GSH levels. Hepaticcontents of GSH in mice were elevated by 14.8 or 31.7%with the treatment of 200 or 400 mg/kg Moutan Cortex, re-spectively (Fig. 1). AAP treatment alone depleted GSH con-tent to 47.5% of the control level (Fig. 1). Pretreatment ofmice with Moutan Cortex protected the GSH depletion pro-duced by AAP. The high-dose Moutan Cortex pretreatmentwas more effective in preventing hepatic GSH depletion thanthe low-dose pretreatment, but there was still 21.7% GSHdepletion in the high-dose group compared with the controlgroup (Fig. 1).
Quinone reductase plays a role in the reduction of quinoneand provides protection against hepatotoxicity caused by the
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Fig. 1. Effect of Moutan Cortex administration on AAP-induced hepaticGSH level in mice. ICR mice were treated with vehicle (control), MoutanCortex (MC) (200 or 400 mg/kg, oral gavage), AAP (400 mg/kg, i.p.),or Moutan Cortex (MC) plus AAP (MC+ AAP). Moutan Cortex wasadministered for 14 days. AAP was administered on day 14 after MoutanCortex exposure. Data shown are mean values with bars indicating theS.D. of 10 mice.∗P < 0.05; control vs. AAP;∗∗P < 0.05; AAP vs.MC + AAP.
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Fig. 2. Effect of Moutan Cortex administration on AAP-induced hepaticNAD(P)H:quinone oxidoreductase (QR) activity in mice. ICR mice weretreated with vehicle (control), Moutan Cortex (MC) (200 or 400 mg/kg,oral gavage), AAP (400 mg/kg, i.p.), or Moutan Cortex (MC) plus AAP(MC + AAP). Moutan Cortex was administered for 14 days. AAP wasadministered on day 14 after Moutan Cortex exposure. Data shown aremean values with bars indicating the S.D. of 10 mice.∗P < 0.05; controlvs. AAP; ∗∗P < 0.05; AAP vs. MC+ AAP.
toxic quinone intermediate (NAPQI) arising from AAP oxi-dation. The effect of Moutan Cortex pretreatment on quinonereductase activity is reported inFig. 2. The administrationof AAP decreased QR activity to 80.1% of the control.Pretreatment of 200 and 400 mg/kg Moutan Cortex beforeAAP administration resulted in 20.2 and 44.1% increases ofAAP-induced decrease of cytosolic QR activity, respectively(Fig. 2).
AAP requires cytochrome P450 2E1-associated bioacti-vaton to produce liver injury. To evaluate another possiblemechanism of hepatoprotection produced by Moutan Cor-tex, the effect of Moutan Cortex treatment of mice on livermicrosomal cytochrome P450 2E1 was examined by mea-suring the oxidation of substrate, aniline andp-nitrophenol,that are selectively metabolized by cytochrome P450 2E1.Table 2 shows that the hydroxylation of aniline was in-
Table 2Effect of Moutan Cortex administration on AAP-induced microsomalaniline andp-nitrophenol hydroxylation activities in mice
a Each value represents the mean± S.D. of 10 mice.∗ P < 0.05; control vs. AAP.∗∗ P < 0.05; AAP vs. Moutan Cortex+ AAP.
418 Y.-H. Shon, K.-S. Nam / Journal of Ethnopharmacology 90 (2004) 415–419
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Fig. 3. Effect of Moutan Cortex administration on AAP-induced DNAfragmentation in mice. ICR mice were treated with vehicle (control),Moutan Cortex (MC) (200 or 400 mg/kg, oral gavage), AAP (400 mg/kg,i.p.), or Moutan Cortex (MC) plus AAP (MC+AAP). Moutan Cortex wasadministered for 14 days. AAP was administered on day 14 after MoutanCortex exposure. Data shown are mean values with bars indicating theS.D. of 10 mice.∗P < 0.05; control vs. AAP;∗∗P < 0.05; AAP vs.MC + AAP.
creased in AAP-treated mice. Moutan Cortex exposure priorto AAP produced statistically significant decrease in ani-line hydroxylase activity relative to AAP alone exposure.Microsomalp-nitrophenol hydroxylase activity was furtherexamined to evaluate the effect of Moutan Cortex treat-ment on the activity of cytochrome P450 2E1. The activityof p-nitrophenol hydroxylase was elevated in AAP-treatedmice. Pre-exposure of 200 and 400 mg/kg Moutan Cortexprior to AAP administration reducedp-nitrophenol hydrox-ylase activity by 35.2 and 55.1%, respectively.
Quantitative evaluation of DNA damage based on a sed-imentation assay has been presented inFig. 3. Fragmenta-tion was assessed by centrifuging liver homogenates and re-acting unsedimented DNA fragments with diphenylamine.Moutan Cortex treatment alone did not alter the integrity ofgenomic DNA. AAP treatment produced significant DNAdamage. A 400 mg/kg of AAP caused 2.1-fold increase ofDNA fragmentation compared with the control. However,Moutan Cortex (400 mg/kg) pre-exposure showed a signifi-cant impact on AAP-induced DNA fragmentation, and bothconcentrations (200 and 400 mg/kg) were effective in reduc-ing DNA damage (Fig. 3).
4. Discussion
The levels of hepatic enzyme, alanine aminotransferase(ALT), primarily reflects the degree of liver damage and hasbeen commonly used a diagnostic marker for hepatotoxicity(Pumford et al., 1997). The high serum ALT levels in mice24 h following AAP exposure (400 mg/kg) clearly indicate
AAP-mediated hepatotoxicity in these animals. Moutan Cor-tex pretreatment protected against AAP-induced liver injury.
Previous studies on the mechanisms of AAP-induced hep-atotoxicity have shown that GSH plays a key role in thedetoxification of the reactive toxic metabolites of AAP, andthat liver necrosis begins when GSH stores are markedly de-pleted (Mitchell et al., 1973). Our results showed that pre-treatment with Moutan Cortex (200 or 400 mg/kg) reducedAAP-induced hepatic GSH depletion. This result is prob-ably due to the inhibition of the bioactivation of AAP byMoutan Cortex resulting in decreased formation of NAPQI.In addition, the components of Moutan Cortex may directlyconjugate with NAPQI, the reactive intermediate.
Another mechanism by which the hepatotoxicant AAPproduces liver injury involves its biotransformation by livermicrosomal cytochrome P450 enzymes to more toxic chem-icals. Early studies have demonstrated that AAP-inducedhepatotoxicity can be modulated by substances which influ-ence cytochrome P450 enzymes activities (Mitchell et al.,1973; Potter et al., 1974). Several cytochrome P450 enzymesare known to play an important role in AAP bioactivationto NAPQI. Ethanol was reported to increase the toxicity ofAAP in mice (Snawder et al., 1994), thus suggesting the in-volvement of cytochrome P450 2E1 in vivo. In vitro studieshave also implicated cytochrome P450 1A2 in addition tocytochrome P450 2E1 in AAP metabolism, although the lat-ter P450 had a lowerKm than cytochrome P450 1A2 (Raucyet al., 1989). Therefore, the effect of Moutan Cortex on hep-atic microsomal cytochrome P450 2E1 activity was exam-ined as a means of protection by Moutan Cortex. We havedemonstrated that AAP treatment significantly increased therates of hepatic microsomal hydroxylation of aniline andp-nitrophenol, both highly associated with the activity of cy-tochrome P450 2E1. Mice hepatic microsomal cytochromeP450 2E1 activity was decreased following treatment withMoutan Cortex. These results demonstrated that there is agood correlation between the decrease in cytochrome P4502E1 activity and protection against AAP-induced hepatotox-icity. In the presence of Moutan Cortex, the transformationof AAP to its reactive metabolite may be inhibited. This in-hibition would offer protection against AAP-induced hepa-totoxicity by inducing an increase in the intracellular con-centration of nonmetabolized AAP, thus preventing the GSHconsumption required by NAPQI.
Stability of the cellular genomic apparatus is constantlychallenged by environmental toxicants, high energy radia-tions, oxidative damage, or factor imbalance, which generateDNA lesions. The studies with AAP have shown that AAPcauses liver Ca2+ to rise, which in turn leads to endonucle-ase activation and DNA fragmentation in the nucleus (Rayet al., 1993; Shen et al., 1992). In this study, Moutan Cortexalone did not change the integrity of the genomic DNA, butMoutan Cortex pre-exposure significantly reduced the DNAfragmenting potency of AAP. Since AAP is known to in-hibit systems that maintain DNA integrity and DNA repairenzyme activity (Dybing et al., 1984), it is possible that the
Y.-H. Shon, K.-S. Nam / Journal of Ethnopharmacology 90 (2004) 415–419 419
capacity of AAP to damage DNA is modulated by MoutanCortex.
Various mechanisms may be involved in Moutan Cortexpretreatment-induced hepatoprotection. The present studydemonstrates that the protective effect of Moutan Cortexagainst AAP appears to be the decreased biotransformationof AAP by the inhibiton of hepatic cytochrome P450 2E1activity, inhibition of the decrease in GSH with a resul-tant increase in the availability of this agent for cell detox-ification, or protection of DNA integrity. However, furtherstudies at the molecular and/or genetic level(s) are neces-sary to explain the protective effects of Moutan Cortex onAAP-induced hepatotoxicity.
Acknowledgements
This work is supported by the Dongguk University re-search fund.
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