Life Sciences 78 (2006) 1378 1384 www.elsevier.

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Effect of bacoside A on brain antioxidant status in cigarette smoke exposed rats

K. Anbarasi a, G. Vani a, K. Balakrishna b, C.SR Shyamala Devi c,*
a c

Department of Biochemistry, University of Madras, Guindy Campus, Chennai-600 025, India b Central Research Institute (Siddha), Chennai-600 106, India Department of Biochemistry, University of Madras, Guindy Campus, Chennai-600 025, India Received 30 March 2005; accepted 11 July 2005

Abstract Free radicals mediated oxidative stress has been implicated in the pathogenesis of smoking-related diseases and antioxidant nutrients are reported to prevent the oxidative damage induced by smoking. Therefore, the present study was conducted to evaluate the antioxidant role of bacoside A (triterpenoid saponin isolated from Bacopa monniera) against chronic cigarette smoking induced oxidative damage in rat brain. Adult male albino rats were exposed to cigarette smoke for a period of 12 weeks and simultaneously administered with bacoside A (10 mg/kg b.w./day, p.o.). Antioxidant status of the brain was assessed from the levels of reduced glutathione, vitamin C, vitamin E, and vitamin A and the activities of superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase. The levels of copper, iron, zinc and selenium in brain and serum ceruloplasmin activity were also measured. Oxidative stress was evident from the diminished levels of both enzymatic and non-enzymatic antioxidants. Alterations in the levels of trace elements with accumulation of copper and iron, and depletion of zinc and selenium were also observed. Bacoside A administration improved the antioxidant status and maintained the levels of trace elements. These results suggest that chronic cigarette smoke exposure enhances oxidative stress, thereby disturbing the tissue defense system and bacoside A protects the brain from the oxidative damage through its antioxidant potential. D 2005 Elsevier Inc. All rights reserved.
Keywords: Antioxidants; Bacoside A; Bacopa monniera; Cigarette smoking; Lipid peroxidation; Oxidative stress; Trace elements

Introduction There is a preponderance of evidence showing a strong association between cigarette smoking and alarming increase in the mortality rate from smoking-related diseases such as pulmonary diseases, cardio and cerebrovascular diseases, cancers, and several others (US DHHS, 1989). The role of free radicals in the pathogenesis of these diseases has been well documented (Church and Pryor, 1985). Formation of free radicals and reactive oxygen species (ROS) is a normal consequence of a variety of biochemical reactions. However,

* Corresponding author. Old No. 62, New No. 66, Second main road, Gandhi Nagar, Adyar, Chennai-600 020, India. Tel.: +91 44 2441 2575; fax: +91 44 2235 2494. E-mail addresses: anbarasii@yahoo.co.in (K. Anbarasi), cssdevi@yahoo.com (C.S.S. Devi). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.07.030

these free radicals are capable of independent existence and can cause oxidative damage to the tissues through lipid peroxidation (Cross et al., 1987). The human body has an inherent synergistic and multilevel defense mechanism, which comprise of two major classes of cellular protection against ROS (Muzakova et al., 2001). The enzymatic part is represented by free radical scavenger enzymes namely superoxide dismutase, catalase and glutathione peroxidase. The non-enzymatic part includes a large number of natural and synthetic antioxidant compounds (e.g. vitamins, thiols etc.) that have the ability to inhibit oxidative stress by scavenging the highly destructive free radical species. The deleterious effects of the free radicals are kept under check by a delicate balance between the rate of their production and the rate of their elimination by these defense systems (Halliwell, 1994). When there is an excessive addition of free radicals from exogenous sources added to the endogenous production, the available tissue defense system

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becomes overwhelmed resulting in oxidative damage to the tissues. A major exogenous source of free radicals is cigarette smoke which is a heterogeneous aerosol consisting of more than 4000 compounds including high concentrations of free radicals, and reactive oxygen and nitrogen species (Stedman, 1968). The obligatory use of the bodys reserve of antioxidants to detoxify the tremendous level of these free radicals in smokers therefore results in severe antioxidant deficiency status, thereby predisposing them to the development of life threatening diseases. Further, this deficiency in smokers may be enhanced by their generally lower intake of both supplementary and dietary antioxidants (Zondervan et al., 1996). When the normal level of antioxidant defense system is insufficient for the eradication of excessive free radicals, administration or supplementation of exogenous antioxidants has a protective role to play (Rekha et al., 2001). Several micronutrients and antioxidants of natural origin have been experimentally proved as effective protective agents against smoking induced oxidative stress (Sohn et al., 1993; Dilsiz et al., 1999; Helen et al., 1999; Koul et al., 2001). In view of the antioxidant property of Bacopa monniera (Tripathi et al., 1996), the effect of bacoside A, which retains the biological activity of Bacopa monniera, on the antioxidant defense system in rat brain has been investigated (Anbarasi et al., 2003). Bacoside A is the dammarene type triterpenoid saponin isolated from the plant Bacopa monniera, which is held in high repute as a potent nerve tonic (Chopra et al., 1956). Bacopa monniera Linn. is used in the indigenous systems of medicine for the treatment of various nervous system ailments such as insomnia, anxiety, epilepsy, hysteria etc. (Nadkarni, 1976). Preclinical and clinical studies have shown that B. monniera improves memory and mental function (Roodenrys et al., 2002; Stough et al., 2001). The plant has been shown as a potent free radical scavenger and antioxidant (Tripathi et al., 1996). Besides it also exhibits vasodilatory (Channa et al., 2003), calcium antagonistic (Dar and Channa, 1999) and muscle relaxant (Dar and Channa, 1997) properties. Preliminary studies indicated that bacosides, the major saponins are responsible for the facilitatory and modulatory effects of B. monniera (Singh et al., 1988). Recently, we have demonstrated the protective effect of bacoside A against smoking induced toxicity in rat brain (Anbarasi et al., 2005a,b). The brain is exposed throughout the life to oxidative stress and a number of diseases of the brain have been hypothesized to involve free radical induced oxidative damage either as a cause or as consequence of the disease processes. This organ is highly susceptible to free radical attack because it generates more of these toxicants per gram of tissue than does any other organ and yet not particularly enriched with protective antioxidants (Arivazhagan et al., 2002). Hence, the present study was undertaken to assess the neuroprotective role of bacoside A against oxidative stress in the brain of rats exposed to cigarette smoke by measuring the enzymatic and non-enzymatic antioxidants, and trace elements.

Materials and methods Chemicals and reagents Reduced glutathione, epinephrine, NADPH, ascorbic acid and a-tocopherol were obtained from Sigma Chemical Company, St. Louis, MO, USA. All other chemicals and reagents used were of reagent grade and highest purity, and obtained from Himedia, Mumbai, India. Standard bacoside A was purchased from Natural Remedies Private Limited, Bangalore, India. Locally available brand of cigarette, Scissors Standard (W.D & H.O. Wills), manufactured by Hyderabad Deccan Cigarette Factory was used in the present study. Bacoside A The plant Bacopa monniera was collected in and around Chennai, India and authenticated by Dr. P. Brindha, Central Research Institute (Siddha), Chennai, India. The dammarane type triterpenoid saponin-bacoside A was isolated from the plant by the standard procedure followed by Singh et al. (1988). The purity of the isolated bacoside A was identified by thin layer chromatography (TLC) and infrared (IR) spectrum analysis using standard bacoside A (data not shown). Aqueous suspension of bacoside A in 1% gum acacia was given orally to the experimental animals at a dosage of 10 mg/kg b.w./day for 12 weeks. Control animals received a corresponding volume of the vehicle suspended in normal saline. Animals Adult male albino rats of Wistar strain (120 200 g) procured from Tamilnadu University of Veterinary and Animal Sciences (TANUVAS), India were used for the present study. The rats were provided with standard pelleted rat feed and water ad libitum. They were acclimatized to the laboratory conditions and maintained under 12 h light and dark cycles at 25 T 2 -C. The experiments were carried out in accordance with the guidelines provided by the Institutional Animal Ethical Committee (No. IAEC 01/037/04). Experimental design The animals were divided into four groups of 6 animals each. Group Icontrol. Group IIrats exposed to cigarette smoke. Group IIIrats administered with bacoside A (10 mg/ kg b.w./day, p.o.). Group IVrats exposed to cigarette smoke and simultaneously administered with bacoside A. Group II and Group IV rats were exposed to cigarette smoke for a period of 12 weeks as described earlier (Anbarasi et al., 2005c). Briefly, the rats were exposed to side stream cigarette smoke in whole body smoke exposure chamber. Cigarette smoke was exposed twice daily, the duration of each exposure was 3 h with an interval of 10 min between each cigarette, using 8 10

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K. Anbarasi et al. / Life Sciences 78 (2006) 1378 1384 Table 2 Levels of reduced glutathione (GSH), vitamin C, vitamin E, and vitamin A in the brain of control and experimental animals Parameters GSH Vitamin C Vitamin E Vitamin A Group I 6.06 T 0.24 1.56 T 0.15 5.21 T 0.30 0.154 T 0.02 Group II 3.82 T 0.28* 0.61 T 0.10* 3.30 T 0.32* 0.102 T 0.01* Group III 6.50 T 0.35@ 1.58 T 0.08NS 5.32 T 0.26NS 0.157 T 0.02NS Group IV 5.92 T 0.20* 1.41 T 0.13* 4.89 T 0.25* 0.146 T 0.02*

cigarettes per day. The same brand of locally available cigarette was used throughout the experiment (Scissors Standard). At the end of experimental period (12 weeks), the animals were killed by cervical decapitation. Blood was collected and serum separated by centrifugation. Brain tissue was excised carefully and immediately washed in ice-cold saline. A 10% (w/v) homogenate was prepared in 0.1 M Tris HCl, pH 7.4 and used for biochemical studies. Biochemical analysis Superoxide dismutase was assayed by the method of Misra and Fridovich (1972). One unit of the enzyme activity is defined as the amount of enzyme required for the autooxidation of epinephrine by 50% per minute. Catalase activity was measured by following decomposition of H2O2 according to the method of Beers and Sizer (1952). The activity of glutathione peroxidase was assayed by measuring the amount of GSH consumed in the reaction mixture by the method of Rotruck et al. (1973). Glutathione reductase, which utilizes NADPH to convert oxidized GSH to reduced form, was assayed by the method of Stall et al. (1969). Protein was estimated by the method of Lowry et al. (1951). Reduced glutathione was measured according to the procedure of Moron et al. (1979) using DTNB reagent. Vitamin C was measured according to the method of Omaye et al. (1979) using DTC reagent. Vitamin E was measured according to the method of Quaife and Dju (1948) using dipyridyl reagent and vitamin A was measured by the method of Otto et al. (1946). Serum ceruloplasmin activity was assayed by following the procedure as described by Ravin (1961). Copper, zinc and iron was estimated using atomic absorption spectrophotometer after digestion with nitric acid and perchloric acid and selenium using coupled atomic emission spectrophotometer and flurometer. Statistical analysis Results are expressed as mean T SD (n = 6). Students t-test was used for the statistical evaluation of the data obtained.

(Values are expressed as mean T SD, n = 6). Unit GSH: Amoles/mg protein. Vitamins C, E, and A: mg/g wet tissue. Statistical comparisons are made between Group I vs. Group II and Group III; Group II vs. Group IV. *p < 0.001, @p < 0.05, NSnon significant.

Statistical comparisons are made between Group I vs. Group II and Group III; Group II vs. Group IV. Results The activities of superoxide dismutase, catalase, glutathione peroxidase, and glutathione reductase in brain of control and experimental animals are summarized in Table 1. A significant decrease ( P < 0.001) in the activities of the antioxidant enzymes was observed in Group II rats exposed to cigarette smoke compared to Group I rats. Group IV rats showed increased ( P < 0.001) activities of these enzymes as compared to Group II rats. Bacoside A per se administered Group III rats showed a significant ( P < 0.01) increase in their activities as compared to Group I rats which shows the enzymatic activation of antioxidants by bacoside A. Table 2 shows the levels of reduced glutathione, vitamin C, vitamin E, vitamin A in the brain of control and experimental animals. Group II rats exposed to cigarette smoke showed a significant decrease ( P < 0.001) in the levels of glutathione and vitamins C, E, and A when compared to control rats. A significant increase ( P < 0.001) in the levels of the nonenzymatic antioxidants was observed in Group IV rats when compared to Group II rats. Group III rats administered with bacoside A alone registered an increase ( P < 0.05) in GSH levels with no significant changes in other parameters as compared with Group I rats.

Table 1 Activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) in the brain of control and experimental animals Parameters SOD CAT GPx GR Group I 2.84 T 0.13 10.26 T 2.2 4.75 T .022 0.81 T 0.03 Group II 1.17 T 0.20* 4.25 T 2.0* 2.19 T 0.34* 0.58 T 0.02* Group III 3.12 T 0.14# 13.52 T 1.20# 5.14 T 0.20# 0.87 T 0.05# Group IV 2.70 T 0.15* 9.78 T 1.80* 4.42 T 0.32* 0.78 T 0.03*

Table 3 Serum ceruloplasmin (CP) activity and the levels of copper (Cu), iron (Fe), zinc (Zn), and selenium (Se) in the brain of control and experimental rats Parameters CP Cu Fe Zn Se Group I 38.42 T 2.9 13.2 T 3.2 268.12 T 15.6 0.89 T 0.02 12.6 T 2.2 Group II 17.53 T 2.5* 24.5 T 2.8* 358.33 T 12.1* 0.57 T 0.01* 5.50 T 2.0* Group III 39.50 T 3.0 12.6 T 3.4NS 263.46 T 14.3NS 0.92 T 0.04NS 12.8 T 3.2NS
NS

Group IV 35.25 T 3.2* 15.0 T 3.0* 282.58 T 15.2* 0.85 T 0.05* 11.8 T 2.5*

(Values are expressed as mean T SD, n = 6). Units: SOD: units/mg protein. CAT: Amoles of H2O2 decomposed/min/mg protein. GPx: Amoles of GSH oxidized/min/mg protein. GR: Amoles of NADPH oxidized/min/mg protein. Statistical comparisons are made between Group I vs. Group II and Group III; Group II vs. Group IV. *p < 0.001, #p < 0.01, NSnon significant.

(Values are expressed as mean T SD, n = 6). Units: CP: units/mg protein. Cu, Fe, Zn, Se: Ag/g wet tissue. Statistical comparisons are made between Group I vs. Group II and Group III; Group II vs. Group IV. *p < 0.001, NSnon significant.

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Serum ceruloplasmin activity and the levels of copper, iron, zinc, and selenium in the brain of control and experimental rats are shown in Table 3. The activity of ceruloplasmin, and the levels of zinc and selenium were significantly decreased ( P < 0.001), while the levels of copper and iron were significantly increased ( P < 0.001) in cigarette smoke exposed to Group II rats compared to Group I rats. The levels of copper and iron were significantly decreased ( P < 0.001) and the levels of zinc, selenium and ceruloplasmin activity were significantly increased ( P < 0.001) in Group IV rats compared to Group II rats. No significant changes were observed in these parameters in Group III rats compared to Group I rats. Discussion The brain is extremely vulnerable to oxidative stress, in part because it is highly enriched with non-heme iron, which is catalytically involved in the production of oxygen free radicals. In addition, the brain contains a relatively high degree of polyunsaturated fatty acids that are particularly good substrates for peroxidation reactions (Halliwell and Gutteridge, 1985). Cigarette smoke is a complex milieu possessing an array of free radicals and ROS, namely hydroxyl, peroxyl, nitric oxide, and superoxide radicals (Pryor, 1997). The sustained release of reactive free radicals from the tar and gas phases of smoke imposes an oxidant stress, promotes lipid peroxidation and consequently perturbs the antioxidant defense system in the blood and tissues of smokers (Pryor and Stone, 1993). Modulation of antioxidant status by increased lipid peroxidation during exposure to cigarette smoke has been reported in various organs (Baskaran et al., 1999; Chitra et al., 2000; Delibas et al., 2003). Our previous finding showed that cigarette smoke exposure increased both basal and induced lipid peroxidation in the rat brain (Anbarasi et al., 2005a). Under an oxidative stress, the antioxidant enzyme levels are increased, in order to cope with the tremendous increase in the production of ROS (Gutteridge and Halliwell, 1994). Acute exposure to cigarette smoke enhances the production of these antioxidant enzymes as a result of adaptive response, which consequently mitigate the damage caused by cigarette smoke (Hilbert and Mohsenin, 1996). An increase in the activities the antioxidant enzymes has been demonstrated upon exposure to cigarette smoke for 4 weeks (Baskaran et al., 1999). However, after prolonged exposure, the toxic effects of cigarette smoke appear to override the adaptive mechanism of the body tissues, as indicated by a decrement in the levels of these enzymes (Hulea et al., 1995). Our results are in corroboration with the above findings, showing decreased activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) in smoke exposed rat brain. SOD is the first enzyme in antioxidant defense that scavenges superoxide radicals to form H2O2 and hence diminishes the toxic effects of the radical. The quinone semiquinone radicals from the tar phase of cigarette smoke are capable of reducing molecular oxygen to superoxide radicals whose excessive generation inactivates this enzyme (Duthie and Arthur, 1994). Hence, a decrease in SOD activity upon

smoke exposure could have resulted from its inactivation by tar phase oxidants. CAT is involved in the detoxification of high concentrations of H2O2, whereas GPx is sensitive to lower concentration. The brain contains less CAT levels and hence GPx has a major role in quenching H2O2 and other peroxides which otherwise will lead to the production of hydroxyl and peroxyl radicals in the presence of iron (Bast and Barr, 1997; Gutteridge and Halliwell, 1994). Inhibition of CAT activity in rat brain and liver by cigarette smoke has been reported (Mendez-Alvarez et al., 1998). The presence and production of the free radicals from smoke lower this enzyme, leading to accumulation of H2O2 and lipid hydroperoxides further worsening the damage (Pryor, 1997). During acute smoke exposure, an increase in CAT activity was observed by Baskaran et al. (1999). However, a decrease in the activity of CAT in the present study suggests the inability of host antioxidant defense to meet the oxidative stress following chronic exposure to cigarette smoke. An increase in GPx activity is expected when the activity of CAT is not sufficient to cope with the oxidative stress. Absence of an augmentation in GPx activity upon smoke exposure in our study has been hypothesized to arise from a decrease in the levels of GSH that is essential for the conjugation of lipid peroxides. GR is an important enzyme for the maintenance of intracellular concentration of reduced glutathione (Gutteridge and Halliwell, 1994) and reduced availability of GSH could have resulted in the reduction of activity of GR upon smoke exposure. Bacoside A administration increased the activities of SOD, CAT, GPx and GR. Bhattacharya et al. (2000) reported increased antioxidant enzymes in rat frontal cortex, striatum and hippocampus upon administration of B. monniera. Enhancement of these enzymes in liver and kidney by B. monniera has been reported under different conditions (Sumathy et al., 2001; Rohini et al., 2004). Hence it is possible that bacoside A might be involved in the activation of enzymic antioxidants. Reduced glutathione (GSH) serves as the brains primary antioxidant defense against prooxidant stress and the level of brain GSH was significantly lowered after exposure to cigarette smoke. Smoking-induced depletion of GSH in kidney, lung and liver has also been reported (Anand et al., 1996; Baskaran et al., 1999). This depletion was directly associated with elevation in brain lipid peroxidation which could be attributed to its protection against ROS generated by smoke, besides its consumption by the antioxidant enzymes GPx and GST (Baskaran et al., 1999). Acetaldehyde, a major aldehyde from the smoke has been shown to deplete the cells of their GSH by conjugating with it, which further makes the cells more vulnerable to peroxidative damage (Nadiger et al., 1987). Hence, the decrease in GSH levels could possibly be related to the inability of host tissue to synthesize GSH that is reflected from decrease in vitamin C, E and A. GSH and these vitamins are tightly linked to each other in a way that it helps to replenish vitamin C which in turn regenerates vitamin E and A (Rani and Panneerselvam, 2001). Ascorbate is the first strong reductant in the aqueous phase that readily reacts with cigarette

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smoke oxidants and affords considerable protection to the cells (Kallner et al., 1981). The lipid-soluble, membrane bound vitamin E reacts with peroxyl radicals present in the smoke and terminates lipid peroxidation (Brown et al., 1997) and vitamin A effectively quenches singlet oxygen (Helen and Vijayammal, 1997). However, smokers are constantly overexposed to free radicals through inhalation of long-lived carbon- and oxygencentered radicals that subsequently deplete the plasma and tissue stores of these micronutrients (Brown et al., 1997; Pamuk et al., 1994). In vitro exposure of plasma to cigarette smoke resulted in the destruction of tocopherols, carotenoids, and retinol (Handelman et al., 1996). The present study also revealed depletion in the levels of non-enzymatic antioxidants such as vitamin C, E and A in the brain of rats exposed to cigarette smoke. Administration of bacoside A increased glutathione levels in the brain. Hepatoprotective and nephroprotective effect of B. monniera following increased GSH levels have been reported (Sumathy et al., 2001; Rohini et al., 2004). Similarly, an increase in GSH levels against smoking induced depletion in the brain indicates neuroprotective role of bacoside A. Bacoside A has been shown to quench superoxide radicals, more effectively than ascorbic acid (Pawar et al., 2001) and effectively scavenge the hydroxyl, peroxyl and nitric oxide radicals (Russo et al., 2003a,b). This could also be the reason for the maintenance of the antioxidant vitamins which otherwise would be spared in scavenging these radicals from smoke. Transition metal complexes are known to play a major role in free radical biology and cigarette tar contains large amounts of metals, complexed to some components of tar such as odiphenols (Cross et al., 1987). The observed increase in the levels of copper and iron in the brain could be due to the mobilization of iron from ferritin and copper from copperbinding protein induced by cigarette smoke exposure, which accelerate the oxidant injury through the formation of hydroxyl radicals via Haber-Weiss/Fenton reaction (Lapenna et al., 1995). Also cigarette smoke exposure resulted in decreased serum ceruloplasmin activity. Ceruloplasmin is a copper binding protein which functions as a ferroxidase that oxidizes iron to Fe3+ and thereby prevents accumulation of free iron. A decrease in serum ceruloplasmin activity has been reported in smokers (Pacht and Davis, 1988). This may in part reflect the increased levels of free iron and copper upon cigarette smoke exposure. It is quite likely that soluble iron and copper are highly toxic to brain. Damaged brain tissue undergoes rapid lipid peroxidation, presumably because metals released by cell disruption are not safely sequestered (Floyd, 1990). The question remains whether this might have contributed to the manifestation of brain damage or it was a consequence of brain damage. B. monniera has been shown to chelate the transition metals, inhibit formation of free radicals, and terminate lipid peroxidation at the initiation level itself (Tripathi et al., 1996). Thus, bacoside A could have protected the brain from cigarette smoke-induced rise in copper and iron as a metal chelator.

The levels of trace elements like zinc and selenium in brain were decreased upon exposure to cigarette smoke. Zinc, the cofactor for the enzyme SOD has been shown to protect the brain during ischemia or hypoglycemia (Choi, 1989). Selenium functions as an important nutrient of the brain and being a component of GPx, it plays a major role in preventing free radical mediated cell damage (Bou-Resli et al., 2002). Cadmium, the heavy metal from tobacco, decreased the bioavailability of selenium and zinc and hence depletes the antioxidant status (Preston, 1991). Zinc and selenium therapy has been found to be effective during cigarette smoking (Al-Bader et al., 1999; Dilsiz et al., 1999). However, alterations in mineral metabolism are secondary to chronic diseases associated with long-term smoking. Administration of bacoside A restored the levels of zinc and selenium, and the activity of ceruloplasmin in cigarette smoke exposed rat brain, probably by preventing cadmium mediated toxicity. Conclusion The above findings show that chronic cigarette smoking induces an oxidative stress on the brain by augmenting lipid peroxidation and diminishing both enzymatic and non-enzymatic antioxidant status. Bacoside A ameliorates cigarette smoking induced peroxidative changes probably through its free radical scavenging, anti-lipid peroxidative and antioxidant activities in the brain tissue. Thus, the results of our investigation suggest that bacoside A can be a potent antioxidant in the brain, an organ highly prone to oxidative stress against chronic smoking induced toxicity and hence may have useful properties as a natural antioxidant supplement, capable of preventing brain damage caused by oxidative stress. However, further studies pertaining to the precise mechanism of action of Bacoside A are warranted. Acknowledgements The financial assistance provided to Ms. K. Anbarasi (Grant No: 9/115(593)/2003, EMR-I, dt 06.05.2003) and Dr. G. Vani (Grant No: 9/115(511)/2000, EMR-I, dt 20.04.2000) by the Council of Scientific and Industrial Research (CSIR), New Delhi is gratefully acknowledged. References
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