- Email: [email protected]
Hepatoprotective and free radical scavenging effects of Nelumbo nucifera
Phytomedicine 10: 165–169, 2003 © Urban & Fischer Verlag http://www.urbanfischer.de/journals/phytomed
Phytomedicine
Hepatoprotective and free radical scavenging effects of Nelumbo nucifera D.-H. Sohn, Y.-C. Kim, S.-H. Oh, E.-J. Park, X. Li, and B.-H. Lee College of Pharmacy and Medicinal Resources Research Center, Wonkwang University, Iksan, Jeonbuk, Korea
Summary Ethanol extracts from Nelumbo nucifera (ENN) seeds were studied for possible antioxidative and hepatoprotective effects. Antioxidative effects were measured spectrophotometrically by reduction of 2,2′-Diphenyl-1-picrylhydrazyl (DPPH) radicals. Hepatoprotective effects were tested using carbon tetrachloride (CCl4) and aflatoxin B1 (AFB1)-induced hepatocyte toxicity models. ENN showed potent free radical scavenging effects with a median inhibition concentration of 6.49 µg/ml. Treatment of hepatocytes with ENN inhibited both the production of serum enzymes and cytotoxicity by CCl4. The genotoxic and cytotoxic effects of AFB1 were also inhibited by ENN in dose-dependent manners. These hepatoprotective effects of ENN against CCl4 and AFB1 might result from its potent antioxidative properties. Key words: Seeds of Nelumbo nucifera, DPPH, carbon tetrachloride, aflatoxin B1, hepatocyte
j Introduction The liver is the first organ to encounter ingested nutrients, drugs, and environmental toxicants that enter the hepatic portal blood from the digestive system and liver function can be detrimentally altered by injury resulting from acute or chronic exposure to toxicants. Carbon tetrachloride (CCl4) and aflatoxin B1 (AFB1) are widely used to produce experimental hepatotoxicity. CCl4 is converted to trichloromethyl-(CCl3·) and then the peroxy-radical (CCl3OO·) by cytochrome P450 enzymes. These free radicals and reactive oxygen species (ROS) then initiate lipid peroxidation and liver damage (Slater, 1984). AFB1 is a hepatotoxic and hepatocarcinogenic compound produced by Aspergillus flavus. A variety of human foods, such as cereals, millets, and oil seeds are susceptible to infection by the fungus, which produces aflatoxins during growth, harvest, transport, and storage. AFB1 is also biotransformed by P450 enzymes to yield an electrophilic epoxide, which attacks DNA to initiate hepatotoxicity and genotoxicity (Eaton and Gallagher, 1994) via oxidative damage (Kodama et al., 1990; Shen et al., 1995, 1996).
While searching for antioxidative agents from natural products containing hepatoprotective properties, we have found some positive effects of Nelumbo nucifera Gaertn. (Nymphaeaceae). N. nucifera is a large aquatic herb widely found in Korea, which has been reported to have pharmacological effects (especially in the rhizome extracts) including hypoglycemic, anti-inflammatory, and antipyretic properties (Mukherjee et al., 1996, 1997a, b). In this study, we report on the protective effect of ethanol extracts of N. nucifera (ENN) against CCl4– and AFB1-induced hepatotoxicity.
j Materials and Methods Chemicals and animals
AFB1, silibin, collagenase type VI, dexamethasone, insulin and [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) were purchased from Sigma Chem. Co. (St. Louis, MO, USA). Williams’ Medium E and fetal bovine serum were products of 0944-7113/03/10/02-03-165 $ 15.00/0
166
D.-H. Sohn et al.
Gibco BRL (Grand Island, NY, USA). Male Fischer 344 rats (180–200 g body weight) were obtained from SLC Inc (Hamamatsu, Japan). Plant material and preparation of ENN
The seeds of N. nucifera were purchased from the herbal medicine co-operative association of Jeonbuk Province, Korea, in June 2000. A voucher specimen (no. WP 280) was deposited at the Herbarium of the College of Pharmacy, Wonkwang University (Korea). One hundred grams of this crude drug was extracted with ethanol under reflux for 3 h and filtrate was evaporated in vacuo to give ethanol extract (1.64 g, 1.64 w/w%).
S. typhimurium Bacterial Mutation Assay
The preincubation method of Maron and Ames (1983) was carried out using S. typhimurium TA 100. For the antimutagenicity assay, overnight culture of the bacteria, S9 mixture (Maron and Ames, 1983), AFB1 and various concentrations of ENN were preincubated at 37 °C for 30 min. S9 mixture was prepared from the pooled livers of Arclor 1254-induced Sprague-Dawley rats and used as the enzyme source for metabolic activation of the mutagen. They were diluted with top agar, plated onto minimal glucose agar plates and incubated further at 37 °C. Histidine revertant colonies were scored after 48 hrs of incubation. Each of the concentrations in an experiment was run in triplicate and they were repeated at least twice with positive and negative controls.
DPPH free radical scavenging activity
Reduction of 2,2′-Diphenyl-1-picrylhydrazyl radical (DPPH) to diphenylpicryl hydrazine by ENN was measured spectrophotometrically at 517 nm according to the method of Ratty et al., (1988). L-Ascorbic acid was used as a reference compound and data were expressed as the percent decrease in the absorbance compared to the control. Liver perfusion and culture of primary rat hepatocytes
Rat liver perfusion was performed according to the method of Seglen (1976). Briefly, after anaesthetization, 250 ml phase I solution (118 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4 and 25 mM NaHCO3; pH 7.4; 37 °C) was perfused through portal vein for 15 min, followed by 250 ml phase II solution (phase I solution plus 3 mM CaCl2, 1.2 mM MgSO4 containing 0.2 mg/ml collagenase; pH 7.4; 37 °C) for 15 min. The liver was mechanically disintegrated and filtered through a 100 mesh filter (Sigma). Hepatocytes were washed three times with prechilled HEPES buffer (140 mM NaCl, 6.7 mM KCl, 1 mM CaCl2, 2.4 mM HEPES; pH 7.4; 4 °C) and resuspended in Williams’ Medium E with 10% FBS, which was maintained during the experiments. The viability of hepatocytes was at least 90%.
Hepatoprotective activity against AFB1
AFB1 and ENN were dissolved in dimethyl sulfoxide and diluted with the culture medium. Hepatocytes (1 × 105 cells/well) were seeded into a collagen coated 24 well plate and preincubated for 3 h. Cells were treated with 1 µM AFB1 and various concentrations of ENN as described in the figure legend. The general viability of cultured cells was determined by reduction of MTT to formazan following 48 h incubation (Monks et al., 1991). Statistical analysis
Data were presented as mean ± standard deviation. Statistical comparisons were made using the student’s t-test. The level of statistical significance was defined as p < 0.05.
Hepatoprotective activity against CCl4
Hepatocyte injury was induced by CCl4 according to the modified method of by Kiso et al. (1983). Hepatocytes were plated at a density of 1.25 × 105 cells/cm2 on 24-well and treated with 5 mM CCl4 and various concentrations of ENN at 18 h after incubation. ENN was dissolved in DMSO and the final concentration of DMSO did not exceed 0.2% of the total volume. After 1.5 h hepatotoxicity was assessed by cellular leakage of aspartate transaminase (AST) and by cell survival rate, which were measured by Autodry Chemistry Analyzer (SPOTCHEMTM SP4410, Arkray, Japan) and MTT assay (Monks et al., 1991), respectively.
Fig. 1. DPPH free radical scavenging activity of ENN. Reduction of DPPH to diphenylpicryl hydrazine by ENN (1–10 µg/ml) was measured spectrophotometrically at 517 nm. Values are expressed as the percent decrease in the absorbance compared to the control. Statistical comparisons were made using the student’s t-test. Data were obtained from 3 independent experiments and all data are significantly different from the control (p < 0.001). Vitamin C (1 mM) was used as a reference compound.
Hepatoprotective and free radical scavenging effects of Nelumbo nucifera
j Results The free radical scavenging effect of ENN was determined by spectrophotometric monitoring of the reduction of DPPH. ENN showed a free radical scavenging effect in a dose-dependent manner with a median inhibition concentration (IC50) of 6.68 µg/ml (Fig. 1). The protective effects of ENN against cytotoxicity induced by CCl4 in primary cultured rat hepatocytes were evaluated by the cellular leakage of aspartate transaminase (AST) and the cell survival rate. Treatment of hepatocytes with CCl4 (5 mM, for 1.5 h) increased the cellular leakage of AST to 14.7-fold and re-
Fig. 2. Hepatoprotective effects of ENN against CCl4. (A) Inhibitory effect of ENN (2.5–500 µg/ml) on cellular leakage of alanine transaminase (AST) induced by 5 mM CCl4 was monitored in rat primary hepatocytes as described in Materials and Methods. (B) Cytoprotective effect of ENN (2.5–500 µg/ml) was tested on 5 mM CCl 4-induced cell death in rat primary hepatocytes as described in Materials and Methods. Statistical comparisons were made using the ANOVA test. Values are expressed as means ± SD of three replicates (*p < 0.05, **p < 0.01, ***p < 0.001, significantly different from CCl4 group). Silibin 200 µg/ml (S200) was used as a reference compound. N means the normal control.
167
duced cell survival rate to 28% of controls. The cellular leakage of AST and the cell death caused by CCl4 were significantly inhibited in dose-dependent manners by ENN concentrations between 10 and 500 µg/ml (Fig. 2). These results clearly show that ENN protects against CCl4-induced cytotoxicity in primary cultured rat hepatocytes.
Fig. 3. Hepatoprotective effects of ENN against AFB1. (A) Antigenotoxic effect of ENN (10–250 µg/plate) against AFB1 (0.1 µg/plate) was monitored using S. typhimurium TA100 in the presence of metabolic activation system as described in ‘Materials and Methods’. Values are average numbers of revertants after subtraction of spontaneous mutants, which has been done in triplicate and repeated at least twice. 2-Amino-fluorene (2-AF; 10 µg/plate) was used as a positive control. (B) Cytoprotective effect of ENN (100–500 µg/ml) was tested on AFB1-induced cell death in primary cultured rat hepatocytes. Hepatocytes were incubated with AFB1 (1 µM) and ENN and the general viability of cultured cells was determined by MTT assay following 48 h incubation. Statistical comparisons were made using the student’s t-test (*p < 0.05, **p < 0.01, *** p < 0.001 compared to AFB1 control). Values are mean ± SD of triplicate samples, which was repeated at least twice.
168
D.-H. Sohn et al.
The protective effect of ENN against hepatotoxin AFB1 was also tested. Its antigenotoxic effect was evaluated using S. typhimurium TA100 in the presence of Aroclor 1254-induced rat liver S9 mixture. As shown in Fig. 3A, AFB1 caused an increase in the number of revertant colonies. Dose-dependent treatment with ENN reduced the genotoxicity of AFB1. The genotoxicity of 0.1 µg/plate of AFB1 was inhibited by 60% in the 100 µg/plate ENN -treated group. At 250 µg/plate of ENN, complete inhibition of AFB1-induced genotoxicity was observed. The cytoprotective effect of ENN was tested on AFB1-induced cell death in primary cultured rat hepatocytes (Fig. 3B). Treatment of hepatocytes with AFB1 (1 µM) decreased the cell survival by 48.9% of controls. ENN showed significant hepatoprotective activity at concentrations of both 250 and 500 µg/ml by 74.5% and 94.6% respectively as compared with AFB1 controls.
j Discussion The hepatoprotective effects of ethanol extracts of Nelumbo nucifera seeds were evaluated by DPPH free radical-scavenging activity, by hepatoprotective activity against CCl4- and AFB1-induced cell death in primary cultured rat hepatocytes and by antigenotoxic activity in S. typhimurium TA100. Several agents initiate hepatotoxicity, such as drugs, toxins, and viruses. Although the direct and indirect mechanisms of hepatotoxicity are diverse, mitochondria are significant targets for many causes of hepatotoxicity. Mitochondrial dysfunction results in impaired bioenergetics and intracellular oxidant stresses, with excessive formation of ROS and peroxynitrite (Jaeschke et al., 2002). DPPH is known to abstract labile hydrogen (Constantin et al., 1990; Matsubara et al., 1991) and the ability to scavenge the DPPH radical is related to the inhibition of lipid peroxidation (Ratty et al., 1988; Rekka and Kourounakis, 1991). As antioxidant action is a complex process, which may prevent the formation or scavenging of free radicals, it was of interest to investigate the interaction of the compounds with the stable free radical DPPH. This interaction expresses the reducing activity of test compounds and indicates their ability to scavenge free radicals (Ratty et al., 1988). CCl4 is known to cause liver injury via metabolic activation by the cytochrome P-450 (CYP) enzyme CYP2E1 (Zangar et al., 2000). During acute liver injury caused by CCl4, monocytes influx into the liver and the subsequent production of ROS contributes to the hepatotoxic effect (Alric et al., 2000). AFB1 is activated by the microsomal cytochrome P450 enzyme system to yield the electrophilic epoxide, which attacks
DNA to initiate hepatotoxicity and genotoxicity (Eaton and Gallagher, 1994). ROS are believed produced in the course of such metabolic processing of AFB1 by cytochrome P450. The enhanced levels of ROS may therefore be responsible for the oxidative damage caused by AFB1, which may ultimately contribute to its cytotoxic and genotoxic effects (Shen et al., 1995, 1996). In conclusion, the protective effect of ENN against CCl4 and AFB1-induced hepatotoxicity in primary rat hepatocytes appears to be related to its free radical scavenging effects and these results support the use of the seeds of Nelumbo nucifera extract as an hepatoprotective agent. Acknowledgements
This research was supported by a grant (PF002105-01) from Plant Diversity Research Center of 21st Century Frontier Research Program funded by Ministry of Science and Technology of Korean government and partially by Wonkwang University in 2002.
j References Alric L, Orfila C, Carrere N, Beraud M, Carrera G, Lepert JC, Duffaut M, Pipy B, Vinel JP (2000) Reactive oxygen intermediates and eicosanoid production by kupffer cells and infiltrated macrophages in acute and chronic liver injury induced in rats by CCl4. Inflamm Res 49: 700–707 Constantin M, Bromont C, Fickat R, Massingham R (1990) Studies on the activity of Bepridil as a scavenger of free radicals. Biochem Pharmacol 40: 1615–1622 Eaton DL, Gallagher EP (1994) Mechanisms of aflatoxin carcinogenesis. Ann Rev Pharmacol Toxicol 34: 135–172 Jaeschke H, Gores GJ, Cederbaum AI, Hinson JA, Pessayre D, Lemasters JJ (2002) Mechanisms of hepatotoxicity. Toxicol Sci 65: 166–176 Kiso Y, Tohkin M, Hikino H (1983) Assay method for antihepatotoxic activity using carbon tetrachloride induced cytotoxicity in primary cultured hepatocytes. Planta Med 49: 222–225 Kodama M, Inoue F, Akao M (1990) Enzymatic and non-enzymatic formation of free radicals from aflatoxin B1. Free Radic Res Commun 10: 137–142 Maron DM, Ames BN (1983) Revised methods for the Salmonella mutagenicity test. Mutat Res 113: 173–215 Matsubara N, Fuchimoto S, Iwagaki H, Nonaka Y, Kimura T, Kashino H, Edamatsu R, Hiramatsu M, Orita K (1991) The possible involvement of free radical scavenging properties in the action of cytokines. Res Commun Chem Pathol Pharmacol 71: 239–242 Monks A, Scudiero D, Skehan P, Shoemaker R, Paull K, Vistica D, Hose C, Langley J, Cronise P, Vaigro-Wolf A, Gray-Goodrich M, Campbell H, Mayo J, Boyd M (1991) Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines. J Natl Cancer Inst 83: 757–766
Hepatoprotective and free radical scavenging effects of Nelumbo nucifera Mukherjee PK, Das J, Sara K, Giri SN, Pal M, Saha BP (1996) Antipyretic activity of Nelumbo nucifera rhizome extract. Ind J Exp Biol 34: 275–276 Mukherjee PK, Saha K, Das J, Pal M, Saha BP (1997a) Studies on the anti-inflammatory activity of rhizomes of Nelumbo nucifera. Planta Med 63: 367–369 Mukherjee PK, Saha K, Pal M, Saha BP (1997b) Effect of Nelumbo nucifera rhizome extract on blood sugar level in rats. J Ethnopharmacol 58: 207–213 Ratty AK, Sunamoto J, Das NP (1988) Interaction of flavonoids with 1,1-diphenyl-2-picrylhydrazyl free radical liposomal membranes and soyabean lipoxygenase 1. Biochem Pharmacol 37: 989–995 Rekka E, Kourounakis PN (1991) Effect of hydroxyethyl rutenosides and related compounds on lipid peroxidation and free radical scavenging activity. Some structural aspects. J Pharm Pharmacol 43: 486–491 Seglen PO (1976) Preparation of isolated rat liver cells. Methods Cell Biol 13: 29–83 Shen HM, Ong CN, Lee BL, Shi CY (1995) Aflatoxin B1-induced 8-hydroxydeoxyguanosine formation in rat hepatic DNA. Carcinogenesis 16: 419–422
169
Shen HM, Shi CY, Shen Y, Ong CN (1996) Detection of elevated reactive oxygen species level in cultured rat hepatocytes treated with aflatoxin B1. Free Radic Biol Med 21: 139–146 Slater TF (1984) Free-radical mechanisms in tissue injury. Biochem J 222: 1–15 Zangar RC, Benson JM, Burnett VL, Springer DL (2000) Cytochrome P450 2E1 is the primary enzyme responsible for low-dose carbon tetrachloride metabolism in human liver microsomes. Chem-Biol Interact 125: 233–243
j Address Byung-Hoon Lee, Ph.D, College of Pharmacy, Wonkwang University, 344-2 Sinyong-dong, Iksan, Jeonbuk 570-749, Korea Tel: ++82-63-850-6806; Fax: ++82-63-856-5606; e-mail: [email protected]
Phytomedicine
Hepatoprotective and free radical scavenging effects of Nelumbo nucifera D.-H. Sohn, Y.-C. Kim, S.-H. Oh, E.-J. Park, X. Li, and B.-H. Lee College of Pharmacy and Medicinal Resources Research Center, Wonkwang University, Iksan, Jeonbuk, Korea
Summary Ethanol extracts from Nelumbo nucifera (ENN) seeds were studied for possible antioxidative and hepatoprotective effects. Antioxidative effects were measured spectrophotometrically by reduction of 2,2′-Diphenyl-1-picrylhydrazyl (DPPH) radicals. Hepatoprotective effects were tested using carbon tetrachloride (CCl4) and aflatoxin B1 (AFB1)-induced hepatocyte toxicity models. ENN showed potent free radical scavenging effects with a median inhibition concentration of 6.49 µg/ml. Treatment of hepatocytes with ENN inhibited both the production of serum enzymes and cytotoxicity by CCl4. The genotoxic and cytotoxic effects of AFB1 were also inhibited by ENN in dose-dependent manners. These hepatoprotective effects of ENN against CCl4 and AFB1 might result from its potent antioxidative properties. Key words: Seeds of Nelumbo nucifera, DPPH, carbon tetrachloride, aflatoxin B1, hepatocyte
j Introduction The liver is the first organ to encounter ingested nutrients, drugs, and environmental toxicants that enter the hepatic portal blood from the digestive system and liver function can be detrimentally altered by injury resulting from acute or chronic exposure to toxicants. Carbon tetrachloride (CCl4) and aflatoxin B1 (AFB1) are widely used to produce experimental hepatotoxicity. CCl4 is converted to trichloromethyl-(CCl3·) and then the peroxy-radical (CCl3OO·) by cytochrome P450 enzymes. These free radicals and reactive oxygen species (ROS) then initiate lipid peroxidation and liver damage (Slater, 1984). AFB1 is a hepatotoxic and hepatocarcinogenic compound produced by Aspergillus flavus. A variety of human foods, such as cereals, millets, and oil seeds are susceptible to infection by the fungus, which produces aflatoxins during growth, harvest, transport, and storage. AFB1 is also biotransformed by P450 enzymes to yield an electrophilic epoxide, which attacks DNA to initiate hepatotoxicity and genotoxicity (Eaton and Gallagher, 1994) via oxidative damage (Kodama et al., 1990; Shen et al., 1995, 1996).
While searching for antioxidative agents from natural products containing hepatoprotective properties, we have found some positive effects of Nelumbo nucifera Gaertn. (Nymphaeaceae). N. nucifera is a large aquatic herb widely found in Korea, which has been reported to have pharmacological effects (especially in the rhizome extracts) including hypoglycemic, anti-inflammatory, and antipyretic properties (Mukherjee et al., 1996, 1997a, b). In this study, we report on the protective effect of ethanol extracts of N. nucifera (ENN) against CCl4– and AFB1-induced hepatotoxicity.
j Materials and Methods Chemicals and animals
AFB1, silibin, collagenase type VI, dexamethasone, insulin and [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) were purchased from Sigma Chem. Co. (St. Louis, MO, USA). Williams’ Medium E and fetal bovine serum were products of 0944-7113/03/10/02-03-165 $ 15.00/0
166
D.-H. Sohn et al.
Gibco BRL (Grand Island, NY, USA). Male Fischer 344 rats (180–200 g body weight) were obtained from SLC Inc (Hamamatsu, Japan). Plant material and preparation of ENN
The seeds of N. nucifera were purchased from the herbal medicine co-operative association of Jeonbuk Province, Korea, in June 2000. A voucher specimen (no. WP 280) was deposited at the Herbarium of the College of Pharmacy, Wonkwang University (Korea). One hundred grams of this crude drug was extracted with ethanol under reflux for 3 h and filtrate was evaporated in vacuo to give ethanol extract (1.64 g, 1.64 w/w%).
S. typhimurium Bacterial Mutation Assay
The preincubation method of Maron and Ames (1983) was carried out using S. typhimurium TA 100. For the antimutagenicity assay, overnight culture of the bacteria, S9 mixture (Maron and Ames, 1983), AFB1 and various concentrations of ENN were preincubated at 37 °C for 30 min. S9 mixture was prepared from the pooled livers of Arclor 1254-induced Sprague-Dawley rats and used as the enzyme source for metabolic activation of the mutagen. They were diluted with top agar, plated onto minimal glucose agar plates and incubated further at 37 °C. Histidine revertant colonies were scored after 48 hrs of incubation. Each of the concentrations in an experiment was run in triplicate and they were repeated at least twice with positive and negative controls.
DPPH free radical scavenging activity
Reduction of 2,2′-Diphenyl-1-picrylhydrazyl radical (DPPH) to diphenylpicryl hydrazine by ENN was measured spectrophotometrically at 517 nm according to the method of Ratty et al., (1988). L-Ascorbic acid was used as a reference compound and data were expressed as the percent decrease in the absorbance compared to the control. Liver perfusion and culture of primary rat hepatocytes
Rat liver perfusion was performed according to the method of Seglen (1976). Briefly, after anaesthetization, 250 ml phase I solution (118 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4 and 25 mM NaHCO3; pH 7.4; 37 °C) was perfused through portal vein for 15 min, followed by 250 ml phase II solution (phase I solution plus 3 mM CaCl2, 1.2 mM MgSO4 containing 0.2 mg/ml collagenase; pH 7.4; 37 °C) for 15 min. The liver was mechanically disintegrated and filtered through a 100 mesh filter (Sigma). Hepatocytes were washed three times with prechilled HEPES buffer (140 mM NaCl, 6.7 mM KCl, 1 mM CaCl2, 2.4 mM HEPES; pH 7.4; 4 °C) and resuspended in Williams’ Medium E with 10% FBS, which was maintained during the experiments. The viability of hepatocytes was at least 90%.
Hepatoprotective activity against AFB1
AFB1 and ENN were dissolved in dimethyl sulfoxide and diluted with the culture medium. Hepatocytes (1 × 105 cells/well) were seeded into a collagen coated 24 well plate and preincubated for 3 h. Cells were treated with 1 µM AFB1 and various concentrations of ENN as described in the figure legend. The general viability of cultured cells was determined by reduction of MTT to formazan following 48 h incubation (Monks et al., 1991). Statistical analysis
Data were presented as mean ± standard deviation. Statistical comparisons were made using the student’s t-test. The level of statistical significance was defined as p < 0.05.
Hepatoprotective activity against CCl4
Hepatocyte injury was induced by CCl4 according to the modified method of by Kiso et al. (1983). Hepatocytes were plated at a density of 1.25 × 105 cells/cm2 on 24-well and treated with 5 mM CCl4 and various concentrations of ENN at 18 h after incubation. ENN was dissolved in DMSO and the final concentration of DMSO did not exceed 0.2% of the total volume. After 1.5 h hepatotoxicity was assessed by cellular leakage of aspartate transaminase (AST) and by cell survival rate, which were measured by Autodry Chemistry Analyzer (SPOTCHEMTM SP4410, Arkray, Japan) and MTT assay (Monks et al., 1991), respectively.
Fig. 1. DPPH free radical scavenging activity of ENN. Reduction of DPPH to diphenylpicryl hydrazine by ENN (1–10 µg/ml) was measured spectrophotometrically at 517 nm. Values are expressed as the percent decrease in the absorbance compared to the control. Statistical comparisons were made using the student’s t-test. Data were obtained from 3 independent experiments and all data are significantly different from the control (p < 0.001). Vitamin C (1 mM) was used as a reference compound.
Hepatoprotective and free radical scavenging effects of Nelumbo nucifera
j Results The free radical scavenging effect of ENN was determined by spectrophotometric monitoring of the reduction of DPPH. ENN showed a free radical scavenging effect in a dose-dependent manner with a median inhibition concentration (IC50) of 6.68 µg/ml (Fig. 1). The protective effects of ENN against cytotoxicity induced by CCl4 in primary cultured rat hepatocytes were evaluated by the cellular leakage of aspartate transaminase (AST) and the cell survival rate. Treatment of hepatocytes with CCl4 (5 mM, for 1.5 h) increased the cellular leakage of AST to 14.7-fold and re-
Fig. 2. Hepatoprotective effects of ENN against CCl4. (A) Inhibitory effect of ENN (2.5–500 µg/ml) on cellular leakage of alanine transaminase (AST) induced by 5 mM CCl4 was monitored in rat primary hepatocytes as described in Materials and Methods. (B) Cytoprotective effect of ENN (2.5–500 µg/ml) was tested on 5 mM CCl 4-induced cell death in rat primary hepatocytes as described in Materials and Methods. Statistical comparisons were made using the ANOVA test. Values are expressed as means ± SD of three replicates (*p < 0.05, **p < 0.01, ***p < 0.001, significantly different from CCl4 group). Silibin 200 µg/ml (S200) was used as a reference compound. N means the normal control.
167
duced cell survival rate to 28% of controls. The cellular leakage of AST and the cell death caused by CCl4 were significantly inhibited in dose-dependent manners by ENN concentrations between 10 and 500 µg/ml (Fig. 2). These results clearly show that ENN protects against CCl4-induced cytotoxicity in primary cultured rat hepatocytes.
Fig. 3. Hepatoprotective effects of ENN against AFB1. (A) Antigenotoxic effect of ENN (10–250 µg/plate) against AFB1 (0.1 µg/plate) was monitored using S. typhimurium TA100 in the presence of metabolic activation system as described in ‘Materials and Methods’. Values are average numbers of revertants after subtraction of spontaneous mutants, which has been done in triplicate and repeated at least twice. 2-Amino-fluorene (2-AF; 10 µg/plate) was used as a positive control. (B) Cytoprotective effect of ENN (100–500 µg/ml) was tested on AFB1-induced cell death in primary cultured rat hepatocytes. Hepatocytes were incubated with AFB1 (1 µM) and ENN and the general viability of cultured cells was determined by MTT assay following 48 h incubation. Statistical comparisons were made using the student’s t-test (*p < 0.05, **p < 0.01, *** p < 0.001 compared to AFB1 control). Values are mean ± SD of triplicate samples, which was repeated at least twice.
168
D.-H. Sohn et al.
The protective effect of ENN against hepatotoxin AFB1 was also tested. Its antigenotoxic effect was evaluated using S. typhimurium TA100 in the presence of Aroclor 1254-induced rat liver S9 mixture. As shown in Fig. 3A, AFB1 caused an increase in the number of revertant colonies. Dose-dependent treatment with ENN reduced the genotoxicity of AFB1. The genotoxicity of 0.1 µg/plate of AFB1 was inhibited by 60% in the 100 µg/plate ENN -treated group. At 250 µg/plate of ENN, complete inhibition of AFB1-induced genotoxicity was observed. The cytoprotective effect of ENN was tested on AFB1-induced cell death in primary cultured rat hepatocytes (Fig. 3B). Treatment of hepatocytes with AFB1 (1 µM) decreased the cell survival by 48.9% of controls. ENN showed significant hepatoprotective activity at concentrations of both 250 and 500 µg/ml by 74.5% and 94.6% respectively as compared with AFB1 controls.
j Discussion The hepatoprotective effects of ethanol extracts of Nelumbo nucifera seeds were evaluated by DPPH free radical-scavenging activity, by hepatoprotective activity against CCl4- and AFB1-induced cell death in primary cultured rat hepatocytes and by antigenotoxic activity in S. typhimurium TA100. Several agents initiate hepatotoxicity, such as drugs, toxins, and viruses. Although the direct and indirect mechanisms of hepatotoxicity are diverse, mitochondria are significant targets for many causes of hepatotoxicity. Mitochondrial dysfunction results in impaired bioenergetics and intracellular oxidant stresses, with excessive formation of ROS and peroxynitrite (Jaeschke et al., 2002). DPPH is known to abstract labile hydrogen (Constantin et al., 1990; Matsubara et al., 1991) and the ability to scavenge the DPPH radical is related to the inhibition of lipid peroxidation (Ratty et al., 1988; Rekka and Kourounakis, 1991). As antioxidant action is a complex process, which may prevent the formation or scavenging of free radicals, it was of interest to investigate the interaction of the compounds with the stable free radical DPPH. This interaction expresses the reducing activity of test compounds and indicates their ability to scavenge free radicals (Ratty et al., 1988). CCl4 is known to cause liver injury via metabolic activation by the cytochrome P-450 (CYP) enzyme CYP2E1 (Zangar et al., 2000). During acute liver injury caused by CCl4, monocytes influx into the liver and the subsequent production of ROS contributes to the hepatotoxic effect (Alric et al., 2000). AFB1 is activated by the microsomal cytochrome P450 enzyme system to yield the electrophilic epoxide, which attacks
DNA to initiate hepatotoxicity and genotoxicity (Eaton and Gallagher, 1994). ROS are believed produced in the course of such metabolic processing of AFB1 by cytochrome P450. The enhanced levels of ROS may therefore be responsible for the oxidative damage caused by AFB1, which may ultimately contribute to its cytotoxic and genotoxic effects (Shen et al., 1995, 1996). In conclusion, the protective effect of ENN against CCl4 and AFB1-induced hepatotoxicity in primary rat hepatocytes appears to be related to its free radical scavenging effects and these results support the use of the seeds of Nelumbo nucifera extract as an hepatoprotective agent. Acknowledgements
This research was supported by a grant (PF002105-01) from Plant Diversity Research Center of 21st Century Frontier Research Program funded by Ministry of Science and Technology of Korean government and partially by Wonkwang University in 2002.
j References Alric L, Orfila C, Carrere N, Beraud M, Carrera G, Lepert JC, Duffaut M, Pipy B, Vinel JP (2000) Reactive oxygen intermediates and eicosanoid production by kupffer cells and infiltrated macrophages in acute and chronic liver injury induced in rats by CCl4. Inflamm Res 49: 700–707 Constantin M, Bromont C, Fickat R, Massingham R (1990) Studies on the activity of Bepridil as a scavenger of free radicals. Biochem Pharmacol 40: 1615–1622 Eaton DL, Gallagher EP (1994) Mechanisms of aflatoxin carcinogenesis. Ann Rev Pharmacol Toxicol 34: 135–172 Jaeschke H, Gores GJ, Cederbaum AI, Hinson JA, Pessayre D, Lemasters JJ (2002) Mechanisms of hepatotoxicity. Toxicol Sci 65: 166–176 Kiso Y, Tohkin M, Hikino H (1983) Assay method for antihepatotoxic activity using carbon tetrachloride induced cytotoxicity in primary cultured hepatocytes. Planta Med 49: 222–225 Kodama M, Inoue F, Akao M (1990) Enzymatic and non-enzymatic formation of free radicals from aflatoxin B1. Free Radic Res Commun 10: 137–142 Maron DM, Ames BN (1983) Revised methods for the Salmonella mutagenicity test. Mutat Res 113: 173–215 Matsubara N, Fuchimoto S, Iwagaki H, Nonaka Y, Kimura T, Kashino H, Edamatsu R, Hiramatsu M, Orita K (1991) The possible involvement of free radical scavenging properties in the action of cytokines. Res Commun Chem Pathol Pharmacol 71: 239–242 Monks A, Scudiero D, Skehan P, Shoemaker R, Paull K, Vistica D, Hose C, Langley J, Cronise P, Vaigro-Wolf A, Gray-Goodrich M, Campbell H, Mayo J, Boyd M (1991) Feasibility of a high-flux anticancer drug screen using a diverse panel of cultured human tumor cell lines. J Natl Cancer Inst 83: 757–766
Hepatoprotective and free radical scavenging effects of Nelumbo nucifera Mukherjee PK, Das J, Sara K, Giri SN, Pal M, Saha BP (1996) Antipyretic activity of Nelumbo nucifera rhizome extract. Ind J Exp Biol 34: 275–276 Mukherjee PK, Saha K, Das J, Pal M, Saha BP (1997a) Studies on the anti-inflammatory activity of rhizomes of Nelumbo nucifera. Planta Med 63: 367–369 Mukherjee PK, Saha K, Pal M, Saha BP (1997b) Effect of Nelumbo nucifera rhizome extract on blood sugar level in rats. J Ethnopharmacol 58: 207–213 Ratty AK, Sunamoto J, Das NP (1988) Interaction of flavonoids with 1,1-diphenyl-2-picrylhydrazyl free radical liposomal membranes and soyabean lipoxygenase 1. Biochem Pharmacol 37: 989–995 Rekka E, Kourounakis PN (1991) Effect of hydroxyethyl rutenosides and related compounds on lipid peroxidation and free radical scavenging activity. Some structural aspects. J Pharm Pharmacol 43: 486–491 Seglen PO (1976) Preparation of isolated rat liver cells. Methods Cell Biol 13: 29–83 Shen HM, Ong CN, Lee BL, Shi CY (1995) Aflatoxin B1-induced 8-hydroxydeoxyguanosine formation in rat hepatic DNA. Carcinogenesis 16: 419–422
169
Shen HM, Shi CY, Shen Y, Ong CN (1996) Detection of elevated reactive oxygen species level in cultured rat hepatocytes treated with aflatoxin B1. Free Radic Biol Med 21: 139–146 Slater TF (1984) Free-radical mechanisms in tissue injury. Biochem J 222: 1–15 Zangar RC, Benson JM, Burnett VL, Springer DL (2000) Cytochrome P450 2E1 is the primary enzyme responsible for low-dose carbon tetrachloride metabolism in human liver microsomes. Chem-Biol Interact 125: 233–243
j Address Byung-Hoon Lee, Ph.D, College of Pharmacy, Wonkwang University, 344-2 Sinyong-dong, Iksan, Jeonbuk 570-749, Korea Tel: ++82-63-850-6806; Fax: ++82-63-856-5606; e-mail: [email protected]