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lipopolysaccharide-induced hepatitis in rats
Journal of Ethnopharmacology 101 (2005) 55–60
Hepatoprotective activity of Tridax procumbens against d-galactosamine/lipopolysaccharide-induced hepatitis in rats Vilwanathan Ravikumar, Kanchi Subramanian Shivashangari, Thiruvengadam Devaki ∗ Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, India Received 23 September 2004; accepted 24 March 2005 Available online 31 May 2005
Abstract The hepatoprotective activity of aerial parts of Tridax procumbens was investigated against d-Galactosamine/Lipopolysaccharide (dGalN/LPS) induced hepatitis in rats. d-GalN/LPS (300 mg/kg body weight/30 g/kg body weight)-induced hepatic damage was manifested by a significant increase in the activities of marker enzymes (aspartate transaminase, alanine transaminase, alkaline phosphatase, lactate dehydrogenase and gamma glutamyl transferase) and bilirubin level in serum and lipids both in serum and liver. Pretreatment of rats with a chloroform insoluble fraction from ethanolic extract of Tridax procumbens reversed these altered parameters to normal values. The biochemical observations were supplemented by histopathological examination of liver sections. Results of this study revealed that Tridax procumbens could afford a significant protection in the alleviation of d-GalN/LPS-induced hepatocellular injury. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Tridax procumbens; d-Galactosamine; Lipopolysaccharide; Hepatitis; Marker enzymes; Lipids
1. Introduction Hepatitis is a common disease in the world especially in the developing countries. Despite, considerable progress in the treatment of liver diseases by oral hepatoprotective agents, search for newer drugs continues because the existing synthetic drugs have several limitations. Hence, there are many researchers of traditional medicines attempting to develop new drugs for hepatitis (Liu, 1989). Tridax procumbens Linn. (Compositeae), a weed found throughout India is employed as indigenous medicine for a variety of ailments including jaundice (Saraf and Dixit, 1991). It is commonly used in Indian traditional medicine as anticoagulant, antifungal and insect repellent; in bronchial catarrh, diarrhoea and dysentery (Ali et al., 2001). Moreover it possesses wound healing activity and promotes hair growth (Saraf et al., 1991). Tridax procumbens is also dispensed as ‘Bhringraj’, which has a great reputation in Ayurvedic ∗
Corresponding author. Tel.: +91 44 22351269; fax: +91 44 22352494. E-mail address: [email protected] (T. Devaki).
0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.03.019
medicines for liver disorders (Pathak et al., 1991). The hepatoprotective action has been demonstrated (Saraf and Dixit, 1991; Pathak et al., 1991). We have reported recently the antioxidant property of Tridax procumbens (Ravikumar et al., 2004). However no reports were available on the protective potential of Tridax procumbens against experimentally induced hepatitis in rats. Thus the aim of the present study is to further evaluate the hepatoprotective activity of Tridax procumbens correlating biochemical and histological changes against d-GalN/LPS-induced hepatitis in rats. d-GalN has been proposed to be hepatotoxic due to its ability to destruct liver cells (Anandan et al., 1999). Its toxicity is of clinical importance because there is a close resemblance between the multifocal necrosis produced by dGalN and the lesion of viral hepatitis in humans (Decker and Keppler, 1972). d-GalN given at the time of endotoxin challenge markedly sensitizes mice and other species to the lethal effects of endotoxin (Galanos et al., 1979). This amino sugar is known to selectively block the transcription and indirectly hepatic protein synthesis (Decker and Keppler, 1974) and as a consequence of endotoxin toxicity, it causes
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fulminant hepatitis within 8 h after administration (Hase et al., 1997). This immunological liver injury model has been used to evaluate the efficacy of several hepatoprotective agents (Kondo et al., 1994) and hence selected as a model for inducing hepatitis in the present investigation.
The rats were anesthetised and sacrificed after the experiment by cervical dislocation. Blood was collected and the serum separated was used for various biochemical assays. The liver tissue excised was washed with ice-cold saline. A portion of the liver was then homogenised in 0.1 M Tris buffer and the homogenate was used for biochemical estimations.
2. Materials and methods
2.3.1. Biological assays The activities of serum aspartate transaminase (AST), alanine transaminase (ALT) (Reitman and Frankel, 1957), alkaline phosphatase (ALP) (King and Armstrong, 1934), lactate dehydrogenase (LDH) (King, 1965), gamma glutamyl transferase (␥-GT) (Rosalki and Rau, 1972) and levels of bilirubin (Malloy and Evelyn, 1987) were assayed. The extraction of serum and tissue lipids (Folch et al., 1957) followed by the estimation of total cholesterol (Zlatkis et al., 1953), triglycerides (Foster and Dunn, 1973), free fatty acids (Falholt et al., 1973) and phospholipids (Zilversmit and Davis, 1950) were carried out.
2.1. Plant material Aerial parts of Tridax procumbens were collected from our University Campus during October–December 2001. The plant was authenticated by Dr. R. Rengasamy, Professor, CAS in Botany, University of Madras, Guindy Campus, Chennai 600 025. 2.1.1. Preparation of plant extract The shade-dried aerial parts were defatted with petroleum ether (60–80 ◦ C) and extracted with 95% ethanol using soxhlet apparatus. After complete removal of the solvent, the residue was further fractionated into chloroform soluble and insoluble portions. The chloroform insoluble fraction was triturated with 2% polyvinylpyrrolidone (PVP) in water to get a suspension of 100 mg/ml (Ravikumar et al., 2004). 2.2. Chemicals d-GalN and LPS (Sero type 011.B4 extracted by phenol water method from Escherichia coli) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals used were of analytical grade. 2.3. Experimental animals Male albino rats of Wistar strain weighing 120–150 g obtained from Tamilnadu Veterinary and Animal Sciences University (TANUVAS), Madhavaram, Chennai, were used for this study. They were acclimatized to animal house conditions and were fed a commercial pellet diet (Hindustan Lever Limited, Bangalore, India) and water ad libitum. The experiments were conducted according to the ethical norms approved by Ministry of Social Justices and Empowerment, Government of India and Institutional Animal Ethics Committee guidelines 360/01/a/CPCSEA dtd. 21.05.01). The animals were divided into four groups of six animals each. Group 1 served as vehicle control and received 2% PVP (1 ml/kg body weight orally) for 10 days. Group 2 rats were given Tridax procumbens extract alone (300 mg/kg body weight orally) for 10 days. Group 3 rats were induced with hepatitis by giving intraperitoneal injections of d-GalN and LPS (300 mg/kg body weight and 30 g/kg body weight) 18 h before the experiment (Omar et al., 1996). Group 4 rats were pretreated with Tridax procumbens extract for 10 days prior to the induction of d-GalN/LPS.
2.3.2. Histopathology Small pieces of liver tissues were collected in 10% formal saline for proper fixation. These tissues were processed and embedded in paraffin wax sections of 5 to 6 microns in thickness were cut and stained with hematoxylin and eosin (Luna, 1966). 2.4. Statistical analysis All the grouped data were statistically evaluated with SPSS (Chicago, IL) 7.5 software. Hypothesis testing methods included one way analysis of variance (ANOVA) followed by least significant difference test. A value of P < 0.05 was considered to indicate statistical significance. All the results were expressed as mean ± S.D. for six animals in each group. 3. Results Rats intoxicated with d-GalN/LPS alone (Group 3) developed hepatocellular damage as evident from a significant elevation (P < 0.05) in the serum activities of AST, ALT, ALP, LDH, ␥-GT and bilirubin level when compared with control (Table 1). There was a significant increase in the levels of cholesterol, triglycerides and free fatty acids in both, serum and tissue of rats treated with d-GalN/LPS when compared with control whereas the levels of phospholipids in both serum and liver (Tables 2 and 3) were found to be decreased. Pretreatment of Tridax procumbens extract afforded a significant protection against d-GalN/LPS-induced liver injury by maintaining the levels to near normal. There was no significant change in the activities of marker enzymes, bilirubin level and lipids in the rats treated with Tridax procumbens extract alone as compared to the control thereby showing the absence of any adverse toxic effects of Tridax procumbens extract.
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Table 1 Levels of serum AST, ALT, ALP, LDH, ␥-GT and bilirubin in control and experimental groups of rats Parameters
AST (IU/l)
ALT (IU/l)
Group 1 Group 2 Group 3 Group 4
113.35 ± 6.79 114.02 ± 5.87 432.75 ± 14.71a 223.53 ± 15.69b
38.61 39.19 146.71 66.48
± ± ± ±
ALP (IU/l)
3.47 1.52 6.10a 2.36b
59.43 60.93 151.32 90.41
± ± ± ±
3.43 2.89 6.06a 3.73b
LDH (IU/l)
␥-GT (IU/l)
110.31 ± 4.94 111.27 ± 6.04 200.24 ± 21.33a 126.59 ± 4.93b
6.48 6.63 14.68 7.98
± ± ± ±
0.0632 0.079a 0.014a 0.077b
Bilirubin (mg/dl) 0.58 ± 0.019 0.57 ± 0.016a 1.07 ± 0.023a 0.74 ± 0.024b
Values are given as mean ± S.D. for groups of six animals each; values are statistically significant at P < 0.05 a P < 0.05 compared with group 1. b P < 0.05 compared with group 3. Table 2 Levels of serum cholesterol, triglycerides, free fatty acids and phospholipids in control and experimental groups of rats Parameters
Cholesterol (mg/dl)
Group 1 Group 2 Group 3 Group 4
84.27 85.32 113.66 91.47
± ± ± ±
5.28 6.08 6.58a 4.89b
Triglycerides (mg/dl) 40.82 41.03 104.82 57.74
± ± ± ±
3.42 2.04 4.89a 2.05b
Free fatty acids (mg/dl) 34.63 35.83 52.02 41.95
± ± ± ±
1.35 2.34 3.78a 2.31b
Phospholipids (mg/dl) 92.85 94.78 59.75 80.64
± ± ± ±
4.24 3.46 2.91a 3.75b
Values are given as mean ± S.D. for groups of six animals each. Values are statistically significant at P < 0.05. a P < 0.05 compared with group 1. b P < 0.05 compared with group 3. Table 3 Levels of cholesterol, triglycerides, free fatty acids and phospholipids in the liver of control and experimental groups of rats Parameters
Cholesterol (mg/dl)
Triglycerides (mg/dl)
Free fatty acids (mg/dl)
Phospholipids (mg/dl)
Group 1 Group 2 Group 3 Group 4
11.56 ± 1.47 12.17 ± 2.56 20.46 ± 0.78a 16.02 ± 1.14b
22.54 ± 1.20 23.39 ± 1.31 45.31 ± 2.34a 34.18 ± 0.71b
0.69 ± 0.015 0.70 ± 0.014 2.27 ± 0.153a 1.43 ± 0.090b
26.49 ± 1.12 25.09 ± 0.85a 19.37 ± 1.33a 24.68 ± 0.38b
Values are given as mean ± S.D. for groups of six animals each. Values are statistically significant at P < 0.05. a P <0.05 compared with group 1. b P < 0.05 compared with group 3.
3.1. Histopathological observation Diffused areas of hepatitis especially in the perivenular region, which extends to the central zone with inflammatory collections, were observed in d-GalN/LPS induced rats (Fig. 1) when compared to control (Fig. 2). Pretreatment with Tridax procumbens extract reversed to a large extent the hepatic lesions produced by d-GalN/LPS as it is evident from the absence of cellular necrosis and inflammatory infiltrate Fig. 2. Photomicrograph of control rat liver section showing normal architecture. Hematoxylin-eosin stain (100×).
around the central zone (Fig. 3) which further confirms the hepatoprotective potential of the extract. The rats, given Tridax procumbens extract alone, did not show any abnormal change in the architecture of the liver (Fig. 4) as compared to the control rats.
Fig. 1. Photomicrograph of d-GalN/LPS-intoxicated rat liver section which shows loss of architecture and cell necrosis (perivenular) extending to the central zone. The cell necrosis with inflammatory collections is more prominent in the central zone than around central vein. Hematoxylin-eosin stain (100×).
4. Discussion Exogenous administration of d-GalN has been found to induce liver damage which closely resembles human viral hepatitis (Taniguchi et al., 2004). The toxicity of d-GalN
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Fig. 3. Photomicrograph of liver section of rat pretreated with Tridax procumbens extract prior to d-GalN/LPS challenge showing central vein surrounded by hepatocytes with sinusoidal dilatation with occasional inflammatory cells. No hepatic necrosis was seen around central vein or in the central zone. Hematoxylin-eosin stain (100×).
results from inhibition of RNA and protein synthesis in the liver (Decker and Keppler, 1972; Konishi et al., 1974). The metabolism of d-GalN may deplete several uracil nucleotides including UDP-glucose, UDP-galactose and UTP (Decker and Keppler, 1974) which are trapped in the formation of uridine-diphosphogalactosamine. Accumulation of UDP-sugar nucleotides (Endo et al., 1992; Manabe et al., 1996) may contribute to the changes in the rough endoplasmic reticulum and to the disturbance of protein metabolism. Further, intense galactosamination of membrane structures is thought to be responsible for loss in the activity of ionic pumps. The impairment in the calcium pump, with consequent increase in the intracellular calcium is considered to be responsible for cell death (Tsai et al., 1997). An evidence of hepatic injury is leakage of cellular enzymes into the plasma. When liver cell plasma membrane is damaged, a variety of enzymes normally located in the cytosol are released into the blood stream. Their estimation in the serum is a useful quantitative marker of the extent and type of hepatocellular damage (Mitra et al., 1998). Liver damage induced by d-GalN generally reflects disturbances of liver cell metabolism which lead to characteristic changes in the serum enzyme activities (Lim et al., 2000). The increased levels of AST, ALT, ALP, LDH and ␥-GT in this study may be interpreted as a result of the liver cell destruc-
Fig. 4. Photomicrograph of liver section of rat treated with Tridax procumbens extract alone for 10 days showing normal liver parenchyma with central vein and cords of hepatocytes. Hematoxylin-eosin stain (100×).
tion or changes in the membrane permeability indicating the severity of hepatocellular damage induced by d-GalN/LPS, which is in accordance with previous reports (Manabe et al., 1996; Mitra et al., 1998; Rastogi et al., 1998; Sreepriya and Devaki, 2001). The rise in ALT activity is almost always due to hepatocellular damage and is usually accompanied by rise in AST (Rao et al., 1989). An increase in ALP reflects the pathological alteration in biliary flow (Plaa and Hewitt, 1989). The release of LDH reflects a non-specific alteration in the plasma membrane integrity and/or permeability as a response to d-GalN (Rastogi et al., 1998). ␥-GT is an enzyme embedded in the hepatocyte plasma membrane, mainly in the canalicular domain, again the liberation of this enzyme to serum indicates damage to the cell and thus injury to the liver. It is important to point out that serum ␥GT activity is considered to be one of the best indicators of liver damage (Bulle et al., 1990). Pretreatment with Tridax procumbens extract attenuated the increased activities of these enzymes in serum caused by d-GalN/LPS. Recovery towards normalisation suggests that Tridax procumbens extract causes parenchymal cell regeneration in liver, thus protecting membrane fragility, thereby, decreasing enzyme leakage. Determination of serum bilirubin represents an index for the assessment of hepatic function and any abnormal increase in the levels of bilirubin in the serum indicate hepatobiliary disease and severe disturbance of hepatocellular function (Martin and Friedman, 1992). Increased levels of bilirubin in this study is in agreement with previous reports showing that d-GalN induced hepatitis is characterised by increased levels of bilirubin in serum (Sree Ramamurthy and Srinivasan, 1993; Maezona et al., 1996). The extract mediated suppression of the increased bilirubin level suggests the possibility of the extract being able to stabilise biliary dysfunction. Tridax procumbens extract also showed protection against serum and liver lipid changes caused by d-GalN/LPS evidencing a broad spectrum of hepatoprotective property. Any liver disease will show an increased blood cholesterol level (Mc Intyre and Rosalki, 1992). The significant increase of cholesterol noted in this study might have been due to the inability of the diseased liver to remove cholesterol from circulation. This finding could be correlated with the results of the previous studies (Cartwright et al., 1982; Black et al., 1983). Hepatocellular damage due to alcohol, virus and drug induced hepatitis causes a modest hypertriglyceridemia (Glickman and Sebesin, 1982), which is due to the biochemical changes inferring with the transport of triglycerides out of liver. Our study also showed an increased accumulation of triglycerides in d-GalN/LPS induced rats which is in agreement with previous reports (Koff et al., 1971; Cartwright et al., 1982). The administration of endotoxic LPS resulted in an increase in the free fatty acid content in rats (Dhuley and Naik, 1998). Accumulation of free fatty acids is a consequence of changes in hepatic lipid metabolism. This is well correlated in our study with the increased levels of free fatty acids due to the administration of d-GalN/LPS, which is responsible for the increment of phospholipase A2 activity as a consequence of
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the high level of intracellular calcium causing the hydrolysis of liver membrane phospholipids and release of arachidonic acid (Kramer, 1993). This might account for the decreased levels of phospholipids observed in the serum and liver of d-GalN/LPS challenged rats. Rats pretreated with Tridax procumbens extract prior to the induction of hepatic damage showed a restoration of the altered lipid levels induced by d-GalN/LPS towards near normalcy thereby showing the modulating effect of Tridax procumbens extract against d-GalN/LPS-induced changes in the lipid levels in rats. This can be well correlated with the protective actions of picroliv (an active fraction from Picrorhiza kurroa), which showed lipid changes against hepatotoxicity induced by paracetamol and d-GalN (Ansari et al., 1991). Thus, the present study confirms the hepatoprotective action of Tridax procumbens against d-GalN/LPS-induced hepatitis in rats. The curative efficacy of Tridax procumbens extract was very promising as evidenced by the reversal of the altered values following administration probably by promoting regeneration of hepatocytes that restore integrity. The hepatoprotective action may be mediated through the inhibition of UDP-sugar derivatives, enhancement of glycoprotein biosynthesis and stabilisation of cell membrane and inhibition of lipid accumulation by its hypolipidemic property. The hepatoprotective property of the extract may be attributed to the presence of flavonoids which are present in the plant. Previous phytochemical investigations of Tridax procumbens described the isolation and structural determination of a flavonoid, procumbenetin (Ali et al., 2001). The inhibitory activity of this flavonoid in free radical production could be related to the hepatoprotective effect (Ravikumar et al., 2004). This indicates that the extract of Tridax procumbens may be used as an effective hepatoprotective agent.
Acknowledgement Dr. T. Devaki wishes to thank University Grants Commission–University With Potential For Excellence (UGC–UWPFE) for providing financial assistance to Mr. V. Ravikumar in the form of Junior Research Fellowship.
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Hepatoprotective activity of Tridax procumbens against d-galactosamine/lipopolysaccharide-induced hepatitis in rats Vilwanathan Ravikumar, Kanchi Subramanian Shivashangari, Thiruvengadam Devaki ∗ Department of Biochemistry, University of Madras, Guindy Campus, Chennai 600025, India Received 23 September 2004; accepted 24 March 2005 Available online 31 May 2005
Abstract The hepatoprotective activity of aerial parts of Tridax procumbens was investigated against d-Galactosamine/Lipopolysaccharide (dGalN/LPS) induced hepatitis in rats. d-GalN/LPS (300 mg/kg body weight/30 g/kg body weight)-induced hepatic damage was manifested by a significant increase in the activities of marker enzymes (aspartate transaminase, alanine transaminase, alkaline phosphatase, lactate dehydrogenase and gamma glutamyl transferase) and bilirubin level in serum and lipids both in serum and liver. Pretreatment of rats with a chloroform insoluble fraction from ethanolic extract of Tridax procumbens reversed these altered parameters to normal values. The biochemical observations were supplemented by histopathological examination of liver sections. Results of this study revealed that Tridax procumbens could afford a significant protection in the alleviation of d-GalN/LPS-induced hepatocellular injury. © 2005 Elsevier Ireland Ltd. All rights reserved. Keywords: Tridax procumbens; d-Galactosamine; Lipopolysaccharide; Hepatitis; Marker enzymes; Lipids
1. Introduction Hepatitis is a common disease in the world especially in the developing countries. Despite, considerable progress in the treatment of liver diseases by oral hepatoprotective agents, search for newer drugs continues because the existing synthetic drugs have several limitations. Hence, there are many researchers of traditional medicines attempting to develop new drugs for hepatitis (Liu, 1989). Tridax procumbens Linn. (Compositeae), a weed found throughout India is employed as indigenous medicine for a variety of ailments including jaundice (Saraf and Dixit, 1991). It is commonly used in Indian traditional medicine as anticoagulant, antifungal and insect repellent; in bronchial catarrh, diarrhoea and dysentery (Ali et al., 2001). Moreover it possesses wound healing activity and promotes hair growth (Saraf et al., 1991). Tridax procumbens is also dispensed as ‘Bhringraj’, which has a great reputation in Ayurvedic ∗
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0378-8741/$ – see front matter © 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jep.2005.03.019
medicines for liver disorders (Pathak et al., 1991). The hepatoprotective action has been demonstrated (Saraf and Dixit, 1991; Pathak et al., 1991). We have reported recently the antioxidant property of Tridax procumbens (Ravikumar et al., 2004). However no reports were available on the protective potential of Tridax procumbens against experimentally induced hepatitis in rats. Thus the aim of the present study is to further evaluate the hepatoprotective activity of Tridax procumbens correlating biochemical and histological changes against d-GalN/LPS-induced hepatitis in rats. d-GalN has been proposed to be hepatotoxic due to its ability to destruct liver cells (Anandan et al., 1999). Its toxicity is of clinical importance because there is a close resemblance between the multifocal necrosis produced by dGalN and the lesion of viral hepatitis in humans (Decker and Keppler, 1972). d-GalN given at the time of endotoxin challenge markedly sensitizes mice and other species to the lethal effects of endotoxin (Galanos et al., 1979). This amino sugar is known to selectively block the transcription and indirectly hepatic protein synthesis (Decker and Keppler, 1974) and as a consequence of endotoxin toxicity, it causes
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fulminant hepatitis within 8 h after administration (Hase et al., 1997). This immunological liver injury model has been used to evaluate the efficacy of several hepatoprotective agents (Kondo et al., 1994) and hence selected as a model for inducing hepatitis in the present investigation.
The rats were anesthetised and sacrificed after the experiment by cervical dislocation. Blood was collected and the serum separated was used for various biochemical assays. The liver tissue excised was washed with ice-cold saline. A portion of the liver was then homogenised in 0.1 M Tris buffer and the homogenate was used for biochemical estimations.
2. Materials and methods
2.3.1. Biological assays The activities of serum aspartate transaminase (AST), alanine transaminase (ALT) (Reitman and Frankel, 1957), alkaline phosphatase (ALP) (King and Armstrong, 1934), lactate dehydrogenase (LDH) (King, 1965), gamma glutamyl transferase (␥-GT) (Rosalki and Rau, 1972) and levels of bilirubin (Malloy and Evelyn, 1987) were assayed. The extraction of serum and tissue lipids (Folch et al., 1957) followed by the estimation of total cholesterol (Zlatkis et al., 1953), triglycerides (Foster and Dunn, 1973), free fatty acids (Falholt et al., 1973) and phospholipids (Zilversmit and Davis, 1950) were carried out.
2.1. Plant material Aerial parts of Tridax procumbens were collected from our University Campus during October–December 2001. The plant was authenticated by Dr. R. Rengasamy, Professor, CAS in Botany, University of Madras, Guindy Campus, Chennai 600 025. 2.1.1. Preparation of plant extract The shade-dried aerial parts were defatted with petroleum ether (60–80 ◦ C) and extracted with 95% ethanol using soxhlet apparatus. After complete removal of the solvent, the residue was further fractionated into chloroform soluble and insoluble portions. The chloroform insoluble fraction was triturated with 2% polyvinylpyrrolidone (PVP) in water to get a suspension of 100 mg/ml (Ravikumar et al., 2004). 2.2. Chemicals d-GalN and LPS (Sero type 011.B4 extracted by phenol water method from Escherichia coli) were obtained from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals used were of analytical grade. 2.3. Experimental animals Male albino rats of Wistar strain weighing 120–150 g obtained from Tamilnadu Veterinary and Animal Sciences University (TANUVAS), Madhavaram, Chennai, were used for this study. They were acclimatized to animal house conditions and were fed a commercial pellet diet (Hindustan Lever Limited, Bangalore, India) and water ad libitum. The experiments were conducted according to the ethical norms approved by Ministry of Social Justices and Empowerment, Government of India and Institutional Animal Ethics Committee guidelines 360/01/a/CPCSEA dtd. 21.05.01). The animals were divided into four groups of six animals each. Group 1 served as vehicle control and received 2% PVP (1 ml/kg body weight orally) for 10 days. Group 2 rats were given Tridax procumbens extract alone (300 mg/kg body weight orally) for 10 days. Group 3 rats were induced with hepatitis by giving intraperitoneal injections of d-GalN and LPS (300 mg/kg body weight and 30 g/kg body weight) 18 h before the experiment (Omar et al., 1996). Group 4 rats were pretreated with Tridax procumbens extract for 10 days prior to the induction of d-GalN/LPS.
2.3.2. Histopathology Small pieces of liver tissues were collected in 10% formal saline for proper fixation. These tissues were processed and embedded in paraffin wax sections of 5 to 6 microns in thickness were cut and stained with hematoxylin and eosin (Luna, 1966). 2.4. Statistical analysis All the grouped data were statistically evaluated with SPSS (Chicago, IL) 7.5 software. Hypothesis testing methods included one way analysis of variance (ANOVA) followed by least significant difference test. A value of P < 0.05 was considered to indicate statistical significance. All the results were expressed as mean ± S.D. for six animals in each group. 3. Results Rats intoxicated with d-GalN/LPS alone (Group 3) developed hepatocellular damage as evident from a significant elevation (P < 0.05) in the serum activities of AST, ALT, ALP, LDH, ␥-GT and bilirubin level when compared with control (Table 1). There was a significant increase in the levels of cholesterol, triglycerides and free fatty acids in both, serum and tissue of rats treated with d-GalN/LPS when compared with control whereas the levels of phospholipids in both serum and liver (Tables 2 and 3) were found to be decreased. Pretreatment of Tridax procumbens extract afforded a significant protection against d-GalN/LPS-induced liver injury by maintaining the levels to near normal. There was no significant change in the activities of marker enzymes, bilirubin level and lipids in the rats treated with Tridax procumbens extract alone as compared to the control thereby showing the absence of any adverse toxic effects of Tridax procumbens extract.
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Table 1 Levels of serum AST, ALT, ALP, LDH, ␥-GT and bilirubin in control and experimental groups of rats Parameters
AST (IU/l)
ALT (IU/l)
Group 1 Group 2 Group 3 Group 4
113.35 ± 6.79 114.02 ± 5.87 432.75 ± 14.71a 223.53 ± 15.69b
38.61 39.19 146.71 66.48
± ± ± ±
ALP (IU/l)
3.47 1.52 6.10a 2.36b
59.43 60.93 151.32 90.41
± ± ± ±
3.43 2.89 6.06a 3.73b
LDH (IU/l)
␥-GT (IU/l)
110.31 ± 4.94 111.27 ± 6.04 200.24 ± 21.33a 126.59 ± 4.93b
6.48 6.63 14.68 7.98
± ± ± ±
0.0632 0.079a 0.014a 0.077b
Bilirubin (mg/dl) 0.58 ± 0.019 0.57 ± 0.016a 1.07 ± 0.023a 0.74 ± 0.024b
Values are given as mean ± S.D. for groups of six animals each; values are statistically significant at P < 0.05 a P < 0.05 compared with group 1. b P < 0.05 compared with group 3. Table 2 Levels of serum cholesterol, triglycerides, free fatty acids and phospholipids in control and experimental groups of rats Parameters
Cholesterol (mg/dl)
Group 1 Group 2 Group 3 Group 4
84.27 85.32 113.66 91.47
± ± ± ±
5.28 6.08 6.58a 4.89b
Triglycerides (mg/dl) 40.82 41.03 104.82 57.74
± ± ± ±
3.42 2.04 4.89a 2.05b
Free fatty acids (mg/dl) 34.63 35.83 52.02 41.95
± ± ± ±
1.35 2.34 3.78a 2.31b
Phospholipids (mg/dl) 92.85 94.78 59.75 80.64
± ± ± ±
4.24 3.46 2.91a 3.75b
Values are given as mean ± S.D. for groups of six animals each. Values are statistically significant at P < 0.05. a P < 0.05 compared with group 1. b P < 0.05 compared with group 3. Table 3 Levels of cholesterol, triglycerides, free fatty acids and phospholipids in the liver of control and experimental groups of rats Parameters
Cholesterol (mg/dl)
Triglycerides (mg/dl)
Free fatty acids (mg/dl)
Phospholipids (mg/dl)
Group 1 Group 2 Group 3 Group 4
11.56 ± 1.47 12.17 ± 2.56 20.46 ± 0.78a 16.02 ± 1.14b
22.54 ± 1.20 23.39 ± 1.31 45.31 ± 2.34a 34.18 ± 0.71b
0.69 ± 0.015 0.70 ± 0.014 2.27 ± 0.153a 1.43 ± 0.090b
26.49 ± 1.12 25.09 ± 0.85a 19.37 ± 1.33a 24.68 ± 0.38b
Values are given as mean ± S.D. for groups of six animals each. Values are statistically significant at P < 0.05. a P <0.05 compared with group 1. b P < 0.05 compared with group 3.
3.1. Histopathological observation Diffused areas of hepatitis especially in the perivenular region, which extends to the central zone with inflammatory collections, were observed in d-GalN/LPS induced rats (Fig. 1) when compared to control (Fig. 2). Pretreatment with Tridax procumbens extract reversed to a large extent the hepatic lesions produced by d-GalN/LPS as it is evident from the absence of cellular necrosis and inflammatory infiltrate Fig. 2. Photomicrograph of control rat liver section showing normal architecture. Hematoxylin-eosin stain (100×).
around the central zone (Fig. 3) which further confirms the hepatoprotective potential of the extract. The rats, given Tridax procumbens extract alone, did not show any abnormal change in the architecture of the liver (Fig. 4) as compared to the control rats.
Fig. 1. Photomicrograph of d-GalN/LPS-intoxicated rat liver section which shows loss of architecture and cell necrosis (perivenular) extending to the central zone. The cell necrosis with inflammatory collections is more prominent in the central zone than around central vein. Hematoxylin-eosin stain (100×).
4. Discussion Exogenous administration of d-GalN has been found to induce liver damage which closely resembles human viral hepatitis (Taniguchi et al., 2004). The toxicity of d-GalN
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Fig. 3. Photomicrograph of liver section of rat pretreated with Tridax procumbens extract prior to d-GalN/LPS challenge showing central vein surrounded by hepatocytes with sinusoidal dilatation with occasional inflammatory cells. No hepatic necrosis was seen around central vein or in the central zone. Hematoxylin-eosin stain (100×).
results from inhibition of RNA and protein synthesis in the liver (Decker and Keppler, 1972; Konishi et al., 1974). The metabolism of d-GalN may deplete several uracil nucleotides including UDP-glucose, UDP-galactose and UTP (Decker and Keppler, 1974) which are trapped in the formation of uridine-diphosphogalactosamine. Accumulation of UDP-sugar nucleotides (Endo et al., 1992; Manabe et al., 1996) may contribute to the changes in the rough endoplasmic reticulum and to the disturbance of protein metabolism. Further, intense galactosamination of membrane structures is thought to be responsible for loss in the activity of ionic pumps. The impairment in the calcium pump, with consequent increase in the intracellular calcium is considered to be responsible for cell death (Tsai et al., 1997). An evidence of hepatic injury is leakage of cellular enzymes into the plasma. When liver cell plasma membrane is damaged, a variety of enzymes normally located in the cytosol are released into the blood stream. Their estimation in the serum is a useful quantitative marker of the extent and type of hepatocellular damage (Mitra et al., 1998). Liver damage induced by d-GalN generally reflects disturbances of liver cell metabolism which lead to characteristic changes in the serum enzyme activities (Lim et al., 2000). The increased levels of AST, ALT, ALP, LDH and ␥-GT in this study may be interpreted as a result of the liver cell destruc-
Fig. 4. Photomicrograph of liver section of rat treated with Tridax procumbens extract alone for 10 days showing normal liver parenchyma with central vein and cords of hepatocytes. Hematoxylin-eosin stain (100×).
tion or changes in the membrane permeability indicating the severity of hepatocellular damage induced by d-GalN/LPS, which is in accordance with previous reports (Manabe et al., 1996; Mitra et al., 1998; Rastogi et al., 1998; Sreepriya and Devaki, 2001). The rise in ALT activity is almost always due to hepatocellular damage and is usually accompanied by rise in AST (Rao et al., 1989). An increase in ALP reflects the pathological alteration in biliary flow (Plaa and Hewitt, 1989). The release of LDH reflects a non-specific alteration in the plasma membrane integrity and/or permeability as a response to d-GalN (Rastogi et al., 1998). ␥-GT is an enzyme embedded in the hepatocyte plasma membrane, mainly in the canalicular domain, again the liberation of this enzyme to serum indicates damage to the cell and thus injury to the liver. It is important to point out that serum ␥GT activity is considered to be one of the best indicators of liver damage (Bulle et al., 1990). Pretreatment with Tridax procumbens extract attenuated the increased activities of these enzymes in serum caused by d-GalN/LPS. Recovery towards normalisation suggests that Tridax procumbens extract causes parenchymal cell regeneration in liver, thus protecting membrane fragility, thereby, decreasing enzyme leakage. Determination of serum bilirubin represents an index for the assessment of hepatic function and any abnormal increase in the levels of bilirubin in the serum indicate hepatobiliary disease and severe disturbance of hepatocellular function (Martin and Friedman, 1992). Increased levels of bilirubin in this study is in agreement with previous reports showing that d-GalN induced hepatitis is characterised by increased levels of bilirubin in serum (Sree Ramamurthy and Srinivasan, 1993; Maezona et al., 1996). The extract mediated suppression of the increased bilirubin level suggests the possibility of the extract being able to stabilise biliary dysfunction. Tridax procumbens extract also showed protection against serum and liver lipid changes caused by d-GalN/LPS evidencing a broad spectrum of hepatoprotective property. Any liver disease will show an increased blood cholesterol level (Mc Intyre and Rosalki, 1992). The significant increase of cholesterol noted in this study might have been due to the inability of the diseased liver to remove cholesterol from circulation. This finding could be correlated with the results of the previous studies (Cartwright et al., 1982; Black et al., 1983). Hepatocellular damage due to alcohol, virus and drug induced hepatitis causes a modest hypertriglyceridemia (Glickman and Sebesin, 1982), which is due to the biochemical changes inferring with the transport of triglycerides out of liver. Our study also showed an increased accumulation of triglycerides in d-GalN/LPS induced rats which is in agreement with previous reports (Koff et al., 1971; Cartwright et al., 1982). The administration of endotoxic LPS resulted in an increase in the free fatty acid content in rats (Dhuley and Naik, 1998). Accumulation of free fatty acids is a consequence of changes in hepatic lipid metabolism. This is well correlated in our study with the increased levels of free fatty acids due to the administration of d-GalN/LPS, which is responsible for the increment of phospholipase A2 activity as a consequence of
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the high level of intracellular calcium causing the hydrolysis of liver membrane phospholipids and release of arachidonic acid (Kramer, 1993). This might account for the decreased levels of phospholipids observed in the serum and liver of d-GalN/LPS challenged rats. Rats pretreated with Tridax procumbens extract prior to the induction of hepatic damage showed a restoration of the altered lipid levels induced by d-GalN/LPS towards near normalcy thereby showing the modulating effect of Tridax procumbens extract against d-GalN/LPS-induced changes in the lipid levels in rats. This can be well correlated with the protective actions of picroliv (an active fraction from Picrorhiza kurroa), which showed lipid changes against hepatotoxicity induced by paracetamol and d-GalN (Ansari et al., 1991). Thus, the present study confirms the hepatoprotective action of Tridax procumbens against d-GalN/LPS-induced hepatitis in rats. The curative efficacy of Tridax procumbens extract was very promising as evidenced by the reversal of the altered values following administration probably by promoting regeneration of hepatocytes that restore integrity. The hepatoprotective action may be mediated through the inhibition of UDP-sugar derivatives, enhancement of glycoprotein biosynthesis and stabilisation of cell membrane and inhibition of lipid accumulation by its hypolipidemic property. The hepatoprotective property of the extract may be attributed to the presence of flavonoids which are present in the plant. Previous phytochemical investigations of Tridax procumbens described the isolation and structural determination of a flavonoid, procumbenetin (Ali et al., 2001). The inhibitory activity of this flavonoid in free radical production could be related to the hepatoprotective effect (Ravikumar et al., 2004). This indicates that the extract of Tridax procumbens may be used as an effective hepatoprotective agent.
Acknowledgement Dr. T. Devaki wishes to thank University Grants Commission–University With Potential For Excellence (UGC–UWPFE) for providing financial assistance to Mr. V. Ravikumar in the form of Junior Research Fellowship.
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