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Environmental Toxicology and Pharmacology 25 (2008) 321–328

Amelioratory effect of Andrographis paniculata Nees 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 2007 Available online 4 November 2007

Abstract The ameliorative properties of bioactive compound andrographolide (ANDRO), aqueous extract of Andrographis paniculata (AE-AP) and vitamin E (vit.E) were tested against nicotine induced liver, kidney, heart, lung and spleen toxicity. A group of male Wistar rats were intraperitoneally administered vehicle, nicotine (1 mg/kg body weight/day), nicotine + ANDRO (250 mg/kg body weight/day), nicotine + AE-AP (250 mg/kg body weight/day) and nicotine + vit.E (50 mg/kg body weight/day) for the period of 7 days. The significantly increased levels of lipid peroxidation, protein oxidation and the decreased antioxidant enzyme status were noted in nicotine treated group as compared to vehicle treated group. ANDRO, AE-AP and vit.E significantly reduced the lipid peroxidation, protein oxidation and increased the antioxidant enzyme status. This indicates A. paniculata and vit.E may act as putative protective agent against nicotine induced tissue injury and may pave a new path to develop suitable drug therapy. © 2007 Elsevier B.V. All rights reserved.
Keywords: Adrographis paniculata; Andrographolide; Nicotine; Oxidative stress; Tissues; Vitamin E

1. Introduction Nicotine, as most biologically active chemical in tobacco smoke, has been the subject of intense scientific scrutiny. Among the most well characterized chemicals found in tobacco and tobacco smoke, are polycyclic aromatic hydrocarbons (PAHs) and the highly addictive alkaloid, nicotine and its metabolites (Campain, 2004). To further complicate the picture, nicotine is converted, during the production of cigarette and chewing

Abbreviations: AE-AP, aqueous extract of Andrographis paniculata; ANDRO, andrographolide; CAT, catalase; DTNB, 5,5-dithio-bis-2-nitrobenzoic acid; EDTA, ethylenediamine tetraacetic acid; FTIR, Fourier transform infra red spectroscopy; GR, glutathione reductase; GSH, reduce glutathione; GSH-Px, glutathione peroxidase; GSSG, oxidized glutathione; HPLC, high pressure liquid chromatography; MDA, malondialdehyde; OFR, oxygen free radicals; PC, protein carbonyls; SOD, superoxide dismutase; TLC, thin layer chromatography. ∗ Corresponding author. Tel.: +91 3222 275329; fax: +91 3222 275329. E-mail address: somenathroy@hotmail.com (S. Roy). 1382-6689/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.etap.2007.10.034

tobacco, into two highly mutagenic nitrosamine, N -nitrosonor nicotine (NNN) and 4-(methylnitrosamine)-1-(3-pyridyl)-1butanone (NNK) and is metabolized into cotinine. These chemicals derivatives also exhibit a wide spectrum of biological activity as compared to parent compound (Campain, 2004). Nicotine has been reported to induce oxidative stress both in vivo and in vitro (Pigeolot et al., 1990). The mechanism of generation of free radicals by nicotine is not clear. But oxidative stress occurs when there are excess free radicals and/or low antioxidant defense, and result in chemical alteration of biomolecules causing structural and functional modification. Oxygen free radicals (OFR) production has been directly linked to oxidation of cellular macromolecules, which may induce a variety of cellular responses through generation of secondary metabolic reactive species (Chiarugi, 2003). Previous reports have shown enhanced lipid peroxidation and inadequate antioxidant status by nicotine. Medicinal plants and their active principles have received greater attention as anti-peroxidative agent (Lee and Park, 2002).

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Andrographolide, the main active constituent of Adrographis paniculata (A. paniculata), has excellent anti-inflammatory, anti-bacterial and anti-viral effects. A. paniculata is an Indian herb, well known as ‘king of bitter’. This bitter herb generally has an affinity with heart and liver. New research has confirmed a host of pharmacological benefits of this herb for its enormous potential in far wide range of diseases. The present study was undertaken to evaluate the amelioratory property of andrographolide on tissue antioxidant status during nicotine induced oxidative stress in male Wistar rats.
2. Materials and methods 2.1. Animals
The weight matched (120–140 g) male Wistar rats were obtained, divided in different groups, housed in polypropylene cage and provided standard pellet diet (Chaulia Equipment and Chemicals, Ganapatinagar, Midnapore, India) and water ad libitum. The animals were maintained under standard conditions of temperature (25 ± 2 ◦ C) and humidity (60 ± 5%) with an alternating 12 h light/dark cycles. Animals were maintained in accordance with the guidelines of the National Institute of Nutrition, Indian Council of Medical Research, Hyderabad, India, and approved by the ethical committee of Vidyasagar University. All the experiments were conduced with the ethical guidelines laid down by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) constituted by the Animal Welfare Division of Government of India on the use of animals in scientific research.

2.5. Tissue extracts preparation
After decapitation, liver, kidney, heart, lung and spleen were excised from rats and washed with cold saline. Washed tissues were immediately immersed in liquid nitrogen and stored at −80 ◦ C. On preparation, tissues were sliced and homogenized in ice cold 50 mM sodium phosphate buffer (pH 7.0) containing 0.1 mM ethylenediamine tetraacetic acid (EDTA) to yield 10% (w/v) homogenate. The homogenates were then centrifuged at 1000 rpm for 10 min at 4 ◦ C. The supernatants were separated and used for enzyme assays and protein determination (Husain et al., 2001).

2.6. Preparation of aqueous extract
The fresh aerial parts of A. paniculata was blended and extracted with distilled water (10:1). The mixture was filtered with Whatman filter paper (No. 1) and concentrated at 38 ◦ C by a rotary evaporator, then allowed to stand at room temperature over night. The filtration and concentration processes were repeated to yield an aqueous solution. This solution was then centrifuged at 2000 × g for 10 min and supernatant was freeze dried to obtain the crude water extract (Zhang and Tan, 1996).

2.7. Isolation of ANDRO by thin layer chromatography (TLC)
A. paniculata powder was homogenized and extracted with distilled water (10:1). The mixture was filtered with Whatman filter paper (No. 1). The solution was then centrifuged at 200 × g for 10 min and supernatant was collected. The supernatant was mixed with ethyl acetate, yielding approximately 0.7% of ethyl acetate fraction. Two gram of ethyl acetate fraction was dissolved in methanol to obtain methanol extract (Zhang and Tan, 1997). The methanol extracts and the reference andrographolide (Sigma Chemical Ltd.) was spotted, separated on TLC plate. The plate was photographed after staining with 5% methanolic sulphuric acid. After this, the parallel band of methanol extract matching with the reference andrographolide was scrapped under ultra violet light (254 nm) and then eluted out from the TLC plate in methanol. Then the methanol was evaporated out by rotary evaporator and used for HPLC as well as biological activity studies.

2.2. Plant materials
A. paniculata Nees herbs were collected from the campus of IIT, Kharagpur, West Bengal, India in July–August, 2005 and air-dried. A voucher specimen has been deposited to the CAL herbarium, Botanical Survey of India, Howrah, India under the accession number IIT-VU/Ap-1.

2.3. Chemicals and reagents
Standard andrographolide, epinephrine, TBA, DNPH, H2 O2 , EDTA, DTNB, NADPH were purchased from Sigma Chemical Co., USA. Nicotine was purchased from Merck, Germany. Other chemicals were procured from Merck Ltd., SRL Pvt. Ltd., Mumbai, India.

2.8. Detection of ANDRO by high performance liquid chromatography (HPLC)
Isolated ANDRO was detected with standard andrographolide (Sigma Chemical 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 volume: 20 l; UV detection at 229 nm (Singha et al., 2007)

2.4. Treatment schedule
The animals were randomized into experimental and control groups and divided into five groups of six animals each. Rats’ in-group ‘A’ served as control, received 5% DMSO in physiological saline. Group ‘B’ animals received nicotine [3-(1-methyl-2-pyrrolidinyl)pyridine, C10 H14 N2 ] 1.0 mg/kg body weight/day (in physiological saline, pH was adjusted at 7.4 prior to injection), rats in group ‘C’ were administered nicotine as in group ‘B’ as well as andrographolide (250 mg/kg body weight/day) in 5% dimethyl sulfoxide (DMSO) with physiological saline, rats in group ‘D’ received nicotine (1.0 mg/kg body weight/day) as well as aqueous extract (250 mg/kg body weight/day) dissolved in 5% DMSO 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 body weight/day) in olive oil. Simultaneously, animals of group ‘A’, ‘B’, ‘C’ and ‘D’ were 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 and duration of nicotine were selected as per reported by many researchers (Chen et al., 2001 and Tuncok et al., 2001) and the dose of ANDRO was chosen as per the previous report (Madav et al., 1995). The experiment was terminated at the end of 7 days and all animals were sacrificed by an intraperitoneal injection of sodium pentobarbital (60–70 mg/kg body weight) (Chandran and Venugopal, 2004).

2.9. Analytical methods
2.9.1. Lipid peroxidation The extent of lipid peroxidation was estimated as the concentration of thiobarbituric acid reactive product malondialdehyde (MDA) by using the method of Ohkawa et al. (1979). One hundred microliters of tissue homogenate was added to 100 l of double-distilled water and 50 l of 8.1% sodium dodecyl sulfate (SDS) and incubated at room temperature for 10 min. Three hundred seventyfive microliters of 20% acetic acid (pH 3.5), along with 375 l of thiobarbituric acid (0.6%), was added to the tissue solution and placed in a boiling water bath for 60 min. After incubation, 250 l of double-distilled water and 1.25 ml of 15:1 butanol–pyridine solution were added to the mixture and centrifuged for 5 min at 2000 × g. The resulting supernatant was removed and measured at 532 nm with the use of the Hitachi U-2000 spectrophotometer. Malondialdehyde concentrations were determined by using 1,1,3,3-tetraethoxypropane as standard. 2.9.2. Protein carbonyl Protein carbonyl (PC) levels were measured according to method described by Reznick and Packer (1994); based on spectrophotometric detection of the

S. Neogy et al. / Environmental Toxicology and Pharmacology 25 (2008) 321–328 reaction of 2,4-dinitrophenylhydrazine with protein carbonyl to form protein hydrazones. Briefly, after precipitation of protein with an equal volume of 1% trichloroacetic acid (TCA), the pellet was resuspended in 10 mM DNPH in 2N HCl or with 2N HCl as control blank. Next after the washing procedure with 1:1 ethanol/ethylacetate, the final palette was dissolved in 6 M Guanidine. The carbonyl group was determined from the absorbance at 370 nm. The result was expressed as micromoles of carbonyl groups per milligram of protein with molar extinction coefficient of 22,000 M−1 cm−1 . 2.9.3. Superoxide dismutase Superoxide dismutase activity was determined at room temperature according to the method of Misra and Fridovich (1972). Ten microliters of tissue homogenate was added to 970 l (0.05 M, pH 10.2, 0.1 mM EDTA) of sodium carbonate buffer. Twenty microliters of 30 mM epinephrine (dissolved in 0.05% acetic acid) was added to the mixture to start the reaction. Superoxide dismutase activity was measured at 480 nm for 4 min on a Hitachi U-2000 spectrophotometer. Activity was expressed as the amount of enzyme that inhibits the oxidation of epinephrine by 50%, which is equal to 1 U/mg of protein. 2.9.4. Catalase Catalase activity was determined at room temperature by using a slightly modified version of Aebi (1984). Ten microliters of ethanol was added to 100 l of tissue homogenate. The tissue mixture was then placed in an ice bath for 30 min and tubes were brought at room temperature, followed by the addition of 10 l of Triton X-100 RS. Ten microliters of the tissue homogenate was added to a cuvette containing 240 l (0.05 M, pH 10.2, 0.1 mM EDTA) of sodium phosphate buffer, and 250 ml of 0.066 M H2 O2 (dissolved in sodium phosphate buffer) was added to start the reaction. Catalase activity was measured at 240 nm for 1 min with the use of the Hitachi U-2000 spectrophotometer. The molar extinction coefficient of 43.6 M cm−1 was used to determine CAT activity. One unit of activity is equal to the millimoles of H2 O2 degraded per minute per milligram of protein. 2.9.5. Reduced glutathione Reduced glutathione estimation was performed by the method of Grifith (1980). The required amount of tissue homogenate was mixed with 12% sulfosalicylic acid and centrifuged at 2000 × g for 15 min to settle the precipitated proteins. 0.1 ml of protein free supernatant, 0.7 ml of 0.3 mM NADPH, 0.1 ml of 6 mM DTNB and 0.48 units of glutathione reductase were combined and the absorbance of 5-thio-2-nitrobenzoic acid (TNB) was read at 412 nm. A standard curve was obtained with standard reduced glutathione. The level of GSH was expressed as microgram per mg protein. 2.9.6. Oxidized glutathione Oxidized glutathione estimation was performed by the method of Grifith (1980). The required amount of tissue homogenate was mixed with 12% sulfosalicylic acid and centrifuged at 2000 × g for 15 min to settle the precipitated proteins. 0.1 ml of protein free supernatant incubated in room temperature with 0.005 ml of 2 M 2-venyl pyridine for 1 h. Following incubation, 0.4 ml of 0.5 mM NADPH, 0.1 ml 6 M DTNB and 0.48 unit of glutathione reductase were added and measured at 412 nm. A standard curve was obtained with standard oxidized glutathione. The level of GSSG was expressed as microgram per mg protein. 2.9.7. Redox ratio (GSH/GSSG) Redox ratio was determined for all the five groups by taking the ratio of reduced glutathione/oxidized glutathione. 2.9.8. 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 7.0), 1 mM EDTA, 1 mM sodium azide, 0.2 mM NADPH, 1 U glutathione reductase and 1 mM reduced glutathione. The sample, after its addition, was allowed to equilibrate for 5 min at 25 ◦ C. The reaction was initiated by adding 0.1 ml of 2.5 mM H2 O2 . Absorbance at 340 nm was recorded for 5 min. Values were expressed as nanomoles of NADPH oxidized to NADP by using the extinction coefficient of 6.2 3 103 M−1 cm−1 at 340 nm. The activity of GSH-Px

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Fig. 1. TLC of methanol extract (T) and standard andrographlide (S) after derivatisation with 5% methanolic sulphuric acid at visible. was expressed in terms of nanomole NADPH consumed/min per milligram of protein.

2.10. Statistical analysis
The data were expressed as mean ± S.E.M. Comparisons of the means of control, nicotine, nicotine with ANDRO, nicotine with AE-AP and nicotine with vit.E group were made by two-way ANOVA with multiple comparison t-test, P < 0.05 as a limit of significance.

3. Results 3.1. Isolation of ANDRO Isolation of ANDRO was carried out by TLC (Fig. 1). The band parallel to reference was eluted out and used for HPLC analysis and supplemented in rats. 3.2. Detection of ANDRO by HPLC The detection of andrographolide was done with standard andrographolide (Sigma Chemical Co., USA) and the peck of both isolated compound and standard andrographolide matches, hence it confirmed the presence of andrographolide (Fig. 2). 3.3. Lipid peroxidation MDA levels were significantly (P < 0.05) increased in liver, kidney, heart, lungs and spleen by 30.21%, 22.42%, 121.43%, 176.92% and 40.40%, respectively, as compared to the control group. In liver, supplementation with ANDRO, AE-AP and vit.E showed significant (P < 0.05) diminution of MDA content by

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Fig. 2. Detection of andrographolide by HPLC. Black: Isolated andrographolide. Blue: Standard andrographolide (Sigma Chemical Co.). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)

13.45%, 16.42% and 17.14%, respectively, as compared to nicotine treated group. In kidney, significantly (P < 0.05) decreased level of MDA was seen after supplementation with ANDRO, AE-AP and vit.E by 10.78%, 13.29% and 15.21%, respectively. ANDRO did not show any significant change. In heart, supplementation with AE-AP and vit.E showed significant (P < 0.05) diminution in MDA levels by 19.75% and 22.18%, respectively, as compared to nicotine treated group. ANDRO did not response against nicotine toxicity in heart. In lungs, MDA level showed a significant (P < 0.05) diminution by 27.89% on ANDRO supplementation to nicotine treated animals. Supplementation with AE-AP and vit.E to nicotine treated group, significantly (P < 0.05) decreased the MDA level by 44.79% and 44.51%, respectively. In spleen, supplementation with ANDRO, AE-AP and vit.E decreased MDA levels by 52.36%, 45.28% and 48.58%, respectively, in comparison to nicotine treated animals (Fig. 3). 3.4. Protein oxidation Protein carbonyl levels were significantly (P < 0.05) elevated by 69.09%, 130.17%, 55.94%, 73.80% and 68.55%, respectively in all the treated tissues (liver, kidney, heart, lung and spleen) in comparison to control.

Supplementation with ANDRO, AE-AP and vit.E significantly (P < 0.05) decreases the liver PC content by 23.66%, 26.52% and 32.97%, respectively, as compared to the nicotine treated group. Renal PC content was significantly decreased by 21.72%, 26.22% and 43.07%, respectively in ANDRO, AE-AP and vit.E supplementation as compared to control animals. In heart, supplementation with AE-AP and vit.E showed significant (P < 0.05) fall in PC level by 27.80% and 31.83%, respectively, as compared to nicotine treated group. ANDRO did not show any significant alteration of PC level in heart against nicotine toxicity. In lung, PC content was significantly (P < 0.05) decreased by 25.57%, 36.52% and 39.26% on ANDRO, AE-AP and vit.E administration to nicotine treated animal. In spleen, all three supplementation (ANDRO, AE-AP and vit.E) significantly (P < 0.05) decreased PC level by 23.50%, 32.83% and 33.58%, respectively as compared to nicotine treated group and the decreased was significant in relation to control (Fig. 4). 3.5. Superoxide dismutase activity The SOD activity of liver was significantly (P < 0.05) reduced by 57.67% due to nicotine treatment in relation to control. Significant (P < 0.05) variation in SOD activity by 43.05% and 33.33%, respectively, was seen on supplementation with AE-AP and vit.E to nicotine treated group. But ANDRO did not increase SOD activity significantly as compared to nicotine treated group. Similarly, in kidney SOD activity was declined significantly (P < 0.05) by 56.41% due to nicotine administration. Supplementation with ANDRO, AE-AP and vit.E to nicotine treated groups showed, 27.05%, 42.35% and 55.29% increase of SOD activity significantly (P < 0.05), respectively. The SOD activity of heart was significantly (P < 0.05) reduced by 46.66% due to nicotine toxicity as compared to

Fig. 3. MDA concentration in liver, kidney, heart, lung and spleen. Values are expressed as mean ± S.E.M., for n = 6. *P < 0.05 compared to control, # P < 0.05 compared to nicotine.

Fig. 4. PC concentration in liver, kidney, heart, lung and spleen. Values are expressed as mean ± S.E.M., for n = 6. *P < 0.05 compared to control, # P < 0.05 compared to nicotine.

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the control group. While administration of ANDRO to nicotine treated rats showed no change in SOD activity, there was a significant (P < 0.05) increase of the latter on AE-AP and vit.E supplementation. Thus SOD activity of the heart, which was previously lowered by nicotine treatment, was increased by 46.78% and 76.78%, respectively on AE-AP and vit.E administration. Similarly, in lungs SOD activity was significantly (P < 0.05) reduced by 51.00% due to nicotine treatment as compared to control group. Though, supplementation with ANDRO produces no significant variation in SOD activity, where as AE-AP and vit.E supplementation significantly (P < 0.05) increase the activity by 65.16% and 79.64%, respectively, as compared to the nicotine treated group. Thus a fall in SOD activity was partly compensated by AE-AP and vit.E administration. The SOD activity of spleen was significantly (P < 0.05) reduced by nicotine administration in relation to the control group. The reduction was as much as 53.07%. ANDRO, AE-AP and vit.E administration to nicotine treated groups significantly (P < 0.05) increases the SOD activity by 74.69%, 78.22% and 83.82%, respectively (Fig. 5). 3.6. Catalase activity In liver CAT activity was decreased by 50.93% due to nicotine administration. A significant rise (P < 0.05) in CAT activity was observed on supplementation with ANDRO, AE-AP and vit.E by 25.41%, 35.49% and 63.16%, respectively, as compared to nicotine treated group. There was a significant fall (P < 0.05) in kidney CAT activity by 38.19% on nicotine administration. ANDRO, AE-AP and vit.E supplementation to nicotine treated groups showed a significant rise (P < 0.05) in CAT activity by 51.48%, 63.75% and 45.83%, respectively. There was a significant (P < 0.05) reduce in CAT activity of heart due to nicotine toxicity by 24.84%. While CAT activity of heart was significantly (P < 0.05) elevated by ANDRO, AEAP and vit.E supplementation by 36.44%, 59.32%, 48.31%, respectively to nicotine treated group.

Fig. 6. CAT activity in liver, kidney, heart, lung and spleen. Values are expressed as mean ± S.E.M., for n = 6. *P < 0.05 compared to control, # P < 0.05 compared to nicotine.

The CAT activity of lungs was significantly (P < 0.05) reduced by 49.25% due to nicotine treatment in relation to control. Significant (P < 0.05) deviation by 46.95% and 48.75% in catalase activity of lungs was seen on supplementation with AE-AP and vit.E to nicotine treated group. But ANDRO did not increase CAT activity significantly as compared to nicotine treated group. Similarly, in spleen catalase activity was declined significantly (P < 0.05) by 51.64% due to nicotine administration. Supplementation with ANDRO, AE-AP and vit.E to nicotine treated groups showed, 40.50%, 60.45% and 69.49% increase of CAT activity, respectively (Fig. 6). 3.7. GSH concentration In liver GSH content was decreased by 62.61% due to nicotine administration. A significant rise in GSH content was observed on supplementation with ANDRO, AE-AP and vit.E by 63.52%, 89.49% and 143.97%, respectively, as compared to nicotine treated group. There was a fall in kidney GSH content by 29.78% on nicotine administration. AE-AP and vit.E supplementation to nicotine treated groups showed a significant rise in GSH content by 28.20% and 35.22%, respectively. The GSH content of heart was significantly (P < 0.05) reduced by 43.09% due to nicotine toxicity as compared to the control group. While administration of ANDRO to nicotine treated rats showed no change in GSH content, there was a significant (P < 0.05) raise on AE-AP and vit.E supplementation. Thus GSH content of the heart, which was lowered by nicotine treatment, was increased by 21.47% and 32.98%, respectively on AE-AP and vit.E supplementation. Similarly, in lungs GSH content was significantly (P < 0.05) reduced by 56.43% due to nicotine toxicity as compared to control group. Supplementation with ANDRO, AE-AP and vit.E significantly (P < 0.05) increase by 31.88%, 35.16% and 49.45%, respectively, as compared to the nicotine treated group. The GSH content of spleen was significantly (P < 0.05) reduced by nicotine administration in relation to the control group. The reduction was as much as 47.38%. ANDRO, AE-AP

Fig. 5. SOD activity in liver, kidney, heart, lung and spleen. Values are expressed as mean ± S.E.M., for n = 6. *P < 0.05 compared to control, # P < 0.05 compared to nicotine.

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Fig. 7. GSH concentration in liver, kidney, heart, lung and spleen. Values are expressed as mean ± S.E.M., for n = 6. *P < 0.05 compared to control, # P < 0.05 compared to nicotine.

Fig. 8. GSSG concentration in liver, kidney, heart, lung and spleen. Values are expressed as mean ± S.E.M., for n = 6. *P < 0.05 compared to control, # P < 0.05 compared to nicotine.

and vit.E administration to nicotine treated groups significantly (P < 0.05) increases the GSH content by 31.15%, 39.64% and 65.04%, respectively (Fig. 7). 3.8. GSSG concentration The GSSG level of liver was significantly (P < 0.05) reduced by 53.16% due to nicotine treatment in comparison to control. Significant (P < 0.05) variation in GSSG level was seen on supplementation with ANDRO, AE-AP and vit.E by 60.55%, 66.97% and 69.72%, respectively, to nicotine treated group. In kidney, GSSG level was declined significantly (P < 0.05) by 51.61% due to nicotine administration. Supplementation with ANDRO, AE-AP and vit.E to nicotine treated groups showed, 50.30%, 73.36% and 104.24% rise of GSSG level, respectively. There was a fall in GSSG level of heart by 48.54%, on nicotine administration. A treatment with AE-AP and vit.E significantly (P < 0.05) increased the GSSG level by 46.23% and 38.67% as compared to nicotine treated group. While administration of ANDRO showed no significant variations. Similarly, in lungs GSSG level was decreased by 78.88% due to nicotine administration. A significant rise in GSSG level of lungs was observed on supplementation with ANDRO, AE-AP and vit.E by 261.76%, 323.53% and 282.35%, respectively, as compared to nicotine treated group. There was a fall in spleen GSSG level by 53.43% on nicotine administration. ANDRO, AE-AP and vit.E supplementation to nicotine treated groups showed a significant rise in GSSG level by 91.80%, 83.61% and 90.16%, respectively (Fig. 8). 3.9. 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 treated group as compared to control animals. The increased was observed in all the tissues studied. The supplemented groups showed the significantly decreased (P < 0.05) the redox ratio in comparison to nicotine treated animals (Fig. 9).

3.10. GSH-Px activity Due to nicotine administration, GSH-Px activity was decreased in liver by 52.55%. A significant rise (P < 0.05) in GSH-Px activity was observed on supplementation with ANDRO, AE-AP and vit.E by 45.06%, 63.44% and 91.98%, respectively, as compared to nicotine treated animals. There was a significant decrease (P < 0.05) in kidney GSH-Px activity in kidney by 50.02% on nicotine administration. Supplementation with ANDRO, AE-AP and vit.E to nicotine treated groups showed a significant rise (P < 0.05) in GSH-Px activity by 36.76%, 59.1% and 78.72%, respectively. As a result of nicotine toxicity, there was a significant (P < 0.05) reduce in GSH-Px activity in heart by 42.68%. On the other hand GSH-Px activity of heart was significantly (P < 0.05) elevated by the supplementation of ANDRO, AE-AP and vit.E by 29.83%, 41.93%, 59.67%, respectively as compared to nicotine treated group. The GSH-Px activity of lungs was significantly (P < 0.05) reduced by 48.19% due to nicotine treatment in relation to control. Significant (P < 0.05) deviation by 62.59%, 72.38% and

Fig. 9. GSH/GSSG ratio in liver, kidney, heart, lung and spleen. Values are expressed as mean ± S.E.M., for n = 6. *P < 0.05 compared to control, # P < 0.05 compared to nicotine.

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Fig. 10. GSH-Px activities in liver, kidney, heart, lung and spleen. Values are expressed as mean ± S.E.M., for n = 6. *P < 0.05 compared to control, # P < 0.05 compared to nicotine.

80.10% in GSH-Px activity of lungs was seen on supplementation with ANDRO, AE-AP and vit.E in comparison to nicotine treated animals. In spleen, GSH-Px activity was declined significantly (P < 0.05) by 42.0% due to nicotine administration. Supplementation with ANDRO, AE-AP and vit.E to nicotine treated groups showed, 32.78%, 51.61% and 56.02% increase of GSH-Px activity, respectively (Fig. 10). 4. Discussion In the present study, the protective effect of A. paniculata Nees has been carried out against nicotine-induced toxicity in liver, kidney, heart, lung and spleen. For this, we have chosen major bioactive metabolite andrographolide (ANDRO), crude aqueous extract (AE-AP) and vit.E as a positive standard. ANDRO was isolated and characterized by TLC and HPLC. It was confirmed that presence of ANDRO in the isolated compound from A. paniculata Nees. Nicotine, a pharmacologically active ingredient in tobacco, is generally regarded to a primary risk factor in the development of cardiovascular disorders, myocardial infraction, stroke, kidney cancer, pulmonary diseases and certain immunological dysfunction (Jung et al., 2001). This highly addictive alkaloid has been reported to induce oxidative stress both in vivo and in vitro (Suleyman et al., 2002). The mechanisms of free radicals generation by nicotine are not clear. However, it has been reported that nicotine disrupts the mitochondrial respiratory chain leading to an increase generation of superoxide anions and hydrogen peroxide (Yildiz et al., 1998). Previous studies have suggested that, superoxide anion and hydrogen peroxide are the main source of nicotine induced free radicals depleting the cellular antioxidants (Wetscher et al., 1995). In this study, significant elevation of malondialdehyde (MDA) and protein carbonyls contents were observed in nicotine induced hepatocytes, myocytes, spleenocytes, renal and lung tissues. Lipid peroxidation is known to disturb the integrity of cellular membranes, leading to the leakage of cytoplasmic

enzymes (Bagchi et al., 1995). Enhanced lipid peroxidation associated with antioxidant depletion in different tissues, may yield a range of toxic aldehydes that are capable of damaging membrane proteins (Husain et al., 2001; Hedley and Chows, 1992). Recent report by our laboratory suggested that, increased lipid peroxidation and decrease antioxidant enzyme status can be an indicator of disease progression of oral cavity cancer patients (Das et al., 2007). PC formation has been proposed to be an earlier marker of protein oxidation (Reznick and Packer, 1994). Oxidative modification of proteins may lead to the structural alteration and functional inactivation of many enzyme proteins (Davies, 1988). Thus the nicotine induced oxidatively modified proteins is due to either excessive oxidation of proteins or decreased capacity to cleanup oxidatively damaged proteins. In the present investigation, significantly increased MDA and PC levels were either partially or completely returned to the control levels, were may be due to the free radicals scavenging properties of the herbal product and vit.E. In our recent report, we have demonstrated that ANDRO is having hepato-renal protective activity by reducing the lipid peroxidation level against ethanol induced toxicity in mice (Singha et al., 2007). The constant exposure of the tissue to the herbal supplementation for the mentioned schedule may have been able to detoxify the nicotine toxicity by modulating the extent of lipid peroxidation and protein oxidation. These modulatory mechanisms may either be a mutual biomolecular interaction among the reactive radicals or by interaction with herbal bioactive molecules. Antioxidant enzymes are considered to be a primary defense that prevents biological macromolecules from oxidative damage. Aerobic cells contain various amounts of two main antioxidant enzymes: superoxide dismutase (SOD) and catalase (CAT). SODs rapidly dismutate superoxide anion (O2 −• ) to less dangerous H2 O2 , which is further degraded by CAT and glutathione peroxidase (GSH-Px) to water and oxygen (Wetscher et al., 1995). The results of the present study showed a significant fall in SOD activities, in the nicotine treated groups. SOD, dismutate O2 −• and the same in turn is a potent inhibitor of CAT (Ashakumari and Vijayammal, 1996). The depletion in SOD activity was may be due to dispose off the free radicals, produced due to nicotine toxicity. Beside this, on nicotine administration, H2 O2 produced by dismutation of superoxide anion, may have been efficiently converted to O2 by CAT and the enzyme activities showed a marked reduction. The depletion of antioxidant enzyme activity was may be due to inactivation of the enzyme proteins by nicotine-induced ROS generation, depletion of the enzyme substrates, and/or down-regulation of transcription and translation processes. Glutathione is an important cellular reductant, which offers protections against free radicals, peroxide and toxic compounds. It is reformed from GSSG by donation of hydrogen from NADPH, the reaction being catalyzed by glutathione rductase (GR) (Meister, 1994 and John, 2003). In this study, a significant fall in GSH level and GSH-Px activity was observed in nicotine treated animals, may be due to enhanced free radical production (as evidence by increase lipid peroxidation and protein oxidation) and apart from catalase, GSH-Px also involved in the removal of H2 O2 . H2 O2 generated due to nicotine toxicity, engage more GSH, which thereby get converted

328

S. Neogy et al. / Environmental Toxicology and Pharmacology 25 (2008) 321–328 Davies, K.J.A., 1988. Proteolytic systems as secondary antioxidant defenses. In: Chow, C.K. (Ed.), Cellular Antioxidant Defense Mechanisms, vol. 2. CRC Press, Boca Raton, FL, p. 25. Grifith, O.W., 1980. Determination of glutathione and glutathione sulfide using glutathione reductase and 2-vinyl pyridine. Anal. Biochem. 106, 207. Hedley, D., Chows, S., 1992. Flow cytometric measurement of lipid peroxidation in vital cells using parinaric acid. Cytometry 13, 686–692. Husain, K., Scott, B.R., Reddy, S.K., Somani, S.M., 2001. Chronic ethanol and nicotine interaction on rat tissue antioxidant defense system. Alcohol 25, 89–97. John, R. Speakman, 2003. Oxidative phosphorylation, mitochondrial proton cycling, free-radical production and aging. Adv. Cell Aging Gerontol. 14, 35–68. Jung, B.H., Chung, S., Shim, C., 2001. Different pharmokinetics of nicotine following intravenous administration of nicotine base and nicotine hydrogen tartrate in rats. J. Control Rel. 77, 183–190. Lee, B.M., Park, K.K., 2002. Beneficial and adverse effects of chemopreventive agents. Mutat. Res. 523–524, 265–270. MacNee, W., Rahman, I., 2001. Is oxidative stress central to the pathogenesis of chronic obstructive pulmonary disease? Trends Mol. Med. 7 (2), 55–62. Madav, H.C., Tripathi, T., Mishra, S.K., 1995. Analgesic, antipyretic and antialcerogenic effects of andrographolide. Indian J. Pharm. Sci. 57 (3), 121–125. Meister, A., 1994. Glutathione, ascorbate and cellular protection. Cancer Res. 54, 1969–1975. Misra, H.P., Fridovich, I., 1972. The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J. Biol. Chem. 247, 3170–3175. Ohkawa, H., Ohishi, N., Yagi, K., 1979. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction. Anal. Biochem. 95, 351–358. Paglia, D.E., Valentine, W.N., 1967. Studies on quantitative and qualitative characterization of erythrocyte glutathione peroxidase. J. Lab. Clin. Med. 70, 158–169. Pigeolot, E., Corbisier, P., Lambert, D., Michiels, C., Raes, M., Zachary, M.O., Ramacle, J., 1990. Glutathin peroxidase, superoxide dismutase and catalase inactivation by peroxides and oxygen derived radicals. Mech. Age. Dev. 51, 283–297. Reznick, A.Z., Packer, L., 1994. Oxidative damage to proteins: spectrophotometric methods for carbonyl assay. Methods Enzymol. 233, 357–363. Singha, P.K., Roy, S., Dey, S., 2007. Protective activity of andrographolide and arabinogalactan proteins from Andrographis paniculata Nees against ethanol induced toxicity in mice. J. Ethnopharmacol. 111, 13–21. Suleyman, H., Gumustakine, K., Taysi, S., Keles, S., Oztasan, N., Aktas, O., Altinkaynak, K., et al., 2002. Beneficial effect of Hippophase rhamnoides L on nicotine induced oxidative stress in rat blood compared with vitamin E. Biol. Pharm. Bull. 25, 1133–1136. Tuncok, Y., Hieda, Y., Keyler, D.E., Brown, S., Ennifar, S., Fattom, A., Pentel, P.R., 2001. Inhibition of nicotine-induced seizures in rats by combining vaccination against nicotine with chronic nicotine infusion. Exp. Clin. Psychopharmacol. 9 (2), 228–234. Wetscher, G.J., Bagchi, M., Bagchi, D., Perdikis, G., Hinder, P.R., Giaser, K., Hinder, R.A., 1995. Free radicals production in nicotine-treated pancreatic tissue. Free Radic. Biol. Med. 18, 877–882. Yildiz, D., Ercal, N., Armstrong, D.W., 1998. Nicotine enantomers and oxidative stress. Toxicology 130, 155–165. Zhang, C.Y., Tan, B.K.H., 1997. Mechanism of cardiovascular activity of Andrographis paniculata in the anaesthetized rats. J. Ethnopharmacol. 56, 97– 101. Zhang, C.Y., Tan, B.K., 1996. Hypotensive activity of aqueous extract of Andrographis paniculata in rats. Clin. Exp. Pharmacol. Physiol. 23 (8), 675–678.

to GSSG in presence of GSH-Px. Hence, the GSH, and GSSG level decreases on nicotine administration. The toxic effects of nicotine may be prevented during ANDRO, AE-AP and vit.E exposure, since it restores redox ratio. In this scientific investigation, supplementation with ANDRO, AE-AP and vit.E were able to compensate the antioxidant enzyme status, more or less near to the control levels. One possible reason behind this findings is may be the anti-oxidative properties of the supplements. The extracts of A. paniculata increase the glutathione level in all tissues. Increased glutathione level may have efficiently scavenged ROS, increased the antioxidant enzyme status and prevent the oxidative damage in all supplemented groups. Vitamins are the most important micronutrients that are known to modulate the defense mechanisms. In the present study, A. paniculata products (ANDRO and AEAP) and vit.E treatment to the nicotine treated groups, showed an increase in the antioxidant enzyme status. This lipid soluble vitamin may have effectively reduced the free radicals production by scavenging the generated OFRs and thus obstruct the peroxidation chain reactions. As a well-acquainted fact, vit.E acts as a cytosolic antioxidant in the lipid domains. The synthesis of glutathione is regulated by oxidants, antioxidants, growth factors (MacNee and Rahman, 2001). In the nicotine treated groups, ANDRO, AE-AP and vit.E may have been able to elevate the glutathione level, which thereby disposes the free radicals, generated by nicotine toxicity and the chain reaction of ROS is prevented. So, a raise in hepatic, renal, cardiac, lung and spleenic antioxidant levels indicate the involvement of A. paniculata products and vit.E in antioxidant defense against nicotine induced oxidative stress mediated tissue injury. References
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