eCommons@AKU Department of Biological & Biomedical Sciences Medical College, Pakistan May 2012 Gastrointestinal stimulant effect of Urginea indica Kunth. and involvement of muscarinic receptors Saima Abbas Samra Bashir Aga Khan University Aslam Khan Aga Khan University Malik Hassan Mehmood Aga Khan University, [email protected]Anwar Gilani Aga Khan University Follow this and additional works at: hp://ecommons.aku.edu/pakistan_s_mc_bbs Part of the Gastroenterology Commons , and the Natural Products Chemistry and Pharmacognosy Commons Recommended Citation Abbas, S., Bashir, S., Khan, A., Mehmood, M., Gilani, A. (2012). Gastrointestinal stimulant effect of Urginea indica Kunth. and involvement of muscarinic receptors. Phytotherapy Research, 26(5), 704-708. Available at: hp://ecommons.aku.edu/pakistan_s_mc_bbs/75
Transcript
eCommons@AKU
Department of Biological & Biomedical Sciences Medical College, Pakistan
May 2012
Gastrointestinal stimulant effect of Urginea indicaKunth. and involvement of muscarinic receptorsSaima Abbas
Follow this and additional works at: http://ecommons.aku.edu/pakistan_fhs_mc_bbs
Part of the Gastroenterology Commons, and the Natural Products Chemistry andPharmacognosy Commons
Recommended CitationAbbas, S., Bashir, S., Khan, A., Mehmood, M., Gilani, A. (2012). Gastrointestinal stimulant effect of Urginea indica Kunth. andinvolvement of muscarinic receptors. Phytotherapy Research, 26(5), 704-708.Available at: http://ecommons.aku.edu/pakistan_fhs_mc_bbs/75
Gastrointestinal Stimulant Effect ofUrginea indicaKunth. and Involvement of Muscarinic Receptors
SaimaAbbas,1 SamraBashir,1,2AslamKhan,2MalikHassanMehmood2 andAnwarulHassanGilani2*1Department of Pharmacy, Bahauddin Zakariya University, Multan, Pakistan2Natural Product Research Unit, Department of Biological and Biomedical Sciences, the Aga Khan University Medical College,Karachi 74800, Pakistan
Urginea indica Kunth. (Family; Liliaceae) was studied for its gastrointestinal stimulant effect to rationalize thetraditional medicinal uses as a digestive aid, stomachic and laxative. The crude aqueous-methanol extract ofUrginea indica bulb (Ui.Cr) was tested on mice and isolated gut preparations. Ui.Cr, which was tested positivefor alkaloids, tannins and coumarins, increased faecal output and accelerated charcoal meal transit in mice (6–12mg/kg, p.o.), similar to that caused by carbachol (10mg/kg). Ui.Cr (0.01–1mg/mL) caused a spasmogenic ef-fect in guinea-pig ileum that was reproduced in rabbit jejunum (0.01–0.3mg/mL) followed by relaxation at ahigher concentration. Like carbachol, the stimulant effect of Ui.Cr was blocked by atropine, suggesting the acti-vation of muscarinic receptors mediating the prokinetic effect. Ui.Cr (0.01–5.0mg/mL) also inhibited K+
Digestive diseases, affecting 70% of the general popula-tion, are the cause of considerable social and economicalimpact (Ouyang and Chen, 2004). Constipation, diarrheaand dyspepsia, in particular, are commonly prevailingdisorders (Mehmood et al., 2010). Medicinal plants areusually preferred to treat these gastrointestinal disorders,because they contain multiple constituents with effectenhancing and/or side-effect-neutralizing potential(Gilani and Rahman, 2005), and, hence, are consideredrelatively safe in prolonged use. Seed husk of Plantagoovata (psyllium husk), for example, is a widely usedremedy equally popular among traditional healers andmodern physicians for digestive disorders and has beenproven effective in relieving both constipation anddiarrhea (Mehmood et al., 2010)Urginea indica Kunth. (Syn: Scilla indica Roxb),
locally known as ‘Jungli-piyaz’ and commonly as ‘In-dian squill’, belongs to the family Liliaceae. The plantis found in drier sub-Himalayan tracts of westernHimalayas; Bihar, Konkan, and on the CoromandelCoast (Kapoor, 1990). The bulb of Urginea indica ispopularly employed as a gastrointestinal stimulant torelieve constipation and indigestion. The plant has alsobeen reported to be used as an anthelmintic, cardiacstimulant, deobstruent, diuretic, emmenagogue and
for the treatment of calculous affections, cough, bron-chitis, asthma, paralytic affection, rheumatism, leprosy,skin diseases, internal pain and scabies (Kirtikar andBasu, 1988; Baquar, 1989; Prajapati et al., 2003). Bulbscrushed or sliced are also applied under the sole of thefeet to prevent burning sensation (Kapoor, 1990;Usmanghani et al., 1997) and are used externally forremoving corns and warts (Kapoor, 1990; Prajapatiet al., 2003).
The plant has been reported to contain glycosidesincluding scillaren-A and scillaren-B (Prajapati et al.,2003). Urginea indica has been studied for its medi-cinal effect as an antifungal (Shenoy et al., 2006),antiangiogenic and pro-apoptotic (Deepak andSalimath, 2006).
Despite its wide medicinal application, Urginea indicahas not been studied extensively to evaluate its medi-cinal uses pertaining to the gastrointestinal stimulanteffect. This study was therefore undertaken to providea pharmacological rationale for the use of Urgineaindica bulb as a laxative, digestive and stomachic.
MATERIALS AND METHODS
Plant material and preparation of crude extract. Thebulbs of Urginea indica were collected fresh from fieldsof Mianwali subsequent to the identification of the plantby an expert taxonomist at the Institute of Pure andApplied Biology, Bahauddin Zakariya University,Multan. A specimen of the plant has been deposited at
* Correspondence to: Dr Anwarul Hassan Gilani, Professor of Pharmacol-ogy, Department of Biological and Biomedical Sciences, The Aga KhanUniversity Medical College, Karachi-74800, Pakistan.E-mail: [email protected]
PHYTOTHERAPY RESEARCHPhytother. Res. 26: 704–708 (2012)Published online 18 October 2011 in Wiley Online Library(wileyonlinelibrary.com) DOI: 10.1002/ptr.3634
the herbarium of the same institute (voucher no P. Fl59–1843). The plant material was washed for any con-taminants and subjected to shade drying. The driedplant material (400 g) was ground into coarse powdersthrough an electrically driven device and the powderwas soaked at room temperature in 70% aqueous-methanol (v/v) for 3 days in amber colored glass bottleswith occasional shaking. The soaked material waspassed through double layered muslin cloth to removevegetative debris and the obtained fluid was subse-quently filtered through filter paper (Williamson et al.,1998). The residue was re-soaked for the next 3 daysand the procedure repeated twice. The filtrates wereevaporated on a rotary evaporator (R-210, Buchi,Switzerland) under reduced pressure (�760mmHg) at37�C to a thick, semi-solid paste of dark brown colour,the crude extract of Urginea indica bulb (Ui.Cr) yielding10%. Ui.Cr was solubilized in distilled water for allin vitro experiments.
Preliminary phytochemical analysis. Qualitative phyto-chemical analysis of Ui.Cr was done for the detectionof alkaloids, saponins, anthraquinones, coumarins,sterols, terpenes and flavonoids by previously describedmethods (Evans, 2006).
Drugs and animals. Acetylcholine chloride (ACh),atropine sulphate and carbachol (CCh) were purchasedfrom Sigma Chemical Co., St Louis, MO, USA.Chemicals used for making physiological salt solutionswere ethylenediamine tetra-acetic acid (EDTA), potas-sium chloride (Sigma Chemical Co.), calcium chloride,glucose, magnesium chloride, magnesium sulphate,potassium dihydrogen phosphate, sodium dihydrogenphosphate sodium chloride and sodium bicarbonate(Merck, Darmstadt, Germany). All chemicals used wereof the analytical grade available. Stock solutions of allthe chemicals were made in distilled water and theirdilutions were made fresh on the day of experiment.The vehicle used for solubilization of drugs had noeffect on tissue contractility in the control experiments.Animals used in the study were rabbits, guinea-pigs
and mice of local breed and either sex; housed at theAnimal House of the Aga Khan University, Karachi,maintained at 23–25�C with 12 h light/dark cycle.Animals had free access to standard diet and tap waterexcept that 24 h before commencement of the experi-ments, food was withdrawn but water was availablead libitum. Experiments were performed in compliancewith the rulings of the Institute of Laboratory AnimalsResources, Commission on Life Sciences, NationalResearch Council (NRC, 1996) and approved by theEthical Committee of the Aga Khan University,Karachi.
Charcoal meal gastrointestinal transit test. The methodpreviously followed by Gaginella et al. (1994) was usedwith slight modifications. Mice (20–30 g) were dividedinto four groups of six mice each and fasted for 24 h be-fore the experiment. One group serving as the negativecontrol was treated with normal saline (10mL/kg, p.o.).Two of the groups were treated with two increasingdoses of the extract (6 and 12mg/kg), serving as the testgroups and the last group was administered carbachol(10mg/kg), a standard cholinergic agent, as the positivecontrol. After 15min, the animals were given orally
0.3mL of freshly prepared charcoal meal (distilledwater suspension containing 10% gum acacia, 10%vegetable charcoal and 20% starch). Following 30minof charcoal administration, the mice were killed by cer-vical dislocation and the abdomen immediately cutopen, to excise the whole small intestine (pylorus regionto caecum). The length of the small intestine and the dis-tance between the pylorus region and the front of thecharcoal meal was measured to obtain the charcoaltransport ratio or percentage.
Laxative activity. The method of Haruna (1997) was fol-lowed for this activity. Mice (20–25 g), fasted for 24 h be-fore the experiment, were placed individually in cageslined with clean filter paper and divided into sevengroups of six animals each to receive the following treat-ments. The first group, acting as the negative control,was administered with saline (10mL/kg, p.o.). The sec-ond and third groups, acting as the test groups, received,orally, 6 and 12mg/kg of the Ui.Cr. The fourth group,serving as the positive control, received carbachol(10mg/kg, p.o.). The fifth, sixth and seventh groupsreceived atropine (10mg/kg) 30min prior to admin-istering the above said doses of Ui.Cr and carbachol, re-spectively, for their laxative effect. The feces productionwas monitored for 6 h in all seven groups for the totalnumber as well as for consistency. The laxative effectof a given treatment was measured as an increase inthe number of total feces over that of the negativecontrol.
Isolated tissue experiments. Isolated tissue experimentswere performed following the methods previouslyemployed in our laboratory (Gilani et al., 2007, 2009).
Guinea-pig ileum. The ileum was dissected and kept inTyrode’s solution. The segments, about 2 cm long, weresuspended individually in a 10mL tissue bath, filled withTyrode’s solution, aerated with carbogen and main-tained at 37�C. The initial tension of 0.7 g was appliedand each tissue was allowed to equilibrate for at least30min before the addition of any drug. Under these ex-perimental conditions, guinea-pig ileum behaves as aquiescent preparation and is considered more usefulfor measuring spasmogenic activity (Gilani et al.,2004). After equilibration, each tissue preparation wasrepeatedly treated with sub-maximal concentrations(0.3 mM) of ACh at 3min intervals until constantresponses were recorded. Stimulant effect of the extractwas determined on resting baseline of the tissue and wasassessed as the percent of the maximum effect producedby the control drug, ACh (1mM).
Rabbit jejunum. To test the gut modulatory effects ofthe plant material, the rabbit jejunum was dissectedout, kept in Tyrode’s solution and cleaned of mesenter-ies. Respective segments of approximately 2 cm lengthwere suspended in tissue baths containing Tyrode’s solu-tion maintained at 37�C and aerated with carbogen(95% O2 and 5% CO2). The composition of the Ty-rode’s solution in mM was: KCl 2.68, NaCl 136.9, MgCl21.05, NaHCO3 11.90, NaH2PO4 0.42, CaCl2 1.8 and glu-cose 5.55. Intestinal responses were recorded isotonicallyusing a transducer (Model 50–6360, HarvardApparatus, Holliston, USA) coupled with a bridge ampli-fier and PowerLab 4/25 data acquisition system attached
705GASTROINTESTINAL STIMULANT EFFECT OF URGINEA INDICA
to a computer running Chart 6 software (AD Instruments,Sydney, Australia). A preload of 1 g was applied. Thetissues were allowed to equilibrate for at least 30minbefore addition of any drug. After equilibration, eachpreparation was repeatedly treated with sub-maximalconcentrations (0.3mM) of ACh with a 5–10min intervalbetween doses to stabilize the preparation. The prepa-ration was considered stable when three responses ofthe same concentration of ACh were found identical.Spasmogenic effects of the test material were comparedwith the ACh evoked maximum contractile responses.For the determination of Ca2+ channel blocking
(CCB) activity, high K+ (80mM) was used to depolarizethe preparations as described by Farre et al. (1991); K+
(80mM) was added to the tissue bath, which produceda sustained contraction. Once a plateau of the inducedcontraction was achieved, the test material was addedin a cumulative fashion to obtain the concentration-dependent inhibitory response (Van-Rossum, 1963). Toconfirm the Ca2+ antagonist activity of the testsubstances, the tissue was allowed to stabilize in normalTyrode’s solution, which was then replaced with Ca2+-free Tyrode’s solution containing EDTA (0.1mM) for30min in order to remove Ca2+ from the tissues. Thissolution was further replaced with K+-rich and Ca2+-freeTyrode’s solution, of the following composition (mM):KCl 50, NaCl 91.04, MgCl2 1.05, NaHCO3 11.90,NaH2PO4 0.42, glucose 5.55 and EDTA 0.1. Followingan incubation period of 30min, control concentration-response curves (CRCs) of Ca2+ were obtained.When the CRCs of Ca2+ were found to be superimpos-able (usually after two cycles), the tissue was pretreatedwith the crude extract for 60min to test the possibleCCB effect. The CRCs of Ca2+ were reconstructed inthe presence of different concentrations of the testmaterial.
Statistical analysis. All the data expressed are mean �standard error of mean (SEM, n=number of experi-ments) and the median effective concentration (EC50)with 95% confidence intervals (CI). The CRCs wereanalysed by non-linear regression using the GraphPadprogram (GraphPAD, SanDiego, CA, USA). The statis-tical parameters applied are the Student’s t-test and one-way analysis of variance (ANOVA) with post hocDunnett’s or Tukey’s test. A value of p< 0.05 wasconsidered significantly different.
RESULTS
Preliminary phytochemical analysis
TheUi.Crwas found to contain alkaloids, tannins and cou-marins, while other classes of phytochemicals were absent.
Effect on charcoal meal transit
The prokinetic effect of Ui.Cr was measured in terms of itseffect on charcoal meal transit in the small intestine ofmice (Fig. 1). Ui.Cr, dose-dependently (6 and 12mg/kg),accelerated charcoal meal travel through the smallintestine, similar to that caused by carbachol. Thedistance travelled by the charcoal meal in the Ui.Cr
treated mice, measured as % of total length of small in-testine, was 80.2� 5.2 and 87.9� 4 with 6 and 12mg/kg,respectively, as opposed to 49.9� 6.1 with saline. Car-bachol, similarly, enhanced the proportion of intestinetravelled by charcoal meal to 74.9%� 6.4.
Laxative effect
The Ui.Cr, when administered orally, showed a laxativeeffect in mice as reflected by the increase in the numberof feces (Fig. 2). The laxative effect was dose-depend-ently mediated at 6 and 12mg/kg and was comparableto carbachol (10mg/kg). The respective total numberof feces passed by the groups of mice treated with Ui.Cr (6 and 12mg/kg) and carbachol (10mg/kg) over 6 hwere 4.3� 0.9, 9.2� 1.4 and 11�1.7. Pretreament ofthe animals with atropine (10mg/kg) blocked the laxa-tive effect of both Ui.Cr and carbachol.
Figure 1. Effect of Urginea indica crude extract (Ui.Cr) and carba-chol (CCh) on charcoal meal transit in mice. Values given aremean � SEM, n=6. *p<0.05 and **p<0.01 vs control.
Figure 2. Laxative effect of Urginea indica crude extract (Ui.Cr)and carbachol (CCh) measured as fecal output in the presenceand absence of atropine. Values are expressed as mean � SEM,n=6. *p<0.05, **p<0.01 and ***p<0.001.
The Ui.Cr caused a concentration-dependent (0.01–1.0mg/mL) contractile effect in guinea-pig ileum, whichwas diminished by atropine (0.1mM)pretreatment (Fig. 3).The magnitude of the contractile effect was 3.86� 1.22%,7.41�1.57%, 22.37� 3.85%, 28.97� 3.64% and 35.71� 3.51% (mean� SEM, n=4) at 0.01, 0.03, 0.1, 0.3 and1.0mg/mL, respectively, compared with the ACh(1.0mM)-evoked maximum contraction. With further in-crement in the concentration, the contractile effect wasabolished, showing a spasmolytic component.
Effect on jejunum
The Ui.Cr caused a concentration-dependent (0.01–0.3mg/mL) stimulant effect in spontaneously contactingrabbit jejunum followed by relaxation at a higher concen-tration as shown in Fig. 4A. The magnitude of the highestcontraction caused by Ui.Cr (0.3mg/mL) in this prepa-ration was 22.86� 4.10% (mean� SEM, n=4) of ACh(1mM)-induced maximum response. Pretreatment of thetissue with atropine (0.1mM) abolished the contractile ef-fect and the relaxant effect became dominant with anEC50 value of 1.01mg/mL (95% CI, 0.72–1.41, n=3) asshown in Fig. 4A.
When tested against high K+-induced contractions,Ui.Cr caused relaxation (Fig. 4A) with an EC50 valueof 0.32mg/mL (0.26–0.39 n= 6). Similarly, verapamilrelaxed both spontaneous and K+ -induced contractionswith EC50 values of 0.035 (0.03–0.4, n= 4) and 0.14 mM(0.10–0.19, n= 4), respectively (Fig. 4B). The Ui.Cr alsoproduced a concentration-dependent (0.3–1.0mg/mL)rightward shift in the Ca2+ CRCs, similar to that causedby verapamil at 0.1 and 0.3 mM (Fig. 4C and D).
DISCUSSION
Urginea indica is used in traditional medicine as a diges-tive, stomachic and laxative; therefore the current studywas undertaken to determine its gut stimulatory effectusing the in vivo and in vitro models to rationalize themedicinal uses. The Ui.Cr was studied on mice for itseffect on the charcoal meal transit through the small gutand the laxative activity. The Ui.Cr dose-dependentlyenhanced the intestinal transit of charcoal and causeda significant increase in the number of feces when admi-nistered orally to mice. The Ui.Cr was found dis-tinctly potent in its gastrointestinal stimulant effectwith comparable potency to carbachol, a standardmuscarinic agonist and gastrointestinal stimulant(Brown and Taylor, 1996). In our previous studies itwas observed that the laxative effect of natural productsis usually mediated through an ACh like mechanism(Gilani et al., 2000, 2004). To evaluate if the gut stimu-lating effect of this plant was also mediated by the in-volvement of a similar pathway, the laxative effect ofUi.Cr was challenged with atropine. Pretreatment ofmice with atropine blocked the laxative effect of Ui.Crsimilar to carbachol, thus suggesting that the gastro-intestinal stimulatory effect of Ui.Cr observed in vivois mediated through activation of muscarinic receptors.Acetylcholine is a neurotransmitter released by theparasympathetic nervous system. It plays an importantphysiological role to regulate the peristaltic movementsof the gut and to release of digestive juices mediatingthis action by stimulation of M3 muscarinic receptorsubtypes and atropine blocks all muscarinic receptorsites (Brown and Taylor, 1996).
To confirm the mode of action, Ui.Cr was furtherstudied on isolated tissue preparations. When tested onguinea-pig ileum, a quiescent preparation considered use-ful for this purpose (Bashir et al., 2006), Ui.Cr produced aspasmogenic effect, which was blocked by atropine likethat of ACh, thus confirming that the stimulatory effectof Ui.Cr was mediated through muscarinic receptor acti-vation. The spasmogenic effect of Urginea indica was
Figure 3. Bar-chart showing the concentration-dependent spas-mogenic effect of crude extract of Urginea indica (Ui.Cr) in theabsence and presence of atropine (0.1mM) in isolated guinea-pigileum. The responses are given as % of acetylcholine (10mM)induced maximum contraction (Ach Max.). The values are shownas mean � SEM, n=4.
Figure 4. The effect of crude extract of Urginea indica (Ui.Cr) andverapamil, respectively, on spontaneous and K+ (80mM)-inducedcontractions (A and B) and Ca2+ concentration-response curves (Cand D) in isolated rabbit jejunum. Values are presented as mean �SEM, n=4–7.
707GASTROINTESTINAL STIMULANT EFFECT OF URGINEA INDICA
further studied on spontaneously contracting rabbit je-junum where the plant extract exhibited a weak atropinesensitive excitatory effect at the lower concentration fol-lowed by an inhibitory effect at the higher concentration,indicating the presence of a combination of gut stimulantand inhibitory constituents in Urginea indica.The relaxant effect of Ui.Cr observed at a high
concentration was mediated through a Ca2+ antagonistlike effect, reflected by the inhibitory effect on K+-inducedcontractions and displacement of Ca2+ curves to the right,similar to that of verapamil, a standard Ca2+-channelblocker (Hamilton et al., 1986). The potency of Ui.Cr forits in vivo gut stimulant effect was comparable to carba-chol, but the maximum stimulant effect as observedin vitro was moderate (approximately 40% of AChinduced maximum response), presenting an interestingpicture. Acetylcholine is one of the most important neuro-transmitters exhibiting normal function in gut motility butcholinergic drugs are not used clinically to relieve consti-pation as they cause side effects such as abdominal crampsdue to their strong effect as opposed to a physiological ef-fect which is short lived (Brown and Taylor, 1996). Themoderate effect of Ui.Cr is considered valuable as beyondthis it could have caused abdominal cramps but the coex-istence of a spasmolytic component is probably meant bynature to offset the excessive stimulation which could havebeen otherwise harmful.
These gastrointestinal stimulant effects of the Urgineaindica extract correlate well with its traditional use inindigestion and constipation (Kirtikar and Basu, 1988;Baquar, 1989; Prajapati et al., 2003), the conditionsresulting from delayed gastrointestinal transit (Tatsutaand Iishi, 1993).
The results of the preliminary phytochemical analysisof the crude extract of Urginea indica revealed the pres-ence of alkaloids, tannins and coumarins compounds inthe plant. Alkaloids have shown a spasmolytic effectthrough blockade of voltage-dependent Ca2+ channels(Khalid et al., 2004; Gilani et al., 2005), but the possiblenature of the cholinergic and anticholinergic constitu-ents in this plant is uncertain.
These investigations indicate that the crude extractUrginea indica possesses gut stimulatory activitymediated through a cholinergic mechanism, which couldbe the possible reason for its use in disorders resultingfrom delayed gastrointestinal transit such as constipa-tion and indigestion.
Conflict of Interest
The authors have declared that there is no conflict ofinterest.
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