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Hepatoprotective effect of Bacoside-A, a major constituent of Bacopa monniera Linn
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Phytomedicine 15 (2008) 901–905 www.elsevier.de/phymed
Hepatoprotective effect of Bacoside-A, a major constituent of Bacopa monniera Linn T. Sumathi, A. Nongbri Department of Medical Biochemistry, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai 600113, Tamil Nadu, India
Abstract Bacoside-A (B-A) was evaluated for its hepatoprotective activity against D-GalN induced liver injury in rats. B-A is a major constituent isolated from the plant Bacopa monniera Linn. B-A (10 mg/kg of body weight) was administered orally once daily for 21 days and then D-GalN (300 mg/kg of body weight) was injected on 21st day after final administration of B-A. B-A reduces the elevated levels of serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), g-glutamyl transferase (g-GT), lactate dehydrogenase (LDH), 50 nucleotidase (50 ND). In addition B-A also significantly restored towards normalization of the decreased levels of Vit-C, and Vit-E induced by D-GalN both in liver and plasma. These results suggest that B-A has hepatoprotective effect against D-GalN induced hepatotxicity in rats. r 2007 Elsevier GmbH. All rights reserved. Keywords: Bacopa monniera; Bacoside-A; D-galactosamine; Hepatotoxicity
Introduction Bacoside-A is a dammarane type triterpenoid saponin isolated from the plant Bacopa monniera (Garay et al., 1996), which is held in high repute as a potent nerve tonic (Chopra et al., 1956). Bacopa monniera Wettst. (syn. Herpestis monniera (Linn) H. B and K family Scrophulariaceae), is a medicinal plant commonly known as Brahmi, 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 Bacopa monniera improves memory and mental function (Roodenrys et al., 2002). The chronic effects of an extract of Bacopa monniera on Corresponding author. Tel.: +91 44 24545861; fax: +91 44 24540709. E-mail address: [email protected] (T. Sumathi).
0944-7113/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2007.11.020
cognitive function in healthy human subjects have been reported (Stough et al., 2001). Bacopa monniera also exhibits potent anti-oxidant (Tripathi et al., 1996), hepatoprotective effect on morphine toxicity (Sumathy et al., 2001), anticancer (Elangovan et al., 1995), antiulcer (Sairam et al., 2002), calcium antagonist (Dar and Channa, 1999),vasodilatory (Channa et al., 2003), smooth muscle relaxant (Dar and Channa, 1997), antiaddictive (Sumathy et al., 2007) and mast cell stabilizing (Samiulla et al., 2001) properties. The structure of B-A was initially deduced as 3-(a-Larabinopyranosyl)–O-b-D-glucopyranosid-10, 20-dihydroxy16-keto-dammarene- (24) mainly by analyzing the acid hydrolysis products of B-A (Chatterji et al., 1965). Preliminary studies with 13C-NMR indicated that B-A was a mixture of saponins in accordance with an earlier report (Kawai and Shibata, 1978) and not a single chemical entity as had been proposed (Chatterji et al., 1965). HPLC studies on B-A however showed it to
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be a mixture of bacoside A3, bacopaside II, jujubogenin isomer of bacopasaponin C and bacopasaponin C (Deepak et al., 2005). Bacoside-A usually co-occurs with Bacoside-B, the latter differing only in optical rotation and probably an isomer of B-A and its structure has not so far been elucidated (Rastogi, 1990). The nootropic activity of the extract has been attributed to the presence of two saponins, namely bacoside A and bacoside B, of which the former is the more important (Singh and Dhawan, 1997). Bacoside A and B themselves have been studied for anti-stress activities (Chowdhuri et al., 2002). Besides, several agronomy related studies on B-A content in Bacopa monniera have been published (Mathur et al., 2001). B-A also protects the brain from cigarette smoking induced membrane damage. Earlier we reported, the hepatoprotective effects of the alcoholic extract of Bacopa monniera on D-GalN induced liver toxicity (Sumathi and Ramakrishnan, 2007). In this communication we report on the protective role of B-A, the major saponin isolated from the plant Bacopa monniera, against D-galactosamine induced liver injury in rats.
Materials and methods Chemicals D-Galactosamine hydrochloride, nicotinamide adenine dinucleotide (NAD+), nicotinamide adenine dinucleotide phosphate (NADP+), 1-chloro 2, 4- dinitrobenzene (CDNB) and 5, 5- dithiobis-2-nitrobenzoic acid (DTNB) were obtained from SRL, India. All other reagents used were analytical grade.
Isolation of Bacoside-A The plant Bacopa monniera was collected in and around Chennai, India, and authenticated by Dr. P. Brinda, Central Research Institute (Siddha), Chennai, India. The dammarane type triterpenoid saponin B-A was isolated from the plant by the standard procedure followed by Singh et al. (1988). Aqueous suspension of B-A with 1% gum acacia was given orally to the animals at a dosage of 10 mg/kg, b.w/day (Anbarasi et al., 2005).
Animals Adult male albino rats of Wister strain weighing 150–200 g were purchased from Tamilnadu Veterinary and Animal Science University, Chennai, India. They were housed in an acrylic fiber cage in a temperature controlled room (temperature 2272 1C) and were maintained on a 12 h light/dark cycle. They were given a solid diet and water ad libitum. Animal studies were
conducted according to the Institute Animal Ethics Committee regulations approved by the committee for the purpose of the Control and Supervision of experiments on Animals (CPCSEA).
Experimental design Rats were randomly divided into four groups, each group containing six animals. Group I served as normal control and received normal saline for 21 days. Group II served as toxic control and received normal saline for 21 days. Group III and IV were administered with B-A (10 mg/kg of body weight) (Anbarasi et al., 2005) orally by intragastral gavage for 21 days. Group II and III were also injected intraperitoneally with D-GalN (300 mg/kg of body weight) on day 21. After 24 h of D-GalN administration, the blood was collected from tail vein under light ether anesthesia. Immediately, after blood withdrawal, all the groups were sacrificed. Liver samples were also collected for biochemical estimations. The blood samples were allowed to clot for 30–40 min, serum was separated by centrifugation at 3000 rpm for 15 min at 37 1C and used for various biochemical parameters. Liver samples were collected and washed with chilled normal saline, weighed and 10% (w/v) liver homogenates were made in ice cold 0.15 M KCl solution using motor driven Teflon pestle. The homogenates were centrifuged at 3000 rpm for 15 min and supernatants were taken for the assay.
Biochemical determinations in serum Alanine transaminase (ALT), aspartate transaminase (AST) (Reitman and Frankel, 1957), alkaline phosphatase (ALP) (King, 1965a), 50 nucleotidase (50 ND) (Luly et al., 1972), lactate dehydrogenase (LDH) (King, 1965b) and g-glutamyl transferase (gGT) (Rasalki and Rau, 1972) activities in the serum were assayed by the reported procedures. The protein content was measured by the methods of Lowry et al. (1951) with BSA as standard.
Non-enzymic antioxidant status Non-enzymic antioxidants such as vitamin C and vitamin E were estimated in plasma and liver tissue according to the method of Omaye et al. (1979) and Desai (1971) respectively.
Statistical analysis Data are expressed as mean7SD. Significance of difference was evaluated using Student’s t-test and po0.05 were considered as significant.
ARTICLE IN PRESS T. Sumathi, A. Nongbri / Phytomedicine 15 (2008) 901–905
rats, the activities of serum marker enzymes were similar to control rats (group I).
Results Effects of B-A on AST, ALT, ALP, gamma-GT, 50 ND and LDH activities Table 1 shows the activities of hepatic marker enzymes viz., AST, ALT, ALP, g-GT, 50 ND and LDH in serum of control and experimental rats. In D-GalN induced rats (group II), the activities of marker enzymes viz., AST (po0.001), ALT (po0.001) ALP (po0.01), g-GT (po0.01), 50 ND (po0.001) and LDH (po0.001) were found to be significantly increased in serum when compared with the control (group I) rats, whereas in BA pretreated (group III) rats these marker enzymes viz., AST (po0.001), ALT (po0.001) ALP (po0.01), g-GT (po0.01), 50 ND (po0.001) and LDH (po0.01) were found to be significantly decreased in serum when compared with the group II rats. In BA alone treated
Table 1.
903
Effects of B-A on the level of plasma and liver vitamin-C and E Table 2 shows the effect of B-A on the levels of plasma and hepatic vitamin-C and vitamin-E in control and experimental rats. The levels of vitamin-C (po0.05) and vitamin-E (po0.01) in plasma and liver tissue were significantly decreased in D-GalN induced rats (group II) when compared with control rats (group I). In B-A pretreated rats the level of above non-enzymic antioxidants were significantly (po0.05) increased to near normal in plasma and liver when compared with DGalN induced rats. B-A alone treated rats (group IV) did not show any changes on the level of non-enzymic antioxidants and resembled the control (group I).
Effect of B-A on hepatic marker enzymes of control and experimental rats
Parameters
Group I Control
Group II D-GalN
Group III B-A+D-GalN
Group IV B-A
AST(IU/L) ALT(IU/L) ALP(IU/L) g-GT(IU/L) 50 ND(IU/L) LDH(IU/L)
68.2878.03 29.4672.69 7.2671.03 5.3270.45 3.2170.29 67.2377.12
152.67711.9a$ 82.5877.27a$ 12.2470.94a# 9.5470.79a# 6.5670.32a$ 136.67712.38a$
75.7275.84b$ 38.6573.91b$ 9.7270.43b# 7.2470.59b# 3.7370.25b$ 79.3277.2b#
75.9577.28 31.9173.86 8.8770.75 6.0970.85 3.5970.31 73.3178.15
Values are expressed as mean7SD; n ¼ 6. $ po0.001; #po0.01. a as compared with group I. b as compared with group II.
Table 2.
Effect of B-A on the levels of plasma and hepatic vitamin-C and vitamin-E of control and experimental rats
Group
Control D-GalN B-A+D-GalN B-A
Plasma
Liver
Vit-C (mg/dl)
Vit-E (mg/dl)
Vit-C (mg/100 g tissue)
Vit-E (mg/100 g tissue)
19370.09 1.2570.06a@ 1.6370.09b@ 1.8970.17
2.3970.17 1.3470.12a# 1.9170.13b@ 2.1870.10
0.7870.07 0.5370.03a@ 0.6770.02b@ 0.6570.04
5.1470.24 3.1970.19a# 4.0370.12b@ 4.9170.17
Values are expressed as mean7SD; n ¼ 6. # po0.01; @po0.05. a as compared with group I. b as compared with group II.
Legend Group I Group-II Group-III Group-IV
-Saline control rats -D-galactosamine induced rats -B-A Pretreated+D-galactosamine induced rats -B-A alone treated rats
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Discussion The present study has demonstrated that B-A has protective effects against liver damage induced by D-GalN in rats. D-GalN-induced hepatotoxicity is well established as similar to viral hepatitis (Decker and Keppler, 1974). Liver damage induced by D-GalN, reflects disturbances of liver cell metabolism, which lead to characteristic changes in the serum enzyme activities. Elevated serum enzymes are indicative of cellular leakage and loss of functional integrity of the cell membrane in liver (Ryan et al., 1990). Hence significant rise in the transaminases levels could be taken as an index of liver damage. In our study, significant increase in the activities of serum enzymes viz., ALT, AST, ALP, g-GT, 50 ND and LDH were observed in D-GalN intoxicated rats. Pretreatment with B-A significantly prevented these increased enzyme activities produced by D-GalN, indicating that B-A has hepatoprotective effect against D-GalN induced liver injury. A detailed mechanism of the hepatotoxicity induced by D-GalN has not been found out but the mechanism might be through immune system or oxidative stress. The a-tocopherol and ascorbic acid are naturally occurring free radical scavengers (Yu, 1994). Both ascorbic acid and a-tocopherol are known to be decreased in liver diseases (Bjorneboe et al., 1987). The decrease in the levels of the a-tocopherol and vitamin-C in our study might be due to their increased utilization for scavenging oxygen-derived radicals. It has been reported that the phenolic OH is essential for both antioxidant activity and free radical kinetics (Priyadarsini et al., 2003). Alanko et al. (1999) reported that compounds with hydroxyl group present in the phenolic ring have peroxyl radical and superoxide scavenging properties. The presence of a hydroxyl group in the phenolic ring of B-A might be a reason for its free radical scavenging properties. A number of reports suggested that the Bacopa monniera has a hepatoprotective and antioxidant potentiality, which could have been possible because of the presence of its active major saponin Bacoside-A. Hence from our result it can be suggested that BacosideA was a compound responsible for the hepatoprotective and antioxidant potential against liver injury caused by D-GalN administration. In conclusion, this study presents strong evidence of hepatoprotective effects of B-A against D-GalN intoxicated rats. Earlier studies have shown that the neuropsychopharmacological effects of the nootropic Bacopa monniera is due to the presence of its major saponins Bacoside-A and B (Singh and Dhawan, 1997). In another study the protective effict of B-A against chronic cigarette smoking has also been reported (Anbarasi et al., 2005). Though several studies have been reported for B-A, its effect on hepatotoxicity is not
yet reported. Hence, in this present communication we report the protective effects of B-A the major dammarane saponin isolated from the plant Bacopa monniera, against D-GalN induced liver toxicity. Further studies are needed for better understanding of the mechanism of action and to evaluate the efficacy of the B-A on liver organelle that are possibly damaged during experimental hepatitis.
References Alanko, J., Riutta, A., Holm, P., Mucha, I., Vapaatalo, H., Metsa-Ketala, T., 1999. Modulation of arachidonic acid metabolism by phenols: relation to their structure and Antioxidant properties. Free Radic. Biol. Med. 26, 193–201. Anbarasi, K., Vani, G., Balakrishna, K., Shyamala Devi, C.S., 2005. Creatine kinase isoenzyme patterns upon chronic exposure to cigarette smoke: protective effect of Bacoside A. Vas. Pharmaocol. 42, 57–61. Bjorneboe, G., Johnsen, J., Bjorneboe, A., Morland, J., Drevon, C.A., 1987. Effect of heavy alcohol consumption on serum concentrationof fat-soluble vitamins and selenium. Alcohol Alcohol 1, 533–537. Channa, S., Dar, A., Yaqoob, M., Anjum, S., Sultani, Z., Rahman, A., 2003. Broncho-vasodilatatory activity of fractions and pure constituents isolated from Bacopa monniera. J. Ethnopharmacol. 86, 27–35. Chatterji, N., Rastogi, R.P., Dhar, M.L., 1965. Chemical examination of Bacopa monniera Wettst.: parti-isolation of chemical constituents. Indian J. Chem. 3, 24–29. Chopra, R.N., Nayar, S.L., Chopra, I.C., 1956. Glossary of Indian Medicinal Plant. CSIR, New Delhi, p. 32. Chowdhuri, D.K., Parmar, D., Kakkar, P., Shukla, R., Seth, P.K., Srimal, R.C., 2002. Anti-stress effects of bacosides of Bacopa monnieri: modulation of HSP to expression, SoI and cytochrome P450 activity in rat brain. Phytother. Res. 16, 639–645. Dar, A., Channa, S., 1997. Relaxant effect of ethanol extract of Bacopa monniera on trachea, pulmonary artery and aorta from rabbit and guinea-pig. Phytother. Res. 11, 323–325. Dar, A., Channa, S., 1999. Calcium antagonistic activity of Bacopa monniera on vascular and intestinal smooth muscles of rabbit and guinea-pig. J. Ethnopharmacol. 66, 167–174. Decker, K., Keppler, D., 1974. Galactosamine hepatitis: key role of the nucleotide deficiency period in the pathogenesis of cell injury and cell death. Rev. Physiol. Biochem. Pharmacol. 71, 77–106. Deepak, M., Sangli, G.K., Arun, P.C., Amit, A., 2005. Quantitative determination of the major saponin mixture Bacoside A in Bacopa monniera by HPLC. Phytochem. Anal. 16, 24–29. Desai, I.D., 1971. Vitamin E analysis methods for animal tissues. Methods Enzymol. 105, 138–147. Elangovan, V., Govindasamy, S., Ramamoorthy, N., Balasubramanian, K., 1995. In vitro studies on the anticancer activity of Bacopa monnieri. Fitoterapia 66, 211–215.
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Garay, S., Mahato, S.B., Ohtani, K., Yamasaki, K., 1996. Dammarane-type triterpenoid saponins from Bacopa monniera. Phytochemistry 42, 815–820. Kawai, K.I., Shibata, S., 1978. Pseudojujubogenin, a new sapogenin from Bacopa monniera. Phytochemistry 17, 287–289. King, J., 1965a. The phosphohydrolyases—acid and alkaline phosphatase. In: Practical Clinical Enzymology. Van D.Nostrand Co. Ltd., London, pp. 191–208. King, J., 1965b. The dehydrogenases or oxido reductaselactate dehydrogenase. In: Practical Clinical Enzymology. Van D. Nostrand Co. Ltd., London, pp. 83–93. Lowry, O.H., Rosebrough, N.J., Forr, A.L., Ramdall, R.J., 1951. Protein measurement with the Folins phenol reagent. J. Biol. Chem. 193, 265–275. Luly, P., Barnaberi, O., Tria, E., 1972. Hormonal control in vitro of plasma membrane bound Na+, K+ ATPase of rat liver. Biochem. Biophys. Acta 282, 447–452. Mathur, S., Gupta, M.M., Kumar, S., 2001. Expression of growth and bacoside A in response to seasonal variation Bacopa monnieri accessions. J. Med. Aroma. Plant Sci. 22/4A–23/1A, 320–326. Nadkarni, K.M., 1976. Indian Materia Medica. Popular Prakashan Pvt. Ltd., Mumbai, pp. 624–625. Omaye, S.T., Turnbull, J.D., Sauberlich, H.E., 1979. Selected methods for the determination of ascorbic acid in animal cells, tissues, and body fluids. Methods Enzymol. 62, 3–11. Priyadarsini, K.I., Maity, D., Naik, G.H., Kumar, M.S., Unnikrishnan, M.K., Satav, J.G., Mohan, H., 2003. Role of phenolic O–H and methylene hydrogen on the free radical reactions and antioxidant activity of curcumin. Free Radic. Bio. Med. 35, 475–484. Rasalki, S.B., Rau, D., 1972. Serum gamma glutamyl transpepdidase activity in alcoholism. Clin. Chem. Acta 39, 41–47. Rastogi, R.P., 1990. Compendium of Indian Medicinal Plants, vol. 1. CSIR, New Delhi, pp. 118–122. Reitman, S., Frankel, A.S., 1957. A colorimetric method for the determination of serum glutamic oxaloacetic and glutamic pyruvic transaminases. Am. J. Clin. Pathol. 28, 53–56. Roodenrys, S., Booth, D., Bulzoni, S., Phipps, A., Micallef, C., Smoker, J., 2002. Chronic effects of Brahmi (Bacopa
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monnieri) on human memory. Neuropsychopharmacology 27, 279–281. Ryan, C.J., Aslam, M., Courtney, J.M., 1990. Transferance of hepatic coma to normal rats from galactosamine treated donors by reverse plasma exchange. Biomat. Artif. Cells Artif. Organs 18, 477–482. Sairam, K., Dorababu, M., Goel, R.K., Bhattacharya, S.K., 2002. Antidepressant activity of standardized extract of Bacopa monniera in experimental models of depression in rats. Phytomedicine 9, 207–211. Samiulla, D.S., Prashanth, D., Amit, A., 2001. Mast cell stabilising activity of Bacopa monnieri. Fitoterapia 72, 284–285. Singh, H.K., Dhawan, B.N., 1997. Neuropsychopharmacological effects of the Ayurvedic nootropic Bacopa monniera Linn (Brahmi). Indian J. Pharmacol. 29, S359–S365. Singh, H.K., Rastogi, R.P., Srimal, R.C., Dhawan, B.N., 1988. Effect of Bacoside A and B on avoidance responses in rats. Phytother. Res. 2, 70–75. Stough, C., Lloyd, J., Clarke, J., Downey, A.L., Hutchison, C.W., Rodgers, T., Nathan, P.J., 2001. The chronic effects of an extract of Bacopa monniera (Brahmi) on cognitive function in healthy human subjects. Psychopharmacology 156, 481–484. Sumathi, T., Ramakrishnan, S., 2007. Hepatoprotective activity of Bacopa monniera on D-Galactosamine induced hepatotoxicity in rats. Nat. Prod. Sci. 13 (3), 195–198. Sumathy, T., Subramanian, S., Govindasamy, S., Balakrishna, K., Veluchamy, G., 2001. Protective role of Bacopa monniera on morphine induced hepatotoxicity in rats. Phytother. Res. 15, 643–645. Sumathy, T., Balakrishnan, K., Veluchamy, G., Niranjali, Devaraj, 2007. Inhibitory effect of Bacopa monniera on morphine induced pharmacological effects in mice. Nat. Prod. Sci. 13 (1), 46–53. Tripathi, Y.B., Chaurasia, S., Tripathi, E., Upadhyay, A., Dubey, G.P., 1996. Bacopa monniera Linn. as an antioxidant: mechanism of action. Indian J. Exp. Biol. 34, 523–526. Yu, B.P., 1994. Cellular defenses against damage from reactive oxygen species. Physiol. Rev. 74, 139–162.
Phytomedicine 15 (2008) 901–905 www.elsevier.de/phymed
Hepatoprotective effect of Bacoside-A, a major constituent of Bacopa monniera Linn T. Sumathi, A. Nongbri Department of Medical Biochemistry, Dr. ALM Post Graduate Institute of Basic Medical Sciences, University of Madras, Taramani Campus, Chennai 600113, Tamil Nadu, India
Abstract Bacoside-A (B-A) was evaluated for its hepatoprotective activity against D-GalN induced liver injury in rats. B-A is a major constituent isolated from the plant Bacopa monniera Linn. B-A (10 mg/kg of body weight) was administered orally once daily for 21 days and then D-GalN (300 mg/kg of body weight) was injected on 21st day after final administration of B-A. B-A reduces the elevated levels of serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), g-glutamyl transferase (g-GT), lactate dehydrogenase (LDH), 50 nucleotidase (50 ND). In addition B-A also significantly restored towards normalization of the decreased levels of Vit-C, and Vit-E induced by D-GalN both in liver and plasma. These results suggest that B-A has hepatoprotective effect against D-GalN induced hepatotxicity in rats. r 2007 Elsevier GmbH. All rights reserved. Keywords: Bacopa monniera; Bacoside-A; D-galactosamine; Hepatotoxicity
Introduction Bacoside-A is a dammarane type triterpenoid saponin isolated from the plant Bacopa monniera (Garay et al., 1996), which is held in high repute as a potent nerve tonic (Chopra et al., 1956). Bacopa monniera Wettst. (syn. Herpestis monniera (Linn) H. B and K family Scrophulariaceae), is a medicinal plant commonly known as Brahmi, 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 Bacopa monniera improves memory and mental function (Roodenrys et al., 2002). The chronic effects of an extract of Bacopa monniera on Corresponding author. Tel.: +91 44 24545861; fax: +91 44 24540709. E-mail address: [email protected] (T. Sumathi).
0944-7113/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.phymed.2007.11.020
cognitive function in healthy human subjects have been reported (Stough et al., 2001). Bacopa monniera also exhibits potent anti-oxidant (Tripathi et al., 1996), hepatoprotective effect on morphine toxicity (Sumathy et al., 2001), anticancer (Elangovan et al., 1995), antiulcer (Sairam et al., 2002), calcium antagonist (Dar and Channa, 1999),vasodilatory (Channa et al., 2003), smooth muscle relaxant (Dar and Channa, 1997), antiaddictive (Sumathy et al., 2007) and mast cell stabilizing (Samiulla et al., 2001) properties. The structure of B-A was initially deduced as 3-(a-Larabinopyranosyl)–O-b-D-glucopyranosid-10, 20-dihydroxy16-keto-dammarene- (24) mainly by analyzing the acid hydrolysis products of B-A (Chatterji et al., 1965). Preliminary studies with 13C-NMR indicated that B-A was a mixture of saponins in accordance with an earlier report (Kawai and Shibata, 1978) and not a single chemical entity as had been proposed (Chatterji et al., 1965). HPLC studies on B-A however showed it to
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be a mixture of bacoside A3, bacopaside II, jujubogenin isomer of bacopasaponin C and bacopasaponin C (Deepak et al., 2005). Bacoside-A usually co-occurs with Bacoside-B, the latter differing only in optical rotation and probably an isomer of B-A and its structure has not so far been elucidated (Rastogi, 1990). The nootropic activity of the extract has been attributed to the presence of two saponins, namely bacoside A and bacoside B, of which the former is the more important (Singh and Dhawan, 1997). Bacoside A and B themselves have been studied for anti-stress activities (Chowdhuri et al., 2002). Besides, several agronomy related studies on B-A content in Bacopa monniera have been published (Mathur et al., 2001). B-A also protects the brain from cigarette smoking induced membrane damage. Earlier we reported, the hepatoprotective effects of the alcoholic extract of Bacopa monniera on D-GalN induced liver toxicity (Sumathi and Ramakrishnan, 2007). In this communication we report on the protective role of B-A, the major saponin isolated from the plant Bacopa monniera, against D-galactosamine induced liver injury in rats.
Materials and methods Chemicals D-Galactosamine hydrochloride, nicotinamide adenine dinucleotide (NAD+), nicotinamide adenine dinucleotide phosphate (NADP+), 1-chloro 2, 4- dinitrobenzene (CDNB) and 5, 5- dithiobis-2-nitrobenzoic acid (DTNB) were obtained from SRL, India. All other reagents used were analytical grade.
Isolation of Bacoside-A The plant Bacopa monniera was collected in and around Chennai, India, and authenticated by Dr. P. Brinda, Central Research Institute (Siddha), Chennai, India. The dammarane type triterpenoid saponin B-A was isolated from the plant by the standard procedure followed by Singh et al. (1988). Aqueous suspension of B-A with 1% gum acacia was given orally to the animals at a dosage of 10 mg/kg, b.w/day (Anbarasi et al., 2005).
Animals Adult male albino rats of Wister strain weighing 150–200 g were purchased from Tamilnadu Veterinary and Animal Science University, Chennai, India. They were housed in an acrylic fiber cage in a temperature controlled room (temperature 2272 1C) and were maintained on a 12 h light/dark cycle. They were given a solid diet and water ad libitum. Animal studies were
conducted according to the Institute Animal Ethics Committee regulations approved by the committee for the purpose of the Control and Supervision of experiments on Animals (CPCSEA).
Experimental design Rats were randomly divided into four groups, each group containing six animals. Group I served as normal control and received normal saline for 21 days. Group II served as toxic control and received normal saline for 21 days. Group III and IV were administered with B-A (10 mg/kg of body weight) (Anbarasi et al., 2005) orally by intragastral gavage for 21 days. Group II and III were also injected intraperitoneally with D-GalN (300 mg/kg of body weight) on day 21. After 24 h of D-GalN administration, the blood was collected from tail vein under light ether anesthesia. Immediately, after blood withdrawal, all the groups were sacrificed. Liver samples were also collected for biochemical estimations. The blood samples were allowed to clot for 30–40 min, serum was separated by centrifugation at 3000 rpm for 15 min at 37 1C and used for various biochemical parameters. Liver samples were collected and washed with chilled normal saline, weighed and 10% (w/v) liver homogenates were made in ice cold 0.15 M KCl solution using motor driven Teflon pestle. The homogenates were centrifuged at 3000 rpm for 15 min and supernatants were taken for the assay.
Biochemical determinations in serum Alanine transaminase (ALT), aspartate transaminase (AST) (Reitman and Frankel, 1957), alkaline phosphatase (ALP) (King, 1965a), 50 nucleotidase (50 ND) (Luly et al., 1972), lactate dehydrogenase (LDH) (King, 1965b) and g-glutamyl transferase (gGT) (Rasalki and Rau, 1972) activities in the serum were assayed by the reported procedures. The protein content was measured by the methods of Lowry et al. (1951) with BSA as standard.
Non-enzymic antioxidant status Non-enzymic antioxidants such as vitamin C and vitamin E were estimated in plasma and liver tissue according to the method of Omaye et al. (1979) and Desai (1971) respectively.
Statistical analysis Data are expressed as mean7SD. Significance of difference was evaluated using Student’s t-test and po0.05 were considered as significant.
ARTICLE IN PRESS T. Sumathi, A. Nongbri / Phytomedicine 15 (2008) 901–905
rats, the activities of serum marker enzymes were similar to control rats (group I).
Results Effects of B-A on AST, ALT, ALP, gamma-GT, 50 ND and LDH activities Table 1 shows the activities of hepatic marker enzymes viz., AST, ALT, ALP, g-GT, 50 ND and LDH in serum of control and experimental rats. In D-GalN induced rats (group II), the activities of marker enzymes viz., AST (po0.001), ALT (po0.001) ALP (po0.01), g-GT (po0.01), 50 ND (po0.001) and LDH (po0.001) were found to be significantly increased in serum when compared with the control (group I) rats, whereas in BA pretreated (group III) rats these marker enzymes viz., AST (po0.001), ALT (po0.001) ALP (po0.01), g-GT (po0.01), 50 ND (po0.001) and LDH (po0.01) were found to be significantly decreased in serum when compared with the group II rats. In BA alone treated
Table 1.
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Effects of B-A on the level of plasma and liver vitamin-C and E Table 2 shows the effect of B-A on the levels of plasma and hepatic vitamin-C and vitamin-E in control and experimental rats. The levels of vitamin-C (po0.05) and vitamin-E (po0.01) in plasma and liver tissue were significantly decreased in D-GalN induced rats (group II) when compared with control rats (group I). In B-A pretreated rats the level of above non-enzymic antioxidants were significantly (po0.05) increased to near normal in plasma and liver when compared with DGalN induced rats. B-A alone treated rats (group IV) did not show any changes on the level of non-enzymic antioxidants and resembled the control (group I).
Effect of B-A on hepatic marker enzymes of control and experimental rats
Parameters
Group I Control
Group II D-GalN
Group III B-A+D-GalN
Group IV B-A
AST(IU/L) ALT(IU/L) ALP(IU/L) g-GT(IU/L) 50 ND(IU/L) LDH(IU/L)
68.2878.03 29.4672.69 7.2671.03 5.3270.45 3.2170.29 67.2377.12
152.67711.9a$ 82.5877.27a$ 12.2470.94a# 9.5470.79a# 6.5670.32a$ 136.67712.38a$
75.7275.84b$ 38.6573.91b$ 9.7270.43b# 7.2470.59b# 3.7370.25b$ 79.3277.2b#
75.9577.28 31.9173.86 8.8770.75 6.0970.85 3.5970.31 73.3178.15
Values are expressed as mean7SD; n ¼ 6. $ po0.001; #po0.01. a as compared with group I. b as compared with group II.
Table 2.
Effect of B-A on the levels of plasma and hepatic vitamin-C and vitamin-E of control and experimental rats
Group
Control D-GalN B-A+D-GalN B-A
Plasma
Liver
Vit-C (mg/dl)
Vit-E (mg/dl)
Vit-C (mg/100 g tissue)
Vit-E (mg/100 g tissue)
19370.09 1.2570.06a@ 1.6370.09b@ 1.8970.17
2.3970.17 1.3470.12a# 1.9170.13b@ 2.1870.10
0.7870.07 0.5370.03a@ 0.6770.02b@ 0.6570.04
5.1470.24 3.1970.19a# 4.0370.12b@ 4.9170.17
Values are expressed as mean7SD; n ¼ 6. # po0.01; @po0.05. a as compared with group I. b as compared with group II.
Legend Group I Group-II Group-III Group-IV
-Saline control rats -D-galactosamine induced rats -B-A Pretreated+D-galactosamine induced rats -B-A alone treated rats
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Discussion The present study has demonstrated that B-A has protective effects against liver damage induced by D-GalN in rats. D-GalN-induced hepatotoxicity is well established as similar to viral hepatitis (Decker and Keppler, 1974). Liver damage induced by D-GalN, reflects disturbances of liver cell metabolism, which lead to characteristic changes in the serum enzyme activities. Elevated serum enzymes are indicative of cellular leakage and loss of functional integrity of the cell membrane in liver (Ryan et al., 1990). Hence significant rise in the transaminases levels could be taken as an index of liver damage. In our study, significant increase in the activities of serum enzymes viz., ALT, AST, ALP, g-GT, 50 ND and LDH were observed in D-GalN intoxicated rats. Pretreatment with B-A significantly prevented these increased enzyme activities produced by D-GalN, indicating that B-A has hepatoprotective effect against D-GalN induced liver injury. A detailed mechanism of the hepatotoxicity induced by D-GalN has not been found out but the mechanism might be through immune system or oxidative stress. The a-tocopherol and ascorbic acid are naturally occurring free radical scavengers (Yu, 1994). Both ascorbic acid and a-tocopherol are known to be decreased in liver diseases (Bjorneboe et al., 1987). The decrease in the levels of the a-tocopherol and vitamin-C in our study might be due to their increased utilization for scavenging oxygen-derived radicals. It has been reported that the phenolic OH is essential for both antioxidant activity and free radical kinetics (Priyadarsini et al., 2003). Alanko et al. (1999) reported that compounds with hydroxyl group present in the phenolic ring have peroxyl radical and superoxide scavenging properties. The presence of a hydroxyl group in the phenolic ring of B-A might be a reason for its free radical scavenging properties. A number of reports suggested that the Bacopa monniera has a hepatoprotective and antioxidant potentiality, which could have been possible because of the presence of its active major saponin Bacoside-A. Hence from our result it can be suggested that BacosideA was a compound responsible for the hepatoprotective and antioxidant potential against liver injury caused by D-GalN administration. In conclusion, this study presents strong evidence of hepatoprotective effects of B-A against D-GalN intoxicated rats. Earlier studies have shown that the neuropsychopharmacological effects of the nootropic Bacopa monniera is due to the presence of its major saponins Bacoside-A and B (Singh and Dhawan, 1997). In another study the protective effict of B-A against chronic cigarette smoking has also been reported (Anbarasi et al., 2005). Though several studies have been reported for B-A, its effect on hepatotoxicity is not
yet reported. Hence, in this present communication we report the protective effects of B-A the major dammarane saponin isolated from the plant Bacopa monniera, against D-GalN induced liver toxicity. Further studies are needed for better understanding of the mechanism of action and to evaluate the efficacy of the B-A on liver organelle that are possibly damaged during experimental hepatitis.
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