Effect of Purified Saponin Mixture from Astragalus corniculatus on Toxicity Models in Isolated Rat Hepatocytes

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1 Pharmaceutical Biology 2008, Vol. 46, No. 12, pp Effect of Purified Saponin Mixture from Astragalus corniculatus on Toxicity Models in Isolated Rat epatocytes Mitka Mitcheva, 1 Magdalena Kondeva-Burdina, 1 Vessela Vitcheva, 1 Ilina Krasteva, 2 and Stefan Nikolov 2 1 Departament of Pharmacology and Toxicology, 2 Departament of Pharmacognosy Faculty of Pharmacy, Medical University, Sofia, Bulgaria Abstract A purified saponin mixture (PSM) isolated from Astragalus corniculatus Bieb. (Fabaceae) was investigated for its protective effect in two models of toxicity, carbon tetrachloride (CCl 4 ) and tert-butyl hydroperoxide (t-bu), using isolated rat hepatocytes. CCl 4 undergoes dehalogenation in the liver endoplasmic reticulum. This process leads to trichlormethyl radical (CCl 3 ) formation, initiation of lipid peroxidation, and measurable toxic effects on the hepatocytes. xidative damage is widely recognized as being involved in the development of many pathological conditions. In our experiment, t-bu was used as a model of oxidative stress. The hepatocytes were incubated with the PSM alone ( µm) and along with CCl 4 (86 µm) and t-bu (75 µm). As a sign of cytotoxicity, cell viability was used. CCl 4 and t-bu significantly decreased hepatocyte viability. ur data indicate that PSM showed lower toxic effects compared to CCl 4 and t-bu and in combination exerted statistically significant protection of cell viability against the toxic agents. Keywords: Isolated hepatocytes, cell viability, carbon tetrachloride toxicity, tert-butyl hydroperoxide toxicity, Astragalus corniculatus, saponins. Introduction Chemical studies on Astragalus species reported the presence of triterpenoid saponins, which exhibited a wide range of biological properties, including immunostimulant, hepatoprotective, antiviral, cardiotonic and analgesic activities (Rios & Waterman, 1997; Verotta & El-Sebakhy, 2001). In previous publications the protective effect of a purified saponin mixture (PSM) from Astragalus corniculatus Bieb. (Fabaceae) against myeloid Graffi tumor in hamsters, expressed by decrease of the tumor transplantation, on tumor growth inhibition and mortality percentage reduction (Krasteva et al., 2004) was reported. The immunostimulating activity on the blood PMNs and pmøs, of PSM, in Graffi-tumor bearing hamsters was also reported (Toshkova et al., 2007). Phytochemical investigations of PSM led to the isolation of three new oleanane-type triterpene saponins (Krasteva et al., 2006, 2007). CCl 4 is widely used as a model of experimental toxicity in rats. Its toxic effects that led to liver injury are mainly due to a cytochrome P450-dependent biotransformation of CCl 4 to free radicals. They initiate the process of lipid peroxidation, which is often the cause of inhibition of enzyme activity (Ferreira et al., 2003). The experimental toxicity induced by CCl 4 is widely used as a model of liver injury in rats. The cellular system of energy supply localized in mitochondria is another target of many hepatotoxic substances causing oxidative stress and is one of the most important mechanisms through which hepatotoxic factors induced apoptotic and necrotic processes (Kroemer et al., 1998). Tert-Butyl hydroperoxide (t-bu) is a chemical with pro-oxidant activity. It has been used as a model of toxicity on which metabolism of different compounds have been elucidated. Based on the information available, the objective of the following study was to investigate the possible protective effect of a purified saponin mixture, extracted from Astragalus corniculatus, using models of CCl 4 - and t-buinduced toxicity in isolated rat hepatocytes. Materials and Methods Chemicals Thin-layer chromatographic study was carried out on Kieselgel 60 F 254 (0.24 mm thick, Merck, Germany) plates. Accepted: May 21, 2008 Address correspondence to: Dr. Ilina Krasteva, Departament of Pharmacognosy, Faculty of Pharmacy, Medical University, 2 Dunav St., Sofia 1000, Bulgaria. Tel: ; Fax: ; ikrasteva@pharmfac.acad.bg DI: / C 2008 Informa UK Ltd.

2 Effect of Saponins on epatotoxicity 867 The spots were visualized by spraying with anisaldehyde/conc 2 S 4, followed by heating at 110 C. Column chromatography was carried out with Sephadex L- 20 (Pharmacia, Sweden) and silica gel 60 ( mesh, Merck, Germany). In our experiments, pentobarbital sodium (Sanofi, France); EPES (Sigma Aldrich, Germany); NaCl, KCl, D-glucose, NaC 3 and CaCl (Merck, Germany); K 2 P 4 (Scharlau Chemie Sa, Spain); MgS (Fluka AG, Germany); collagenase from Clostridium histolyticum type IV (Sigma Aldrich); EGTA (Sigma Aldrich); carbon tetrachloride (Merck, Germany); tert-butyl hydroperoxide (Sigma, Germany) and Trypan blue (Merck, Germany) were used. Plant material Astragalus corniculatus was collected in July 1999 in Northern Bulgaria. The plant was identified by Dr. D. Pavlova from the Department of Botany, Faculty of Biology, Sofia University, where voucher specimen has been deposited (S95265). Preparation of PSM The air-dried powdered aerial parts of Astragalus corniculatus were extracted with 50% Et. The extract was filtrated, concentrated and successively treated with CCl 3 and EtAc, respectively. The aqueous residue was dissolved in Me and precipitated in Me 2 C yielding crude saponin mixture. Part of this was purified by column chromatography on silica gel and Sephadex L-20 to afford several fractions, one of which containing mixture of three saponins (1, 2 and 3). This fraction was used for the pharmacological study. The saponins were further isolated from the fraction by preparative thin-layer chromatography (TLC) on silica gel and identified as new oleananetype triterpene saponins: 3β--[-4-oxo-pentopyranosyl- (1 2)-β-D-glucopyranosyl]-21α-hydrox- yolean-12-ene- 28-oic acid; 21α-hydroxyolean-12-ene-28-oic acid 3β-4- oxo-pentopyranoside and 19α hydrox- yolean-12-ene-28, 21β olide 3-β-D-xylopyranoside (Figure 1). The structures of the isolated compounds were elucidated by chemical and spectral methods. Details of the isolating and identifying of the saponins have been published previously (Krasteva et al., 2006, 2007). Animals Male Wistar rats (body weight g) were used. Rats were housed in plexiglass cages (3 per cage) in a 12/12 light/dark cycle, temperature 20 ± 2 C. Food and water were provided ad libitum. Animals were purchased from the National Breeding Center, Sofia, Bulgaria. All performed procedures were approved by the Institutional Animal Care Committee and were in accordance with European Union Guidelines for animal experimentation. Isolation and incubation of hepatocytes Rats were anesthetized with sodium pentobarbital (0.2 ml/100 g). In situ liver perfusion and cell isolation were performed as described by Fau et al. (1992), with modifications (Mitcheva et al., 2006). After portal catheterization, the liver was perfused with 100 ml EPES buffer (p = 7.85) mm EDTA, followed by 200 ml EPES buffer (p = 7.85), without any addition, and finally 200 ml EPES buffer containing collagenase type IV (50 mg/200 ml) and 7 mm CaCl 2 (p = 7.85). The liver was excised, minced into small pieces and hepatocytes were dispersed in 60 ml Krebs-Ringer-bicarbonate (KRB) buffer (p = 7.35) + 1% bovine serum albumin. After filtration, the hepatocytes were centrifuged and washed out with KRB buffer. Cells were counted under the microscope and cell viability was assessed by Trypan blue exclusion (0.05%) (Fau et al., 1992). Initial viability averaged 89%. Cells were diluted with KRB to make a suspension of about hepatocytes/ml. Incubations, performed in a 5% C % 2 atmosphere, were carried out in 25 ml Erlenmeyer flasks. Each flask contained 3 ml of the cell suspension (i.e., hepatocytes/ml). The cells were incubated in vitro with the toxic agents CCl 4 (86 µm) and t- Bu (75 µm) alone and after pre-incubation with PSM ( µm). Statistical analysis Statistical analysis was performed by applying Student s t-test, with P<0.05 considered statistically significant. All results (n = 4) are expressed as mean ± SD. Results In the first series of experiments hepatocytes were incubated with PSM in four decreasing concentrations ( µm). The results are shown in Table 1. The values were compared with control hepatocytes. The data indicate that cell viability was decreased by PSM in the concentration-dependent manner. The most prominent effect was observed at concentration 100 µm cell viability was decreased by 32% (p < 0.01). The effects of the toxic agents CCl 4 and t-bu on cell viability are shown in Tables 2 and 3, respectively. CCl 4 decreased cell viability by 61% (p < 0.01). After cell incubation with t-bu viability was reduced by 77% (p < 0.01). Results were compared to the control hepatocytes.

3 868 M. Mitcheva et al. C R R= R= 2 1 C 3 Figure 1. Structures of saponins 1 3. The effect of PSM in CCl 4 and t-bu models of toxicity is shown in Tables 2 and 3, respectively. The values were compared to hepatocytes incubated with the toxic agents. PSM statistically significantly reduced the damage caused by the hepatotoxins and preserved cell viability in a concentration dependent manner. The most visible was the effect of PSM at the highest concentration 100 µm. Discussion Isolated hepatocytes provide the opportunity to evaluate the effects by direct interactions of the studied compounds with endogenous factors. epatocytes are a convenient in vitro model to investigate xenobiotic biotransformation and to elucidate possible mechanisms of toxic stress and its protection. Isolated liver cells are used as a suitable model for evaluation of the cytoprotective effects of some prospective biologically active compounds, both newly synthesized and plant isolated. It has been recognized for some time the protective and antioxidant effects of different Astragalus species. Zhang et al. (1992a) have reported the antioxidant effect of Astragalus membranaceus extract and its component cycloastragenol-xylosyl-glucoside on lipid peroxidation, in vivo/in vitro. In their study, ong et al. (1994) discussed

4 Effect of Saponins on epatotoxicity 869 Table 1. Effect of PSM from Astragalus corniculatus (100, 10, 0.1, and 0.01 µm) on cell viability in isolated rat hepatocytes. Group TB exclusion (%) Control 82 ± 4.2 PMS 100 µm 56 ± 5.4 PMS 10 µm 65 ± 6.7 PMS 0.1 µm 71 ± 4.7 PMS 0.01 µm 75 ± 6.2 p<0.05, p<0.01 versus control. in vitro antioxidant effect of water extract from Astragalus on rat heart mitochondria. Rate et al. (1998) carried out randomized study including 40 adult patients with chronic hepatitis C being treated with Astragalus mixture for 6 months. A significant improvement of liver enzymes was observed. There are data in the literature about 43 patients with myocardial infarction treated with Astragalus (Rios & Waterman, 1997). The treatment led to reduction of the free radical formation in erythrocytes, decrease of plasma lipid peroxidation and increase of SD levels that suggests antioxidant activity (llinger & Brunk, 1995). In relation to these data, our study was designed to investigate the effects of PSM, isolated from Astragalus corniculatus, using models of CCl 4 - and t-bu-induced hepatotoxicity in freshly isolated rat hepatocytes. The effect of PSM, per se, was examined as well. The results showed that PSM itself exerted some toxic effects (Table 1), manifested by a decrease of cell viability. These effects were not as prominent as the toxic effects of CCl 4 and t-bu. It is known that CCl 4 is bio-activated by CYP2E1, as well as CYP2B1 and possibly CYP3A, to form the trichlormethyl radical ( CCl 3 ), which initiates the chain reaction of lipid peroxidation (Weber et al., 2003). Preincubation of the hepatocytes with PSM resulted in protection against CCl 4 toxicity (Table 2). PSM, in the model of CCl 4 -induced hepatotoxicity, statistically significant preserved the cell viability. This effect was the most visible at the highest concentration 100 µm. ur data are supported by the results of Zhang et al. (1992b). In their in vivo study the liver protective effect of the saponins, isolated from A. membranaceus and As- Table 2. Effect of PSM from Astragalus corniculatus, in combination with CCl 4, on cell viability in isolated rat hepatocytes. Group TB exclusion (%) Control 82 ± µm CCl 4 32 ± µm CCl µm PMS 76 ± µm CCl µm PMS 56 ± µm CCl µm PMS 47 ± µm CCl µm PMS 39 ± 6.1 p<0.05, p<0.01, p<0.001 versus control. + p<0.05, ++ p<0.01, +++ p<0.001 versus CCl 4. Table 3. Effect of PSM from Astragalus corniculatus, in combination with t-bu, on cell viability in isolated rat hepatocytes. Group TB exclusion (%) Control 82 ± µm t-bu 19 ± µm t-bu µm PMS 63 ± µm t-bu + 10 µm PMS 50 ± µm t-bu µm PMS 46 ± µm t-bu µm PMS 41 ± p<0.05, p<0.01 versus control; ++ p<0.001 versus t- Bu. tragalus sieversianus against chemical injury, induced by CCl 4, D-galactosamine and acetaminophen in mice were reported. Another model, used for oxidative stress, is t-bu. The metabolism of t-bu to free radicals undergoes through several steps. In microsomal suspension, in the absence of NADP, it has been shown to undergo oneelectron oxidation to a peroxyl radical (reaction 1), whereas in the presence of NADP it has been shown to undergo one-electron reduction to an alkoxyl radical (reaction 2). In isolated mitochondria and intact cells, the t-bu has been shown to undergo β-scission to the methyl radical (reaction 3). All these radicals cause lipid peroxidation process (llinger & Brunk, 1995; Bae Jin & Lee, 2003; Zhang et al., 1992b; Donnell & Burkit, 1994). (C 3 ) 3 C (C 3 ) 3 C +e + + (reaction 1) (C 3 ) 3 C + e (C 3 ) 3 C + (reaction 2) (C 3 ) 3 C (C 3 ) 2 C + C 3 (reaction 3) In our study, hepatocytes, pre-incubated with PSM, were significantly protected against t-bu toxicity and again the most effective was the concentration 100 µm(table3). In both hepatotoxic models (CCl 4 and t-bu), PSM, isolated from Astragalus corniculatus, protected cell viability and this protection was more prominent in t-bu model. Considering the experimental data of our study, we discuss a possible influence of PSM on the metabolism, taking place in hepatocytes. Regarding the toxic mechanisms of the both chemical agents (CCl 4 and t-bu), we could suggest that Astragalus corniculatus interferes on the level of cytochrome P-450 system, related to the metabolism of CCl 4 and on the mitochondrial level, related to the t-bu biotransformation. Acknowledgements The authors would like to thank the Medical Science Committee for funding this research (Project 38/2004) through Concurs Grant 2004.

5 870 M. Mitcheva et al. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. References Bae Jin A, Lee JY (2003): The effect of chondroitin sulfate against CCl 4 -induced hepatotoxicity. Biol Pharm Bull 26: Fau D, Berson A, Eugene D, Fromenty B, Fisch C, Pessayre D (1992): Mechanism for the hepatotoxicity of the antiandrogen, nilutamide. Evidence suggesting that redox cycling of this nitroaromatic drug leads to oxidative stress in isolated hepatocytes. J Pharm Exp Ther 263: Ferreira M, Nunes, Leal L, Pessoa, Lemos T, Viana G (2003): Antioxidant effects in the quinine fraction from Auxemma oncocalyx Taub. Biol Pharm Bull 26: ong C, Lo YC, Tan FC, Wei Y, Chen CF (1994): Astragalus membranaceus and Polygonum multiflorum protect rat heart mitochondria against lipid peroxidation. Am J Chin Med 22: Krasteva I, Nikolov S, Kaloga M (2007). A new saponin lactone from Astragalus corniculatus. Nat Prod Res 21: Krasteva I, Nikolov S, Kaloga M, Mayer G (2006): Triterpenoid saponins from Astragalus corniculatus. Z Naturforsch 61b: Krasteva I, Toshkova R, Nikolov S (2004): Protective effect of Astragalus corniculatus saponins against myeloid Graffi tumor in hamsters. Phytoter Res 18: Kroemer G, Dallaporta B, Resche-Rigon M (1998): The mitochondrial death/life regulator in apoptosis and necrosis. Annu Rev Physiol 60: Mitcheva M, Kondeva M, Vitcheva V, Nedialkov P, Kitanov G (2006): Effect of benzophenones from ypericum annulatum on carbon tetrachloride-induced toxicity in freshly isolated rat hepatocytes. Redox Report 11: 3 8. Donnell V, Burkit MJ (1994): Mitochondrial metabolism of a hydroperoxide to free radicals in human endothelial cells: An electron spin resonance spin trapping investigation. Biochem J 304: llinger K, Brunk UT (1995): Cellular injury induced by oxidative stress is mediated through lysosome damage. Free Radic Biol Med 19: Rate R, Bensoussan A, Fan Y (1998): Preliminary report of randomized, double-blind, placebo-controlled trail of a Chinese herbal medicine preparation C-100 in the treatment of chronic hepatitis C. J Gastro ep 13: Rios LJ, Waterman PG (1997): A review of the pharmacology and toxicology of Astragalus. Phytoter Res 11: Toshkova RA, Krasteva, N, Wesselinova W, Ivanov, I, Nikolov, SD (2007) Influence of purified saponin mixture from Astragalus corniculatus Bieb. on phagocytic cells in Graffi tumor bearing hamsters. J Ethnopharmacol 109: Verotta L, El-Sebakhy N (2001): Cycloartane and oleanane saponins from Astragalus sp. In: Studies in Natural Products Chemistry (Bioactive Natural Products, Part F), Elsevier Science, Karachi, pp Weber LW, Boll M, Stampfl A (2003): epatotoxicity and mechanism of action of haloalkanes: Carbon tetrachloride as a toxicological model. Crit Rev Toxicol 33: Zhang Y, Gong Z, Zheng J, Liu X, Shen B (1992a): Effect of Astragalus membranaceus, injection component XGA on lipid peroxides level in mice. Acad Med Shanghai 19: Zhang YJ, Shen J, Zhu S, uang D, Ding Y, Zhang X (1992b): Effects of Astragalus (ASI, SK) on experimental liver injury. Acta Pharm Sinica 27:

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