Environmental Toxicology and Pharmacology 25 (2008) 321–328
Amelioratory effect of Andrographis paniculataNees on liver, kidney, heart, lung and spleen
during nicotine induced oxidative stress
Sreeparna Neogy, Subhasis Das, Santanu Kar Mahapatra,Nirjal Mandal, Somenath Roy ∗
Immunology and Microbiology Laboratory, Department of Human Physiology with Community Health,Vidyasagar University, Midnapore 721102, West Bengal, India
Received 1 June 2007; received in revised form 21 October 2007; accepted 23 October 2007Available online 4 November 2007
bstract
The ameliorative properties of bioactive compound andrographolide (ANDRO), aqueous extract of Andrographis paniculata (AE-AP) and vitamin(vit.E) were tested against nicotine induced liver, kidney, heart, lung and spleen toxicity. A group of male Wistar rats were intraperitoneally
dministered vehicle, nicotine (1 mg/kg body weight/day), nicotine + ANDRO (250 mg/kg body weight/day), nicotine + AE-AP (250 mg/kg bodyeight/day) and nicotine + vit.E (50 mg/kg body weight/day) for the period of 7 days. The significantly increased levels of lipid peroxidation,rotein oxidation and the decreased antioxidant enzyme status were noted in nicotine treated group as compared to vehicle treated group. ANDRO,
E-AP and vit.E significantly reduced the lipid peroxidation, protein oxidation and increased the antioxidant enzyme status. This indicates A.aniculata and vit.E may act as putative protective agent against nicotine induced tissue injury and may pave a new path to develop suitable drugherapy.
Nicotine, as most biologically active chemical in tobaccomoke, has been the subject of intense scientific scrutiny. Amonghe most well characterized chemicals found in tobacco andobacco smoke, are polycyclic aromatic hydrocarbons (PAHs)
nd the highly addictive alkaloid, nicotine and its metabolitesCampain, 2004). To further complicate the picture, nicotines converted, during the production of cigarette and chewing
obacco, into two highly mutagenic nitrosamine, N′-nitrosonoricotine (NNN) and 4-(methylnitrosamine)-1-(3-pyridyl)-1-utanone (NNK) and is metabolized into cotinine. Thesehemicals derivatives also exhibit a wide spectrum of biologicalctivity as compared to parent compound (Campain, 2004).
Nicotine has been reported to induce oxidative stress both inivo and in vitro (Pigeolot et al., 1990). The mechanism of gener-tion of free radicals by nicotine is not clear. But oxidative stressccurs when there are excess free radicals and/or low antioxi-ant defense, and result in chemical alteration of biomoleculesausing structural and functional modification. Oxygen free rad-cals (OFR) production has been directly linked to oxidationf cellular macromolecules, which may induce a variety ofellular responses through generation of secondary metaboliceactive species (Chiarugi, 2003). Previous reports have shown
nhanced lipid peroxidation and inadequate antioxidant statusy nicotine.
Medicinal plants and their active principles have receivedreater attention as anti-peroxidative agent (Lee and Park, 2002).
ndrographolide, the main active constituent of Adrographisaniculata (A. paniculata), has excellent anti-inflammatory,nti-bacterial and anti-viral effects. A. paniculata is an Indianerb, well known as ‘king of bitter’. This bitter herb generallyas an affinity with heart and liver. New research has confirmedhost of pharmacological benefits of this herb for its enor-ous potential in far wide range of diseases. The present studyas undertaken to evaluate the amelioratory property of andro-rapholide on tissue antioxidant status during nicotine inducedxidative stress in male Wistar rats.
. Materials and methods
.1. Animals
The weight matched (120–140 g) male Wistar rats were obtained, dividedn different groups, housed in polypropylene cage and provided standardellet diet (Chaulia Equipment and Chemicals, Ganapatinagar, Midnapore,ndia) and water ad libitum. The animals were maintained under standardonditions of temperature (25 ± 2 ◦C) and humidity (60 ± 5%) with an alter-ating 12 h light/dark cycles. Animals were maintained in accordance withhe guidelines of the National Institute of Nutrition, Indian Council of Med-cal Research, Hyderabad, India, and approved by the ethical committee ofidyasagar University. All the experiments were conduced with the ethi-al guidelines laid down by the Committee for the Purpose of Control andupervision of Experiments on Animals (CPCSEA) constituted by the Animalelfare Division of Government of India on the use of animals in scientific
esearch.
.2. Plant materials
A. paniculata Nees herbs were collected from the campus of IIT, Kharagpur,est Bengal, India in July–August, 2005 and air-dried. A voucher specimen has
een deposited to the CAL herbarium, Botanical Survey of India, Howrah, Indiander the accession number IIT-VU/Ap-1.
.3. Chemicals and reagents
Standard andrographolide, epinephrine, TBA, DNPH, H2O2, EDTA, DTNB,ADPH were purchased from Sigma Chemical Co., USA. Nicotine was pur-hased from Merck, Germany. Other chemicals were procured from Merck Ltd.,RL Pvt. Ltd., Mumbai, India.
.4. Treatment schedule
The animals were randomized into experimental and control groups andivided into five groups of six animals each. Rats’ in-group ‘A’ served as control,eceived 5% DMSO in physiological saline. Group ‘B’ animals received nicotine3-(1-methyl-2-pyrrolidinyl)pyridine, C10H14N2] 1.0 mg/kg body weight/dayin physiological saline, pH was adjusted at 7.4 prior to injection), rats inroup ‘C’ were administered nicotine as in group ‘B’ as well as andrographolide250 mg/kg body weight/day) in 5% dimethyl sulfoxide (DMSO) with physio-ogical saline, rats in group ‘D’ received nicotine (1.0 mg/kg body weight/day)s well as aqueous extract (250 mg/kg body weight/day) dissolved in 5%MSO with physiological saline and animals of group ‘E’ treated with nicotine
1.0 mg/kg body weight/day) along with oral dose of vitamin E (50 mg/kg bodyeight/day) in olive oil. Simultaneously, animals of group ‘A’, ‘B’, ‘C’ and ‘D’ere received olive oil orally. All the treatments were done intraperitoneally
i.p.) except vitamin E and olive oil for the period of 7 days. The dose anduration of nicotine were selected as per reported by many researchers (Chen
t al., 2001 and Tuncok et al., 2001) and the dose of ANDRO was chosen aser the previous report (Madav et al., 1995). The experiment was terminated athe end of 7 days and all animals were sacrificed by an intraperitoneal injectionf sodium pentobarbital (60–70 mg/kg body weight) (Chandran and Venugopal,004).
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.5. Tissue extracts preparation
After decapitation, liver, kidney, heart, lung and spleen were excised fromats and washed with cold saline. Washed tissues were immediately immersedn liquid nitrogen and stored at −80 ◦C. On preparation, tissues were slicednd homogenized in ice cold 50 mM sodium phosphate buffer (pH 7.0) con-aining 0.1 mM ethylenediamine tetraacetic acid (EDTA) to yield 10% (w/v)omogenate. The homogenates were then centrifuged at 1000 rpm for 10 min at◦C. The supernatants were separated and used for enzyme assays and proteinetermination (Husain et al., 2001).
.6. Preparation of aqueous extract
The fresh aerial parts of A. paniculata was blended and extracted with dis-illed water (10:1). The mixture was filtered with Whatman filter paper (No. 1)nd concentrated at 38 ◦C by a rotary evaporator, then allowed to stand at roomemperature over night. The filtration and concentration processes were repeatedo yield an aqueous solution. This solution was then centrifuged at 2000 × g for0 min and supernatant was freeze dried to obtain the crude water extract (Zhangnd Tan, 1996).
.7. Isolation of ANDRO by thin layer chromatography (TLC)
A. paniculata powder was homogenized and extracted with distilled water10:1). The mixture was filtered with Whatman filter paper (No. 1). The solutionas then centrifuged at 200 × g for 10 min and supernatant was collected. The
upernatant was mixed with ethyl acetate, yielding approximately 0.7% of ethylcetate fraction. Two gram of ethyl acetate fraction was dissolved in methanolo obtain methanol extract (Zhang and Tan, 1997). The methanol extracts andhe reference andrographolide (Sigma Chemical Ltd.) was spotted, separatedn TLC plate. The plate was photographed after staining with 5% methanoliculphuric acid. After this, the parallel band of methanol extract matching withhe reference andrographolide was scrapped under ultra violet light (254 nm)nd then eluted out from the TLC plate in methanol. Then the methanol wasvaporated out by rotary evaporator and used for HPLC as well as biologicalctivity studies.
.8. Detection of ANDRO by high performance liquidhromatography (HPLC)
Isolated ANDRO was detected with standard andrographolide (Sigmahemical Ltd.) in Water HPLC at 229 nm in � Bondapak C-18 column
3.9 mm × 300 mm). The conditions are as follows:Mobile phase: methanol:water (60:40), v/v; flow rate: 1 ml/ min; injection
olume: 20 �l; UV detection at 229 nm (Singha et al., 2007)
.9. Analytical methods
.9.1. Lipid peroxidationThe extent of lipid peroxidation was estimated as the concentration of thio-
arbituric acid reactive product malondialdehyde (MDA) by using the method ofhkawa et al. (1979). One hundred microliters of tissue homogenate was added
o 100 �l of double-distilled water and 50 �l of 8.1% sodium dodecyl sulfateSDS) and incubated at room temperature for 10 min. Three hundred seventy-ve microliters of 20% acetic acid (pH 3.5), along with 375 �l of thiobarbituriccid (0.6%), was added to the tissue solution and placed in a boiling waterath for 60 min. After incubation, 250 �l of double-distilled water and 1.25 mlf 15:1 butanol–pyridine solution were added to the mixture and centrifugedor 5 min at 2000 × g. The resulting supernatant was removed and measured at32 nm with the use of the Hitachi U-2000 spectrophotometer. Malondialde-yde concentrations were determined by using 1,1,3,3-tetraethoxypropane as
tandard.
.9.2. Protein carbonylProtein carbonyl (PC) levels were measured according to method described
y Reznick and Packer (1994); based on spectrophotometric detection of the
ogy and Pharmacology 25 (2008) 321–328 323
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eaction of 2,4-dinitrophenylhydrazine with protein carbonyl to form proteinydrazones. Briefly, after precipitation of protein with an equal volume of 1%richloroacetic acid (TCA), the pellet was resuspended in 10 mM DNPH in 2NCl or with 2N HCl as control blank. Next after the washing procedure with:1 ethanol/ethylacetate, the final palette was dissolved in 6 M Guanidine. Thearbonyl group was determined from the absorbance at 370 nm. The result wasxpressed as micromoles of carbonyl groups per milligram of protein with molarxtinction coefficient of 22,000 M−1cm−1.
.9.3. Superoxide dismutaseSuperoxide dismutase activity was determined at room temperature accord-
ng to the method of Misra and Fridovich (1972). Ten microliters of tissueomogenate was added to 970 �l (0.05 M, pH 10.2, 0.1 mM EDTA) of sodiumarbonate buffer. Twenty microliters of 30 mM epinephrine (dissolved in 0.05%cetic acid) was added to the mixture to start the reaction. Superoxide dismutasectivity was measured at 480 nm for 4 min on a Hitachi U-2000 spectrophotome-er. Activity was expressed as the amount of enzyme that inhibits the oxidationf epinephrine by 50%, which is equal to 1 U/mg of protein.
.9.4. CatalaseCatalase activity was determined at room temperature by using a slightly
odified version of Aebi (1984). Ten microliters of ethanol was added to 100 �lf tissue homogenate. The tissue mixture was then placed in an ice bath for0 min and tubes were brought at room temperature, followed by the addition of0 �l of Triton X-100 RS. Ten microliters of the tissue homogenate was addedo a cuvette containing 240 �l (0.05 M, pH 10.2, 0.1 mM EDTA) of sodiumhosphate buffer, and 250 ml of 0.066 M H2O2 (dissolved in sodium phosphateuffer) was added to start the reaction. Catalase activity was measured at 240 nmor 1 min with the use of the Hitachi U-2000 spectrophotometer. The molarxtinction coefficient of 43.6 M cm−1 was used to determine CAT activity. Onenit of activity is equal to the millimoles of H2O2 degraded per minute perilligram of protein.
.9.5. Reduced glutathioneReduced glutathione estimation was performed by the method of Grifith
1980). The required amount of tissue homogenate was mixed with 12% sul-osalicylic acid and centrifuged at 2000 × g for 15 min to settle the precipitatedroteins. 0.1 ml of protein free supernatant, 0.7 ml of 0.3 mM NADPH, 0.1 mlf 6 mM DTNB and 0.48 units of glutathione reductase were combined and thebsorbance of 5-thio-2-nitrobenzoic acid (TNB) was read at 412 nm. A standardurve was obtained with standard reduced glutathione. The level of GSH wasxpressed as microgram per mg protein.
.9.6. Oxidized glutathioneOxidized glutathione estimation was performed by the method of Grifith
1980). The required amount of tissue homogenate was mixed with 12% sul-osalicylic acid and centrifuged at 2000 × g for 15 min to settle the precipitatedroteins. 0.1 ml of protein free supernatant incubated in room temperature with.005 ml of 2 M 2-venyl pyridine for 1 h. Following incubation, 0.4 ml of 0.5 mMADPH, 0.1 ml 6 M DTNB and 0.48 unit of glutathione reductase were addednd measured at 412 nm. A standard curve was obtained with standard oxi-ized glutathione. The level of GSSG was expressed as microgram per mgrotein.
.9.7. Redox ratio (GSH/GSSG)Redox ratio was determined for all the five groups by taking the ratio of
educed glutathione/oxidized glutathione.
.9.8. Glutathione peroxidase (GSH-Px)The GSH-Px activity was measured by the method of Paglia and Valentine
1967). The reaction mixture contained 50 mM potassium phosphate buffer (pH.0), 1 mM EDTA, 1 mM sodium azide, 0.2 mM NADPH, 1 U glutathione reduc-
ase and 1 mM reduced glutathione. The sample, after its addition, was allowedo equilibrate for 5 min at 25 ◦C. The reaction was initiated by adding 0.1 mlf 2.5 mM H2O2. Absorbance at 340 nm was recorded for 5 min. Values werexpressed as nanomoles of NADPH oxidized to NADP by using the extinc-ion coefficient of 6.2 3 103 M−1 cm−1 at 340 nm. The activity of GSH-Px
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ig. 1. TLC of methanol extract (T) and standard andrographlide (S) aftererivatisation with 5% methanolic sulphuric acid at visible.
as expressed in terms of nanomole NADPH consumed/min per milligram ofrotein.
.10. Statistical analysis
The data were expressed as mean ± S.E.M. Comparisons of the means ofontrol, nicotine, nicotine with ANDRO, nicotine with AE-AP and nicotine withit.E group were made by two-way ANOVA with multiple comparison t-test,< 0.05 as a limit of significance.
. Results
.1. Isolation of ANDRO
Isolation of ANDRO was carried out by TLC (Fig. 1). Theand parallel to reference was eluted out and used for HPLCnalysis and supplemented in rats.
.2. Detection of ANDRO by HPLC
The detection of andrographolide was done with stan-ard andrographolide (Sigma Chemical Co., USA) and theeck of both isolated compound and standard andrographolideatches, hence it confirmed the presence of andrographolide
Fig. 2).
.3. Lipid peroxidation
MDA levels were significantly (P < 0.05) increased in liver,idney, heart, lungs and spleen by 30.21%, 22.42%, 121.43%,
76.92% and 40.40%, respectively, as compared to the controlroup.
In liver, supplementation with ANDRO, AE-AP and vit.Ehowed significant (P < 0.05) diminution of MDA content by
324 S. Neogy et al. / Environmental Toxicology a
Fig. 2. Detection of andrographolide by HPLC. Black: Isolated andro-gir
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rapholide. Blue: Standard andrographolide (Sigma Chemical Co.). (Fornterpretation of the references to colour in this figure legend, the reader iseferred to the web version of the article.)
3.45%, 16.42% and 17.14%, respectively, as compared to nico-ine treated group.
In kidney, significantly (P < 0.05) decreased level of MDAas seen after supplementation with ANDRO, AE-AP and vit.Ey 10.78%, 13.29% and 15.21%, respectively. ANDRO did nothow any significant change.
In heart, supplementation with AE-AP and vit.E showed sig-ificant (P < 0.05) diminution in MDA levels by 19.75% and2.18%, respectively, as compared to nicotine treated group.NDRO did not response against nicotine toxicity in heart.In lungs, MDA level showed a significant (P < 0.05) diminu-
ion by 27.89% on ANDRO supplementation to nicotine treatednimals. Supplementation with AE-AP and vit.E to nicotinereated group, significantly (P < 0.05) decreased the MDA levely 44.79% and 44.51%, respectively.
In spleen, supplementation with ANDRO, AE-AP and vit.Eecreased MDA levels by 52.36%, 45.28% and 48.58%, respec-ively, in comparison to nicotine treated animals (Fig. 3).
.4. Protein oxidation
Protein carbonyl levels were significantly (P < 0.05) elevatedy 69.09%, 130.17%, 55.94%, 73.80% and 68.55%, respectivelyn all the treated tissues (liver, kidney, heart, lung and spleen) inomparison to control.
ig. 3. MDA concentration in liver, kidney, heart, lung and spleen. Values arexpressed as mean ± S.E.M., for n = 6. *P < 0.05 compared to control, #P < 0.05ompared to nicotine.
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Supplementation with ANDRO, AE-AP and vit.E signifi-antly (P < 0.05) decreases the liver PC content by 23.66%,6.52% and 32.97%, respectively, as compared to the nicotinereated group.
Renal PC content was significantly decreased by 21.72%,6.22% and 43.07%, respectively in ANDRO, AE-AP and vit.Eupplementation as compared to control animals.
In heart, supplementation with AE-AP and vit.E showedignificant (P < 0.05) fall in PC level by 27.80% and 31.83%,espectively, as compared to nicotine treated group. ANDRO didot show any significant alteration of PC level in heart againsticotine toxicity.
In lung, PC content was significantly (P < 0.05) decreasedy 25.57%, 36.52% and 39.26% on ANDRO, AE-AP and vit.Edministration to nicotine treated animal.
In spleen, all three supplementation (ANDRO, AE-AP andit.E) significantly (P < 0.05) decreased PC level by 23.50%,2.83% and 33.58%, respectively as compared to nicotinereated group and the decreased was significant in relation toontrol (Fig. 4).
.5. Superoxide dismutase activity
The SOD activity of liver was significantly (P < 0.05) reducedy 57.67% due to nicotine treatment in relation to control. Sig-ificant (P < 0.05) variation in SOD activity by 43.05% and3.33%, respectively, was seen on supplementation with AE-APnd vit.E to nicotine treated group. But ANDRO did not increaseOD activity significantly as compared to nicotine treated group.
Similarly, in kidney SOD activity was declined significantlyP < 0.05) by 56.41% due to nicotine administration. Supple-entation with ANDRO, AE-AP and vit.E to nicotine treated
The SOD activity of heart was significantly (P < 0.05)educed by 46.66% due to nicotine toxicity as compared to
ig. 4. PC concentration in liver, kidney, heart, lung and spleen. Values arexpressed as mean ± S.E.M., for n = 6. *P < 0.05 compared to control, #P < 0.05ompared to nicotine.
ogy and Pharmacology 25 (2008) 321–328 325
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he control group. While administration of ANDRO to nico-ine treated rats showed no change in SOD activity, there was aignificant (P < 0.05) increase of the latter on AE-AP and vit.Eupplementation. Thus SOD activity of the heart, which was pre-iously lowered by nicotine treatment, was increased by 46.78%nd 76.78%, respectively on AE-AP and vit.E administration.
Similarly, in lungs SOD activity was significantly (P < 0.05)educed by 51.00% due to nicotine treatment as compared toontrol group. Though, supplementation with ANDRO produceso significant variation in SOD activity, where as AE-AP andit.E supplementation significantly (P < 0.05) increase the activ-ty by 65.16% and 79.64%, respectively, as compared to theicotine treated group. Thus a fall in SOD activity was partlyompensated by AE-AP and vit.E administration.
The SOD activity of spleen was significantly (P < 0.05)educed by nicotine administration in relation to the controlroup. The reduction was as much as 53.07%. ANDRO, AE-APnd vit.E administration to nicotine treated groups significantlyP < 0.05) increases the SOD activity by 74.69%, 78.22% and3.82%, respectively (Fig. 5).
.6. Catalase activity
In liver CAT activity was decreased by 50.93% due to nicotinedministration. A significant rise (P < 0.05) in CAT activity wasbserved on supplementation with ANDRO, AE-AP and vit.Ey 25.41%, 35.49% and 63.16%, respectively, as compared toicotine treated group.
There was a significant fall (P < 0.05) in kidney CAT activ-ty by 38.19% on nicotine administration. ANDRO, AE-AP andit.E supplementation to nicotine treated groups showed a sig-ificant rise (P < 0.05) in CAT activity by 51.48%, 63.75% and5.83%, respectively.
There was a significant (P < 0.05) reduce in CAT activity of
eart due to nicotine toxicity by 24.84%. While CAT activityf heart was significantly (P < 0.05) elevated by ANDRO, AE-P and vit.E supplementation by 36.44%, 59.32%, 48.31%,
espectively to nicotine treated group.
ig. 5. SOD activity in liver, kidney, heart, lung and spleen. Values are expresseds mean ± S.E.M., for n = 6. *P < 0.05 compared to control, #P < 0.05 comparedo nicotine.
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ig. 6. CAT activity in liver, kidney, heart, lung and spleen. Values are expresseds mean ± S.E.M., for n = 6. *P < 0.05 compared to control, #P < 0.05 comparedo nicotine.
The CAT activity of lungs was significantly (P < 0.05)educed by 49.25% due to nicotine treatment in relation to con-rol. Significant (P < 0.05) deviation by 46.95% and 48.75% inatalase activity of lungs was seen on supplementation withE-AP and vit.E to nicotine treated group. But ANDRO didot increase CAT activity significantly as compared to nicotinereated group.
Similarly, in spleen catalase activity was declined signif-cantly (P < 0.05) by 51.64% due to nicotine administration.upplementation with ANDRO, AE-AP and vit.E to nicotine
reated groups showed, 40.50%, 60.45% and 69.49% increasef CAT activity, respectively (Fig. 6).
.7. GSH concentration
In liver GSH content was decreased by 62.61% due to nicotinedministration. A significant rise in GSH content was observedn supplementation with ANDRO, AE-AP and vit.E by 63.52%,9.49% and 143.97%, respectively, as compared to nicotinereated group.
There was a fall in kidney GSH content by 29.78% on nicotinedministration. AE-AP and vit.E supplementation to nicotinereated groups showed a significant rise in GSH content by8.20% and 35.22%, respectively.
The GSH content of heart was significantly (P < 0.05)educed by 43.09% due to nicotine toxicity as compared tohe control group. While administration of ANDRO to nicotinereated rats showed no change in GSH content, there was a sig-ificant (P < 0.05) raise on AE-AP and vit.E supplementation.hus GSH content of the heart, which was lowered by nicotine
reatment, was increased by 21.47% and 32.98%, respectivelyn AE-AP and vit.E supplementation.
Similarly, in lungs GSH content was significantly (P < 0.05)educed by 56.43% due to nicotine toxicity as compared toontrol group. Supplementation with ANDRO, AE-AP andit.E significantly (P < 0.05) increase by 31.88%, 35.16% and
9.45%, respectively, as compared to the nicotine treated group.
The GSH content of spleen was significantly (P < 0.05)educed by nicotine administration in relation to the controlroup. The reduction was as much as 47.38%. ANDRO, AE-AP
326 S. Neogy et al. / Environmental Toxicology and Pharmacology 25 (2008) 321–328
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The GSH-Px activity of lungs was significantly (P < 0.05)reduced by 48.19% due to nicotine treatment in relation to con-trol. Significant (P < 0.05) deviation by 62.59%, 72.38% and
ig. 7. GSH concentration in liver, kidney, heart, lung and spleen. Values arexpressed as mean ± S.E.M., for n = 6. *P < 0.05 compared to control, #P < 0.05ompared to nicotine.
nd vit.E administration to nicotine treated groups significantlyP < 0.05) increases the GSH content by 31.15%, 39.64% and5.04%, respectively (Fig. 7).
.8. GSSG concentration
The GSSG level of liver was significantly (P < 0.05) reducedy 53.16% due to nicotine treatment in comparison to con-rol. Significant (P < 0.05) variation in GSSG level was seen onupplementation with ANDRO, AE-AP and vit.E by 60.55%,6.97% and 69.72%, respectively, to nicotine treated group.
In kidney, GSSG level was declined significantly (P < 0.05)y 51.61% due to nicotine administration. Supplementation withNDRO, AE-AP and vit.E to nicotine treated groups showed,0.30%, 73.36% and 104.24% rise of GSSG level, respectively.
There was a fall in GSSG level of heart by 48.54%, on nicotinedministration. A treatment with AE-AP and vit.E significantlyP < 0.05) increased the GSSG level by 46.23% and 38.67%s compared to nicotine treated group. While administration ofNDRO showed no significant variations.Similarly, in lungs GSSG level was decreased by 78.88% due
o nicotine administration. A significant rise in GSSG level ofungs was observed on supplementation with ANDRO, AE-APnd vit.E by 261.76%, 323.53% and 282.35%, respectively, asompared to nicotine treated group.
There was a fall in spleen GSSG level by 53.43% on nicotinedministration. ANDRO, AE-AP and vit.E supplementation toicotine treated groups showed a significant rise in GSSG levely 91.80%, 83.61% and 90.16%, respectively (Fig. 8).
.9. Redox ratio (GSH/GSSG ratio)
As a result of nicotine toxicity, a significant increase
P < 0.05) in the redox ratio was observed in nicotine treatedroup as compared to control animals. The increased wasbserved in all the tissues studied. The supplemented groupshowed the significantly decreased (P < 0.05) the redox ratio inomparison to nicotine treated animals (Fig. 9).
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ig. 8. GSSG concentration in liver, kidney, heart, lung and spleen. Values arexpressed as mean ± S.E.M., for n = 6. *P < 0.05 compared to control, #P < 0.05ompared to nicotine.
.10. GSH-Px activity
Due to nicotine administration, GSH-Px activity wasecreased in liver by 52.55%. A significant rise (P < 0.05)n GSH-Px activity was observed on supplementation withNDRO, AE-AP and vit.E by 45.06%, 63.44% and 91.98%,
espectively, as compared to nicotine treated animals.There was a significant decrease (P < 0.05) in kidney GSH-Px
ctivity in kidney by 50.02% on nicotine administration. Sup-lementation with ANDRO, AE-AP and vit.E to nicotine treatedroups showed a significant rise (P < 0.05) in GSH-Px activityy 36.76%, 59.1% and 78.72%, respectively.
As a result of nicotine toxicity, there was a significantP < 0.05) reduce in GSH-Px activity in heart by 42.68%. On thether hand GSH-Px activity of heart was significantly (P < 0.05)levated by the supplementation of ANDRO, AE-AP and vit.Ey 29.83%, 41.93%, 59.67%, respectively as compared to nico-
ig. 9. GSH/GSSG ratio in liver, kidney, heart, lung and spleen. Values arexpressed as mean ± S.E.M., for n = 6. *P < 0.05 compared to control, #P < 0.05ompared to nicotine.
S. Neogy et al. / Environmental Toxicology a
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0.10% in GSH-Px activity of lungs was seen on supplementa-ion with ANDRO, AE-AP and vit.E in comparison to nicotinereated animals.
In spleen, GSH-Px activity was declined significantlyP < 0.05) by 42.0% due to nicotine administration. Supplemen-ation with ANDRO, AE-AP and vit.E to nicotine treated groupshowed, 32.78%, 51.61% and 56.02% increase of GSH-Px activ-ty, respectively (Fig. 10).
. Discussion
In the present study, the protective effect of A. paniculataees has been carried out against nicotine-induced toxicity in
iver, kidney, heart, lung and spleen. For this, we have chosenajor bioactive metabolite andrographolide (ANDRO), crude
queous extract (AE-AP) and vit.E as a positive standard.NDRO was isolated and characterized by TLC and HPLC.
t was confirmed that presence of ANDRO in the isolated com-ound from A. paniculata Nees.
Nicotine, a pharmacologically active ingredient in tobacco, isenerally regarded to a primary risk factor in the development ofardiovascular disorders, myocardial infraction, stroke, kidneyancer, pulmonary diseases and certain immunological dysfunc-ion (Jung et al., 2001). This highly addictive alkaloid has beeneported to induce oxidative stress both in vivo and in vitroSuleyman et al., 2002). The mechanisms of free radicals gener-tion by nicotine are not clear. However, it has been reported thaticotine disrupts the mitochondrial respiratory chain leading ton increase generation of superoxide anions and hydrogen per-xide (Yildiz et al., 1998). Previous studies have suggested that,uperoxide anion and hydrogen peroxide are the main source oficotine induced free radicals depleting the cellular antioxidantsWetscher et al., 1995).
In this study, significant elevation of malondialdehyde
MDA) and protein carbonyls contents were observed in nicotinenduced hepatocytes, myocytes, spleenocytes, renal and lungissues. Lipid peroxidation is known to disturb the integrityf cellular membranes, leading to the leakage of cytoplasmic
ftit
nd Pharmacology 25 (2008) 321–328 327
nzymes (Bagchi et al., 1995). Enhanced lipid peroxidation asso-iated with antioxidant depletion in different tissues, may yieldrange of toxic aldehydes that are capable of damaging mem-rane proteins (Husain et al., 2001; Hedley and Chows, 1992).ecent report by our laboratory suggested that, increased lipideroxidation and decrease antioxidant enzyme status can be anndicator of disease progression of oral cavity cancer patientsDas et al., 2007). PC formation has been proposed to be anarlier marker of protein oxidation (Reznick and Packer, 1994).xidative modification of proteins may lead to the structural
lteration and functional inactivation of many enzyme proteinsDavies, 1988). Thus the nicotine induced oxidatively modi-ed proteins is due to either excessive oxidation of proteins orecreased capacity to cleanup oxidatively damaged proteins. Inhe present investigation, significantly increased MDA and PCevels were either partially or completely returned to the con-rol levels, were may be due to the free radicals scavengingroperties of the herbal product and vit.E. In our recent report,e have demonstrated that ANDRO is having hepato-renal pro-
ective activity by reducing the lipid peroxidation level againstthanol induced toxicity in mice (Singha et al., 2007). The con-tant exposure of the tissue to the herbal supplementation for theentioned schedule may have been able to detoxify the nicotine
oxicity by modulating the extent of lipid peroxidation and pro-ein oxidation. These modulatory mechanisms may either be a
utual biomolecular interaction among the reactive radicals ory interaction with herbal bioactive molecules.
Antioxidant enzymes are considered to be a primary defensehat prevents biological macromolecules from oxidative dam-ge. Aerobic cells contain various amounts of two mainntioxidant enzymes: superoxide dismutase (SOD) and catalaseCAT). SODs rapidly dismutate superoxide anion (O2
−•) to lessangerous H2O2, which is further degraded by CAT and glu-athione peroxidase (GSH-Px) to water and oxygen (Wetschert al., 1995). The results of the present study showed a significantall in SOD activities, in the nicotine treated groups. SOD, dis-utate O2
−• and the same in turn is a potent inhibitor of CATAshakumari and Vijayammal, 1996). The depletion in SODctivity was may be due to dispose off the free radicals, producedue to nicotine toxicity. Beside this, on nicotine administration,2O2 produced by dismutation of superoxide anion, may haveeen efficiently converted to O2 by CAT and the enzyme activ-ties showed a marked reduction. The depletion of antioxidantnzyme activity was may be due to inactivation of the enzymeroteins by nicotine-induced ROS generation, depletion of thenzyme substrates, and/or down-regulation of transcription andranslation processes. Glutathione is an important cellular reduc-ant, which offers protections against free radicals, peroxide andoxic compounds. It is reformed from GSSG by donation ofydrogen from NADPH, the reaction being catalyzed by glu-athione rductase (GR) (Meister, 1994 and John, 2003). In thistudy, a significant fall in GSH level and GSH-Px activity wasbserved in nicotine treated animals, may be due to enhanced
ree radical production (as evidence by increase lipid peroxida-ion and protein oxidation) and apart from catalase, GSH-Px alsonvolved in the removal of H2O2. H2O2 generated due to nico-ine toxicity, engage more GSH, which thereby get converted
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28 S. Neogy et al. / Environmental Toxico
o GSSG in presence of GSH-Px. Hence, the GSH, and GSSGevel decreases on nicotine administration. The toxic effects oficotine may be prevented during ANDRO, AE-AP and vit.Exposure, since it restores redox ratio.
In this scientific investigation, supplementation withNDRO, AE-AP and vit.E were able to compensate the antiox-
dant enzyme status, more or less near to the control levels. Oneossible reason behind this findings is may be the anti-oxidativeroperties of the supplements. The extracts of A. paniculatancrease the glutathione level in all tissues. Increased glutathioneevel may have efficiently scavenged ROS, increased the antiox-dant enzyme status and prevent the oxidative damage in allupplemented groups. Vitamins are the most important micronu-rients that are known to modulate the defense mechanisms. Inhe present study, A. paniculata products (ANDRO and AE-P) and vit.E treatment to the nicotine treated groups, showed
n increase in the antioxidant enzyme status. This lipid solubleitamin may have effectively reduced the free radicals produc-ion by scavenging the generated OFRs and thus obstruct theeroxidation chain reactions. As a well-acquainted fact, vit.Ects as a cytosolic antioxidant in the lipid domains. The synthe-is of glutathione is regulated by oxidants, antioxidants, growthactors (MacNee and Rahman, 2001). In the nicotine treatedroups, ANDRO, AE-AP and vit.E may have been able to elevatehe glutathione level, which thereby disposes the free radicals,enerated by nicotine toxicity and the chain reaction of ROSs prevented. So, a raise in hepatic, renal, cardiac, lung andpleenic antioxidant levels indicate the involvement of A. panic-lata products and vit.E in antioxidant defense against nicotinenduced oxidative stress mediated tissue injury.
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