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Hepatoprotection of Phyllanthus maderaspatensis against experimentally induced liver injury in rats
Fitoterapia 78 (2007) 134 – 141 www.elsevier.com/locate/fitote
Hepatoprotection of Phyllanthus maderaspatensis against experimentally induced liver injury in rats V.V. Asha ⁎, M.S. Sheeba, V. Suresh, P.J. Wills Molecular Ethnopharmacology Lab, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala 695014, India Received 30 June 2005; accepted 11 October 2006 Available online 16 November 2006
Abstract The hexane extract of Phyllanthus maderaspatensis (200 and 100 mg/kg) showed significant hepatoprotection on carbon tetrachloride and thioacetamide induced liver damage in rats. The protective effect was evident from serum biochemical parameters and histopathological analysis. Rats treated with P. maderaspatensis remarkably prevented the elevation of serum AST, ALT and LDH and liver lipid peroxides in CCl4 and thioacetamide treated rats. Hepatic glutathione levels significantly increased by the treatment with the extracts. Histopathological changes induced by CCl4 and thioacetamide were also significantly reduced by the extract treatment. The activity of hexane extracts of P. maderaspatensis was comparable to that of silymarin, the reference hepatoprotective drug. © 2006 Elsevier B.V. All rights reserved. Keywords: Phyllanthus maderaspatensis; Carbon tetrachloride; Thioacetamide; Hepatoprotection; Silymarin
1. Introduction The genus Phyllanthus (Euphorbiaceae) is widely distributed in most tropical and subtropical countries, and has long been used in folk medicine to treat kidney and urinary bladder disturbances, intestinal disorders, diabetes and hepatitis B infection [1]. There is a large number of natural, mostly plant products, frequently originating from traditional Indian or Chinese medicine, endowed with hepatoprotective activity, many of them acting as radical scavengers, others as enzyme inhibitors, and some as mitogens [2–4]. Whole plant extracts of Phyllanthus maderaspatensis are used to treat liver disorders by traditional healers in India. P. maderaspatensis was screened for their ability to protect rats from paracetamol induced liver damage [5]. In our recent studies, using the same noxious agent, the anti-hepatotoxic action of the n-hexane extract of P. maderaspatensis (at a dose of 6.25 mg/kg) was found to be better than silymarin, a standard hepatoprotective drug [6]. In the present investigation, the hepatoprotective activity of the n-hexane extract of P. maderaspatensis 200 mg and 100 mg/kg dose was further evaluated against two different toxins, CCl4 and thioacetamide.
⁎ Corresponding author. Tel.: +91 471 2345899; fax: +91 471 2348096. E-mail address: [email protected] (V.V. Asha). 0367-326X/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2006.10.009
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135
In our preliminary phytochemical analysis of this most promising extract, we show that chemical components like flavanoids, fatty acids, saponins, and indole derivatives are found in this plant. The esterified sample from the hexane extract shows the fatty acids components in the mixture; they are oleic acid, linoleic acid, myristic acid, palmitic acid and stearic acid. 2. Experimental 2.1. General Carbon tetrachloride (CCl4) was purchased from Merck India Ltd, Mumbai. Thioacetamide, corn oil, silymarin and LDH kit was purchased from the Sigma Chemical Co., St. Louis, MO., USA. AST and ALT assay kits were purchased from Dialab, Austria. All other chemicals and reagents used were of highest purity grade. 2.2. Plant P. maderaspatensis whole plant, collected from Chennai, India, during October 2004 was authenticated in the Regional Research Institute, Poojapura, Thiruvananthapuram where a voucher specimen (ETHNO.6) has been deposited. 2.3. Preparation of extract Fresh whole plant cleaned, dried at room temperature and powdered was Soxhlet extracted with n-hexane. The solvent was evaporated in vacuo giving an extract (yield 6%). The extract suspended in 5% Tween 80 was stored at − 20 °C. 2.4. Animals Wistar rats of either sex (120–160 g) were used. Animals were maintained on standard environmental condition and fed with a standard pellet diet and water ad libitum. Animal studies were approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and conducted according to the regulations of Institutional Animal Ethics Committee. 2.5. Carbon tetrachloride induced hepatotoxicity Animals were divided into five groups consisting of six rats each. Animals of groups I and II received 5% Tween 80 for eight consecutive days. The rats of groups III and IV received P. maderaspatensis n-hexane extract orally at a dose of 200 mg/kg and 100 mg/kg respectively while animals in group V received silymarin at a dose of 50 mg/kg orally for 8 days. Animals of groups II to V were treated with carbon tetrachloride (100 μl/100 g mixed 1:1 in corn oil) on the seventh day. Animals were killed under ether anesthesia after 48 h of hepatotoxin administration. 2.6. Thioacetamide induced hepatotoxicity Five groups consisting of six animals each were included. Group I was normal control, Group II was thioacetamide control, both received 5% Tween 80 for eight days. Groups III–V received P. maderaspatensis n-hexane extracts at an oral dose of 200 mg/kg and 100 mg/kg and silymarin at an oral dose of 50 mg/kg body weight respectively for 8 days. Groups II–V received a single dose of thioacetamide (100 mg/kg; s.c) on the seventh day. Animals were killed 48 h after thioacetamide administration. 2.7. Biological assays Serum marker enzymes such as serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), serum lactate dehydrogenase (LDH) [7–9] were estimated. Blood was collected from the neck blood vessels and kept for 30 min at 4 °C. Serum was separated by centrifugation at 2500 rpm for 15 min at 4 °C. The livers were removed rapidly and cut into separate portions for hepatic glutathione, lipid peroxidation estimation and histopathological
136
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studies. Liver tissues were homogenized on ice in 0.15 M KCl. The homogenates were centrifuged at 3000 rpm for 15 min at 4 °C and the supernatants were taken for lipid peroxidation assay. For the estimation of glutathione, liver tissues were homogenized in 0.2 M phosphate buffer and centrifuged at 2500 rpm for 10 min at 4 °C and supernatants were taken for the assay. ALT, AST and LDH were assayed by automatic analyzer. Glutathione and lipid peroxidation levels were estimated spectrophotometrically. 2.7.1. Estimation of reduced glutathione (GSH) Reduced glutathione (GSH) was estimated by the reaction with DTNB that gives a yellow coloured complex with absorption maximum at 412 nm [10]. 2.7.2. Estimation of lipid peroxides Lipid peroxidation in liver was measured by the formation of malondialdehyde (MDA) and measured by the thiobarbituric acid reactive substance (TBARS) method [11]. 2.8. Histopathological studies Small pieces of liver, fixed in 10% neutral buffered formalin were processed for embedding in paraffin. Sections of 5–6 μm were cut and stained with hematoxylin and eosin. 2.9. Statistical analysis All data were represented as Mean ± SD. Analysis of variance was (ANOVA) used for the statistical analysis of data. Tukey HSD (Tukeys post-hoc test) was used for determining significance. Results with P ≤ 0.05 were considered as statistically significant. 3. Results In carbon tetrachloride control rats, a marked increase in serum AST, ALT and LDH activities was observed in comparison with normal control rats, indicating liver damage (Table 1). The treatment of n-hexane extracts of P. maderaspatensis at a dose of 200 and 100 mg/kg showed a significant decrease of AST, ALT and LDH in CCl4 intoxicated rats. Standard control drug, silymarin at a dose of 50 mg/kg also prevented the elevation of serum enzymes. In rats administered with CCl4 alone a significant reduction of the hepatic glutathione (GSH) level was observed. Treatment with n-hexane extract at 200 and 100 mg/kg exhibited a significant increase in hepatic glutathione levels by 93.4 and 87.5% respectively. Silymarin treated rats remarkably prevented the lowering of hepatic GSH by 88%. A significant increase in tissue MDA level was observed in CCl4 alone treated rats. However, pre-treatment of the rats with extract at 200 and 100 mg/kg reduced significantly by 93 and 86.2% the MDA elevated by CCl4 treatment. In silymarin treated rats, MDA levels were remarkably reduced by 90.2%. The normal architecture of liver (Fig. 1A) was completely lost in rats treated with CCl4 (Fig. 1B) with the appearance of vacuolated hepatocytes and degenerated nuclei. Vacuolization, fatty changes and necrosis of hepatocytes were severe in the centrilobular region and these changes were also observed in areas other than the centrilobular regions. The livers of rats treated with P. maderaspatensis n-hexane extract at a dose of 200 mg/kg (Fig. 1C), 100 mg/kg (Fig. 1D) or silymarin 50 mg/kg (Fig. 1E) showed a significant recovery from CCl4-induced liver damage as evident Table 1 Effect of P. maderaspatensis n-hexane extract on serum AST, ALT and LDH in CCl4 intoxicated rats Treatment
AST (IU/l)
ALT (IU/l)
LDH (IU/l)
Normal control CCl4 control (100 μl/100 g) P. maderaspatensis (200 mg/kg) + CCl4 P. maderaspatensis (100 mg/kg) + CCl4 Silymarin (50 mg/kg) + CCl4
154.6 ± 8.2 1021.9 ± 88.8⁎ 182.2 ± 10.1⁎⁎ 253.9 ± 15.0⁎⁎ 271.8 ± 17.7⁎⁎
54.1 ± 4.4 616.4 ± 40.5⁎ 92.1 ± 8.2⁎⁎ 117.9 ± 11.9⁎⁎ 158.7 ± 14.7⁎⁎
159.2 ± 5.5 1072.7 ± 98.3⁎ 192.1 ± 11.3⁎⁎ 237.5 ± 23.5⁎⁎ 235.7 ± 25.9⁎⁎
⁎P ≤ 0.05 compared to values for normal rats. ⁎⁎P ≤ 0.05 compared to CCl4 control. Values are Mean ± S.D, N = 6.
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from normal hepatocytes with well defined nuclei. Vacuolization and fatty degeneration were remarkably prevented by the treatment with extracts and silymarin. In thioacetamide treated rats, the levels of AST, ALT and LDH were significantly elevated. Treatment with n-hexane extracts of P. maderaspatensis at a dose of 200, 100 mg/kg and silymarin (50 mg/kg) showed a significant decrease in AST, ALT and LDH (Table 2). Treatment with 200 and 100 mg/kg n-hexane extract exhibited a significant increase, 91 and 88.8% respectively in hepatic glutathione levels. Silymarin treated rats also prevented the lowering of hepatic GSH by 84.2%. A significant increase in tissue MDA level was observed in thioacetamide alone treated rats. However, thioacetamide induced
Fig. 1. Effect of P. maderaspatensis n-hexane extract on histopathological changes that occurred in rats during carbon tetrachloride intoxication (hematoxylin and eosin, ×200). (A) Normal control; (B) CCl4 control, (C) P. maderaspatensis n-hexane extract (200 mg/kg); (D) P. maderaspatensis n-hexane extract (100 mg/kg); (E) silymarin (50 mg/kg).
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Fig. 1 (continued ).
elevation of tissue MDA concentration was lowered by 90 and 82.6% in rats treated with n-hexane extract at a dose of 200 and 100 mg/kg respectively which was comparable to silymarin (88.6%). The liver sections of normal control animals showed hepatic cells with well preserved cytoplasm, prominent nucleus and central vein (Fig. 2A). In rats treated with thioacetamide (Fig. 2B), the normal architecture of liver was completely lost with the appearance of centrilobular necrosis with tiny vacuoles, lymphocytes infiltration of the periportal area and fatty changes were observed. The animals administered with n-hexane extract at 200 and 100 mg/kg (Fig. 2C and D) or silymarin (Fig. 2E) showed a significant recovery from thioacetamide induced liver damage as evident from normal hepatic architectural pattern with mild hepatitis. 4. Discussion and conclusion Carbon tetrachloride proves highly useful as an experimental model for the study of acute hepatic injury [12]. CCl4induced liver damage is mainly attributed to the toxic metabolites, mainly trichloromethyl radical, for the initiation of Table 2 Effect of P. maderaspatensis n-hexane extract on serum AST, ALT and LDH in thioacetamide intoxicated rats Treatment
AST (IU/l)
ALT (IU/l)
LDH (IU/l)
Normal control Thioacetamide control (100 mg/kg) P. maderaspatensis (200 mg/kg) + thioacetamide P. maderaspatensis (100 mg/kg) + thioacetamide Silymarin (50 mg/kg) + thioacetamide
156.7 ± 5.8 1977.8 ± 63.6⁎ 263.2 ± 11.7⁎⁎ 307.9 ± 14.5⁎⁎ 343.4 ± 20.7⁎⁎
57.5 ± 5.4 1374.9 ± 49.9⁎ 163.4 ± 11.5⁎⁎ 214.4 ± 15.9⁎⁎ 238.6 ± 18.0⁎⁎
159.6 ± 5.8 2007.0 ± 83.8⁎ 296.7 ± 14.2⁎⁎ 329.2 ± 18.3⁎⁎ 353.2 ± 24.1⁎⁎
⁎P ≤ 0.05 compared to values for normal rats. ⁎⁎P ≤ 0.05 compared to thioacetamide control. Values are Mean ± S.D, N = 6.
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CCl4 dependant lipid peroxidation. Antioxidants and radical scavengers have been used to study the mechanism of CCl4 toxicity as well as to protect liver cells from CCl4-induced damage by breaking the chain reaction of lipid peroxidation [13]. Also, thioacetamide is a potent hepatotoxin and its toxicity is due to the formation of thioacetamide5-oxide which is responsible for the change in cell permeability, increased intracellular concentration of Ca2+, increase in nuclear volume and enlargement of nucleoli and also inhibits mitochondrial activity that leads to cell death [14]. Rats treated with both hepatotoxins showed elevated levels of AST, ALT, LDH and liver lipid peroxides. The cellular
Fig. 2. Effect of P. maderaspatensis n-hexane extract on histopathological changes that occurred in rats during thioacetamide intoxication (hematoxylin and eosin, ×200). (A) Normal control; (B) thioacetamide control, (C) P. maderaspatensis n-hexane extract (200 mg/kg); (D) P. maderaspatensis n-hexane extract (100 mg/kg); (E) silymarin (50 mg/kg).
140
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Fig. 2 (continued ).
antioxidant, reduced glutathione (GSH), was decreased. The scavenging effect of P. maderaspatensis n-hexane extract on free radicals produced by liver was observed when rats were treated with the extracts as evidenced from lowered MDA levels. The protective effect of P. maderaspatensis n-hexane extract was further substantiated by histopathological assessment, where necrotic and infiltrative changes were observed. The results of this study confirm the significant anti-hepatotoxic activity of P. maderaspatensis n-hexane extract. Further clinical studies are needed to evaluate the real therapeutic value of this natural extract. Acknowledgements The authors express sincere thanks to Dr. M. Radhakrishna Pillai, Director, Rajiv Gandhi Center for Biotechnology, for his interest and encouragement in this work and also gratefully acknowledge the Department of Biotechnology, Government of India, for providing the funds for doing this project. The authors are indebted to Mr. K S Sabulal and Miss Ciji Varghese for technical assistance. References [1] [2] [3] [4] [5] [6] [7]
Calixto JB, Santos ARS, Filho VC, Yunes RA. Med Res Rev 1998;18:225. Fadhel ZA, Amran S. Phytother Res 2002;16:S28. Jeong HG, You HJ, Park SJ, Moon AR, Chung YC, Kang SK, et al. Pharmacol Res 2002;46:221. He Q, Osuchowski MF, Johnson VJ, Sharma RP. Planta Med 2002;68:676. Asha VV, Pushpangadan P. Fitoterapia 1998;69:255. Asha VV, Akhila S, Wills PJ, Subramoniam A. J Ethnopharmacol 2004;92:67. Bergmeyer HU, Bowes Jr GN, Horder M, Moss DW. Clin Chim Acta 1976;70:F19.
V.V. Asha et al. / Fitoterapia 78 (2007) 134–141 [8] [9] [10] [11] [12] [13] [14]
Bergmeyer HU. Clin Chim Acta 1980;105:147. Amador E, Dorfman LE, Wacker WE. Clin Chem 1963;9:391. Moron MS, Depierre JW, Mannervik B. Biochim Biophys Acta 1979;582:67. Ohkawa H, Ohishi N, Yagi K. Anal Biochem 1979;95:351. Clawson GA. Pathol Immunopathol Res 1989;8:104. Weber LWD, Boll M, Stampfl A. Crit Rev Toxicol 2003;33:105. Bruck R, Aeed H, Avni Y, Shirin H, Matas Z, Shahmurov M, et al. J Hepatol 2004;40:86.
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Hepatoprotection of Phyllanthus maderaspatensis against experimentally induced liver injury in rats V.V. Asha ⁎, M.S. Sheeba, V. Suresh, P.J. Wills Molecular Ethnopharmacology Lab, Rajiv Gandhi Centre for Biotechnology, Thiruvananthapuram, Kerala 695014, India Received 30 June 2005; accepted 11 October 2006 Available online 16 November 2006
Abstract The hexane extract of Phyllanthus maderaspatensis (200 and 100 mg/kg) showed significant hepatoprotection on carbon tetrachloride and thioacetamide induced liver damage in rats. The protective effect was evident from serum biochemical parameters and histopathological analysis. Rats treated with P. maderaspatensis remarkably prevented the elevation of serum AST, ALT and LDH and liver lipid peroxides in CCl4 and thioacetamide treated rats. Hepatic glutathione levels significantly increased by the treatment with the extracts. Histopathological changes induced by CCl4 and thioacetamide were also significantly reduced by the extract treatment. The activity of hexane extracts of P. maderaspatensis was comparable to that of silymarin, the reference hepatoprotective drug. © 2006 Elsevier B.V. All rights reserved. Keywords: Phyllanthus maderaspatensis; Carbon tetrachloride; Thioacetamide; Hepatoprotection; Silymarin
1. Introduction The genus Phyllanthus (Euphorbiaceae) is widely distributed in most tropical and subtropical countries, and has long been used in folk medicine to treat kidney and urinary bladder disturbances, intestinal disorders, diabetes and hepatitis B infection [1]. There is a large number of natural, mostly plant products, frequently originating from traditional Indian or Chinese medicine, endowed with hepatoprotective activity, many of them acting as radical scavengers, others as enzyme inhibitors, and some as mitogens [2–4]. Whole plant extracts of Phyllanthus maderaspatensis are used to treat liver disorders by traditional healers in India. P. maderaspatensis was screened for their ability to protect rats from paracetamol induced liver damage [5]. In our recent studies, using the same noxious agent, the anti-hepatotoxic action of the n-hexane extract of P. maderaspatensis (at a dose of 6.25 mg/kg) was found to be better than silymarin, a standard hepatoprotective drug [6]. In the present investigation, the hepatoprotective activity of the n-hexane extract of P. maderaspatensis 200 mg and 100 mg/kg dose was further evaluated against two different toxins, CCl4 and thioacetamide.
⁎ Corresponding author. Tel.: +91 471 2345899; fax: +91 471 2348096. E-mail address: [email protected] (V.V. Asha). 0367-326X/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2006.10.009
V.V. Asha et al. / Fitoterapia 78 (2007) 134–141
135
In our preliminary phytochemical analysis of this most promising extract, we show that chemical components like flavanoids, fatty acids, saponins, and indole derivatives are found in this plant. The esterified sample from the hexane extract shows the fatty acids components in the mixture; they are oleic acid, linoleic acid, myristic acid, palmitic acid and stearic acid. 2. Experimental 2.1. General Carbon tetrachloride (CCl4) was purchased from Merck India Ltd, Mumbai. Thioacetamide, corn oil, silymarin and LDH kit was purchased from the Sigma Chemical Co., St. Louis, MO., USA. AST and ALT assay kits were purchased from Dialab, Austria. All other chemicals and reagents used were of highest purity grade. 2.2. Plant P. maderaspatensis whole plant, collected from Chennai, India, during October 2004 was authenticated in the Regional Research Institute, Poojapura, Thiruvananthapuram where a voucher specimen (ETHNO.6) has been deposited. 2.3. Preparation of extract Fresh whole plant cleaned, dried at room temperature and powdered was Soxhlet extracted with n-hexane. The solvent was evaporated in vacuo giving an extract (yield 6%). The extract suspended in 5% Tween 80 was stored at − 20 °C. 2.4. Animals Wistar rats of either sex (120–160 g) were used. Animals were maintained on standard environmental condition and fed with a standard pellet diet and water ad libitum. Animal studies were approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA) and conducted according to the regulations of Institutional Animal Ethics Committee. 2.5. Carbon tetrachloride induced hepatotoxicity Animals were divided into five groups consisting of six rats each. Animals of groups I and II received 5% Tween 80 for eight consecutive days. The rats of groups III and IV received P. maderaspatensis n-hexane extract orally at a dose of 200 mg/kg and 100 mg/kg respectively while animals in group V received silymarin at a dose of 50 mg/kg orally for 8 days. Animals of groups II to V were treated with carbon tetrachloride (100 μl/100 g mixed 1:1 in corn oil) on the seventh day. Animals were killed under ether anesthesia after 48 h of hepatotoxin administration. 2.6. Thioacetamide induced hepatotoxicity Five groups consisting of six animals each were included. Group I was normal control, Group II was thioacetamide control, both received 5% Tween 80 for eight days. Groups III–V received P. maderaspatensis n-hexane extracts at an oral dose of 200 mg/kg and 100 mg/kg and silymarin at an oral dose of 50 mg/kg body weight respectively for 8 days. Groups II–V received a single dose of thioacetamide (100 mg/kg; s.c) on the seventh day. Animals were killed 48 h after thioacetamide administration. 2.7. Biological assays Serum marker enzymes such as serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), serum lactate dehydrogenase (LDH) [7–9] were estimated. Blood was collected from the neck blood vessels and kept for 30 min at 4 °C. Serum was separated by centrifugation at 2500 rpm for 15 min at 4 °C. The livers were removed rapidly and cut into separate portions for hepatic glutathione, lipid peroxidation estimation and histopathological
136
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studies. Liver tissues were homogenized on ice in 0.15 M KCl. The homogenates were centrifuged at 3000 rpm for 15 min at 4 °C and the supernatants were taken for lipid peroxidation assay. For the estimation of glutathione, liver tissues were homogenized in 0.2 M phosphate buffer and centrifuged at 2500 rpm for 10 min at 4 °C and supernatants were taken for the assay. ALT, AST and LDH were assayed by automatic analyzer. Glutathione and lipid peroxidation levels were estimated spectrophotometrically. 2.7.1. Estimation of reduced glutathione (GSH) Reduced glutathione (GSH) was estimated by the reaction with DTNB that gives a yellow coloured complex with absorption maximum at 412 nm [10]. 2.7.2. Estimation of lipid peroxides Lipid peroxidation in liver was measured by the formation of malondialdehyde (MDA) and measured by the thiobarbituric acid reactive substance (TBARS) method [11]. 2.8. Histopathological studies Small pieces of liver, fixed in 10% neutral buffered formalin were processed for embedding in paraffin. Sections of 5–6 μm were cut and stained with hematoxylin and eosin. 2.9. Statistical analysis All data were represented as Mean ± SD. Analysis of variance was (ANOVA) used for the statistical analysis of data. Tukey HSD (Tukeys post-hoc test) was used for determining significance. Results with P ≤ 0.05 were considered as statistically significant. 3. Results In carbon tetrachloride control rats, a marked increase in serum AST, ALT and LDH activities was observed in comparison with normal control rats, indicating liver damage (Table 1). The treatment of n-hexane extracts of P. maderaspatensis at a dose of 200 and 100 mg/kg showed a significant decrease of AST, ALT and LDH in CCl4 intoxicated rats. Standard control drug, silymarin at a dose of 50 mg/kg also prevented the elevation of serum enzymes. In rats administered with CCl4 alone a significant reduction of the hepatic glutathione (GSH) level was observed. Treatment with n-hexane extract at 200 and 100 mg/kg exhibited a significant increase in hepatic glutathione levels by 93.4 and 87.5% respectively. Silymarin treated rats remarkably prevented the lowering of hepatic GSH by 88%. A significant increase in tissue MDA level was observed in CCl4 alone treated rats. However, pre-treatment of the rats with extract at 200 and 100 mg/kg reduced significantly by 93 and 86.2% the MDA elevated by CCl4 treatment. In silymarin treated rats, MDA levels were remarkably reduced by 90.2%. The normal architecture of liver (Fig. 1A) was completely lost in rats treated with CCl4 (Fig. 1B) with the appearance of vacuolated hepatocytes and degenerated nuclei. Vacuolization, fatty changes and necrosis of hepatocytes were severe in the centrilobular region and these changes were also observed in areas other than the centrilobular regions. The livers of rats treated with P. maderaspatensis n-hexane extract at a dose of 200 mg/kg (Fig. 1C), 100 mg/kg (Fig. 1D) or silymarin 50 mg/kg (Fig. 1E) showed a significant recovery from CCl4-induced liver damage as evident Table 1 Effect of P. maderaspatensis n-hexane extract on serum AST, ALT and LDH in CCl4 intoxicated rats Treatment
AST (IU/l)
ALT (IU/l)
LDH (IU/l)
Normal control CCl4 control (100 μl/100 g) P. maderaspatensis (200 mg/kg) + CCl4 P. maderaspatensis (100 mg/kg) + CCl4 Silymarin (50 mg/kg) + CCl4
154.6 ± 8.2 1021.9 ± 88.8⁎ 182.2 ± 10.1⁎⁎ 253.9 ± 15.0⁎⁎ 271.8 ± 17.7⁎⁎
54.1 ± 4.4 616.4 ± 40.5⁎ 92.1 ± 8.2⁎⁎ 117.9 ± 11.9⁎⁎ 158.7 ± 14.7⁎⁎
159.2 ± 5.5 1072.7 ± 98.3⁎ 192.1 ± 11.3⁎⁎ 237.5 ± 23.5⁎⁎ 235.7 ± 25.9⁎⁎
⁎P ≤ 0.05 compared to values for normal rats. ⁎⁎P ≤ 0.05 compared to CCl4 control. Values are Mean ± S.D, N = 6.
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from normal hepatocytes with well defined nuclei. Vacuolization and fatty degeneration were remarkably prevented by the treatment with extracts and silymarin. In thioacetamide treated rats, the levels of AST, ALT and LDH were significantly elevated. Treatment with n-hexane extracts of P. maderaspatensis at a dose of 200, 100 mg/kg and silymarin (50 mg/kg) showed a significant decrease in AST, ALT and LDH (Table 2). Treatment with 200 and 100 mg/kg n-hexane extract exhibited a significant increase, 91 and 88.8% respectively in hepatic glutathione levels. Silymarin treated rats also prevented the lowering of hepatic GSH by 84.2%. A significant increase in tissue MDA level was observed in thioacetamide alone treated rats. However, thioacetamide induced
Fig. 1. Effect of P. maderaspatensis n-hexane extract on histopathological changes that occurred in rats during carbon tetrachloride intoxication (hematoxylin and eosin, ×200). (A) Normal control; (B) CCl4 control, (C) P. maderaspatensis n-hexane extract (200 mg/kg); (D) P. maderaspatensis n-hexane extract (100 mg/kg); (E) silymarin (50 mg/kg).
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Fig. 1 (continued ).
elevation of tissue MDA concentration was lowered by 90 and 82.6% in rats treated with n-hexane extract at a dose of 200 and 100 mg/kg respectively which was comparable to silymarin (88.6%). The liver sections of normal control animals showed hepatic cells with well preserved cytoplasm, prominent nucleus and central vein (Fig. 2A). In rats treated with thioacetamide (Fig. 2B), the normal architecture of liver was completely lost with the appearance of centrilobular necrosis with tiny vacuoles, lymphocytes infiltration of the periportal area and fatty changes were observed. The animals administered with n-hexane extract at 200 and 100 mg/kg (Fig. 2C and D) or silymarin (Fig. 2E) showed a significant recovery from thioacetamide induced liver damage as evident from normal hepatic architectural pattern with mild hepatitis. 4. Discussion and conclusion Carbon tetrachloride proves highly useful as an experimental model for the study of acute hepatic injury [12]. CCl4induced liver damage is mainly attributed to the toxic metabolites, mainly trichloromethyl radical, for the initiation of Table 2 Effect of P. maderaspatensis n-hexane extract on serum AST, ALT and LDH in thioacetamide intoxicated rats Treatment
AST (IU/l)
ALT (IU/l)
LDH (IU/l)
Normal control Thioacetamide control (100 mg/kg) P. maderaspatensis (200 mg/kg) + thioacetamide P. maderaspatensis (100 mg/kg) + thioacetamide Silymarin (50 mg/kg) + thioacetamide
156.7 ± 5.8 1977.8 ± 63.6⁎ 263.2 ± 11.7⁎⁎ 307.9 ± 14.5⁎⁎ 343.4 ± 20.7⁎⁎
57.5 ± 5.4 1374.9 ± 49.9⁎ 163.4 ± 11.5⁎⁎ 214.4 ± 15.9⁎⁎ 238.6 ± 18.0⁎⁎
159.6 ± 5.8 2007.0 ± 83.8⁎ 296.7 ± 14.2⁎⁎ 329.2 ± 18.3⁎⁎ 353.2 ± 24.1⁎⁎
⁎P ≤ 0.05 compared to values for normal rats. ⁎⁎P ≤ 0.05 compared to thioacetamide control. Values are Mean ± S.D, N = 6.
V.V. Asha et al. / Fitoterapia 78 (2007) 134–141
139
CCl4 dependant lipid peroxidation. Antioxidants and radical scavengers have been used to study the mechanism of CCl4 toxicity as well as to protect liver cells from CCl4-induced damage by breaking the chain reaction of lipid peroxidation [13]. Also, thioacetamide is a potent hepatotoxin and its toxicity is due to the formation of thioacetamide5-oxide which is responsible for the change in cell permeability, increased intracellular concentration of Ca2+, increase in nuclear volume and enlargement of nucleoli and also inhibits mitochondrial activity that leads to cell death [14]. Rats treated with both hepatotoxins showed elevated levels of AST, ALT, LDH and liver lipid peroxides. The cellular
Fig. 2. Effect of P. maderaspatensis n-hexane extract on histopathological changes that occurred in rats during thioacetamide intoxication (hematoxylin and eosin, ×200). (A) Normal control; (B) thioacetamide control, (C) P. maderaspatensis n-hexane extract (200 mg/kg); (D) P. maderaspatensis n-hexane extract (100 mg/kg); (E) silymarin (50 mg/kg).
140
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Fig. 2 (continued ).
antioxidant, reduced glutathione (GSH), was decreased. The scavenging effect of P. maderaspatensis n-hexane extract on free radicals produced by liver was observed when rats were treated with the extracts as evidenced from lowered MDA levels. The protective effect of P. maderaspatensis n-hexane extract was further substantiated by histopathological assessment, where necrotic and infiltrative changes were observed. The results of this study confirm the significant anti-hepatotoxic activity of P. maderaspatensis n-hexane extract. Further clinical studies are needed to evaluate the real therapeutic value of this natural extract. Acknowledgements The authors express sincere thanks to Dr. M. Radhakrishna Pillai, Director, Rajiv Gandhi Center for Biotechnology, for his interest and encouragement in this work and also gratefully acknowledge the Department of Biotechnology, Government of India, for providing the funds for doing this project. The authors are indebted to Mr. K S Sabulal and Miss Ciji Varghese for technical assistance. References [1] [2] [3] [4] [5] [6] [7]
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