Hepatoprotective and antioxidant effect of Polygala chinensis L. whole plant against CCl4 induced hepatotoxicity in rats

Background and objectives: Carbon tetrachloride (CCl4) is one of the most commonly used hepatotoxins in the experimental study of liver diseases. Liver diseases are still a world wide health problem. Unfortunately, conventional and synthetic drugs used in the treatment of liver diseases are inadequate and sometimes can have serious side effects. There are numerous plants and polyherbal formulations claimed to have hepatoprotective activity. The ethanol extract of whole plant of Polygala chinensis protect the liver from CCl4 hepatotoxins.Methods: The CCl4 intoxicated adult male albino rats were treated with the ethanol extract of whole plant of Polygala chinensis in doses of 100 and 200 mg/kg orally for 14 days. The rats were sacrificed at the end of the 14 days. Biochemical parameters and antioxidant activities were carried out in the serum of control, CCl4 intoxicated and drug treated rats.Result: CCl4 intoxicated rats showed significant elevation in serum enzymes, bilurubin and lipid peroxidation of the liver tissues and reduction in serum total protein, superoxide dismutase, catalase, reduced glutathione and glutathione peroxidase activity. Treatment with ethanol extract of Polygala chinensis whole plant altered the above parameters to the levels of near normal. All the above results were comparable with the standard drug silymarin (100 mg/kg) treated group. Conclusion: The present study ascertains that the ethanol extract of Polygala chinensis whole plant possesses significant hepatoprotective activity.

bronchitis. In traditional Chinese medicine, Polygala is used for a variety of purposes including the promotion to sleep and calming the spirit. Polygala considered as a powerful tonic herb 3 that can help to develop the mind and aid in creative thinking. Biological activities such as antidiabetic, anti-inflammatory and antioxidant activities were reported 4,5,6 . However, so far there is no systematic study on hepatoprotective activity has been reported in the literature. Hence, the aim of the present study was to investigate the hepatoprotective activity of P. chinensis whole plant extract on CCl 4 induced liver toxicity in rats.

Plant material
The well grown whole plant of Polygala chinensis L. was collected from Vadavalli, Coimbatore, Tamil Nadu. The collected plants were identified by the Botanical Survey of India, Coimbatore. A voucher specimen (VOCB 1348)was retained in Ethnopharmacology unit, Research Department of Botany, V.O.Chidambaram College, Tuticorin for further reference.

2.2.Preparation of plant extracts for phytochemical screening and hepatoprotective studies
The whole plant was dried under shade and then powdered with a mechanical grinder to obtain a coarse powder, which was then subjected to extraction in a Soxhlet apparatus using ethanol. The extract was subjected to qualitative test for the identification of various phytochemical constituents as per standard procedures 7,8,9 . The ethanol extracts were concentrated in a rotary evaporator. The concentrated extract was weighed to calculate the yield of ethanol (10.20%). The concentrated ethanol extract were used for hepatoprotective studies.

2.3.Animals
Normal healthy male Wistar albino rats (180-240 g) were used for the present investigation. Animals were housed under standard environmental conditions at room temperature (25±2°C) and light and dark (12:12 h). Rats were fed with standard pellet diet (Goldmohur brand, MS Hindustan lever Ltd., Mumbai, India) and water ad libitum. Study was carried out as per IAFC approval no:82/PHARMA/SCRI, 2010.

2.4.Acute Toxicity Studies
Acute oral toxicity study was performed as per OECD-423 guidelines (acute toxic class method), albino rats (n=6) of either sex selected by random sampling were used for acute toxicity study 10 . The animals were kept fasting for overnight and provided only with water, after which the extracts were administered orally at 5mg/kg body weight by gastric intubations and observed for 14 days. If mortality was observed in two out of three animals, then the dose administered was assigned as toxic dose. If mortality was observed in one animal, then the same dose was repeated again to confirm the toxic dose. If mortality was not observed, the procedure was repeated for higher doses such as 50, 100 and 2000 mg/kg body weight.

2.7.Statistical Analysis
The data were expressed as the mean ± S.E.M. The difference among the means has been analyzed by one-way ANOVA. p<0.05and p<0.01 were considered as statistical significance using SPSS Software.

Results
The ethanol extract of whole plant of Polygala chinensis subjected for phytochemical study showed the presence of alkaloids, coumarin, glycosides, flavonoids, saponins, steroids, phenols, tannins and xanthoproteins. The ethanol extract did not show any sign and symptoms of toxicity and mortality upto 2000 mg/kg dose. The effect of ethanol extract of P. chinensis on serum total protein, albumin, globulin, A/G ratio, serum transaminases and alkaline phosphatases in CCl 4 intoxicated rats are summarized in Table 1. There was a significant (p< 0.01) increase in serum GOT, GPT and ALP levels in CCl 4 intoxicated group (Group II) compared to the normal control group (Group I). The total protein and albumin levels were significantly (p< 0.01) decreased to 6.18 g/dl and 3.28 g/dl in CCl 4 intoxicated rats from the levels of 7.98 g/dl and 4.88 g/dl respectively in normal group. Ethanol extract of P. chinensis whole plant at the dose of 100 and 200 mg/Kg orally significantly decreased the elevated serum marker enzymes and reversed the altered total protein and albumin to almost normal level. The effect of ethanol extract of P. chinensis on total, conjugated and unconjugated bilirubin is shown in Table 2.  (11) www.ssjournals.com A significant elevation of total, conjugated and unconjugated bilirubin in the serum of CCl 4 intoxicated group (Group II) when compared to normal control (Group I). The ethanol extract of P. chinensis at the dose 100 and 200 mg/kg reduced the levels of total, conjugated and unconjugated bilirubin (Group III and Group IV). The decreases in the concentration of total bilirubin, conjugated bilirubin and unconjugated bilirubin were found to be greater in standard silymarin (Group V) followed by Group IV and Group III ( Table 2).
Lipid peroxidation level was significantly (p<0.01) increased and glutathione peroxidase, glutathione reductase, superoxide dismutase and catalase activity were significantly (p< 0.01) decreased in CCl 4 intoxicated rats when compared with those of the animals in normal control group. Rats treated with ethanol extract of P. chinensis at the doses of 100 and 200 mg/kg significantly decreased the elevated lipid peroxidation levels and restored the altered glutathione peroxidase, glutathione reductase, superoxide dismutase and catalase levels towards the normal levels in a dose dependent manner. The results are well comparable with silymarin (standard drug) treated group.

Discussion
It is well established that CCl 4 induces hepatotoxicity by metabolic activation; therefore it selectively causes toxicity in liver cells maintaining semi-normal metabolic function. CCl 4 is bio-transformed by the cytochrome P 450 system in the endoplasmic reticulum to produce trichloromethyl free radical (CCl 3 ). Trichloromethyl free radical then combined with cellular lipids and proteins in the presence of oxygen to form a trichloromethylperoxyl radical, which may attack lipids on the membrane of endoplasmic reticulum faster than trichloromethyl free radical. Thus, trichloromethyl free radicals lead to elicit lipid peroxidation, the destruction of Ca 2+ homeostasis and finally, results in cell death 17,18 . These results in changes of structures of the endoplasmic reticulum and other membrane, loss of enzyme, metabolic enzyme activation, reduction of protein synthesis and loss of glucose-6-phosphate activation, leading to liver damage 19,20 . Hepatotoxic compounds like CCl 4 are known to cause marked elevation in serum enzyme activities. In the present study, treatment with P. chinensis whole plant extract attenuated the increase in the activities of SGOT, SGPT and ALP produced by CCl 4 indicating that Polygala chinensis whole plant extract protects liver injury induced by CCl 4 towards normalization. Silymarin, a prototype hepatoprotective agent also showed similar changes.
Bilirubin is the main bile pigment that is form the breakdown of heme in the red blood cells. It is transported to the liver where it is secreted by the liver into the bile. Conjugation of bilirubin is a prerequisite for its excretion into the bile 21 . Malfunctioning of the liver was evidenced by the significant increase (p< 0.01) in the level of unconjugated bilirubin in the serum of the group treated with only CCl 4 when compared to normal control. Increase in the level of unconjugated bilirubin in the blood may result from a defect in the function of the liver to conjugate the bilirubin being produced. The significant reduction (p< 0.05) of unconjugated bilirubin level in the serum when CCl 4 was simultaneously administrated with the ethanol extract of P. chinensis when compared with the administration of CCl 4 alone indicates that the conjugating function of the liver was improved. The reduction of the unconjugated bilirubin level by the ethanol extract suggest that the extracts may activate the constitutive androstance receptor (CAR) which is a key regulator in bilirubin clearance in the liver 22 . The primary function of CAR is the bilirubin clearance pathway is to direct cooridnate response to elevated levels of bilirubin by increasing the hepatic expressive of each component of the pathway 23 .
The ability of simultaneous administration of CCl 4 with ethanol extract of P. chinensis to significantly reduce (p< 0.01) the level of serum total bilirubin when compared with that of the CCl 4 treated group suggests the potential of the extract is clearing bilirubin from the serum when its level elevated. Since the results obtained for the serum total protein and albumin concentrations followed the same trend, it thus implicated the same mechanism by which the ethanol extract of P. chinensis exerts its effect on these parameters. The administration of CCl 4 alone may adversely interfere with protein metabolism probably by inhibiting the synthesis of proteins. Administration of ethanol extract of P. chinensis whole plant reversed these changes may be by increasing protein synthesis. This indicates the hepatoprotective activity of P. chinensis whole plant against damage by CCl 4. Stimulation of protein synthesis has been advanced as a contributory hepatoprotective mechanism which accelerates regeneration of cells 24 .
Intake of CCl 4 results in excessive generation of free radicals. Free radicals are the reactive oxygen species (ROS) which are known to cause oxidative damage to number of molecule in cell, including membrane-lipids, proteins and nucleic acids 25 . In the present study the hepatic cellular injury might be due to increased oxidative stress and thereby leading to lipid peroxidation. The level of lipid peroxidation in the CCl 4 treated rats was assessed by measuring the levels of TBARS in the liver tissues 26 .
The increased TBARS levels in the liver of CCl 4 treated animals indicate enhanced lipid peroxidation leading to tissue injury. The cellular antioxidant defense mechanism, which includes scavenging activities of enzymes viz., SOD, CAT and GPx plays an important role in scavenging toxic intermediates of reactive oxygen species. During hepatotoxicity these enzymes might be functionally impaired due to excess generation of free radicals creating oxidative imbalance.
SOD is metalloprotein catalyzing the dismutation of superoxide anion to hydrogen and oxygen. Numerous studies have shown the importance of SOD in protecting cells against oxidative stress 27 . The SOD activity could be decreased in tissue during CCl 4 injection. This decrease could be due to the feedback inhibition or oxidative inactivation of enzyme protein due to excess ROS generation 28 .
CAT, hemeprotein, catalyzes the reduction of hydrogen peroxides 29 , acts as preventive antioxidant and plays an important role in protection against the deleterious effects of lipid peroxidation 30 .The activity levels of catalase in tissue decreased in CCl 4 treated animals might be due to the inhibition of CAT activity, which is suggestive to enhanced synthesis of O 2 is a powerful inhibitor of catalase 31 .
GPx is an enzyme with selenium in the form of selenocysteine and can catalyze the reduction of hydrogen peroxide and hydroperoxidases to non toxic products: GPx has a well-established role in protecting cells against oxidative injury. GPx is non-specific for H 2 O 2 and lipid peroxide generated during CCl 4 treatment which is efficiently scavenged by GPx activity. The depression of this enzyme activity reflects perturbations in normal oxidative mechanism during CCl 4 treatment.
The cellular antioxidant defense enzymes viz., SOD, CAT and GPx were significantly reduced in the CCl 4 treated rats. This might lead to decreased antioxidant defense and increased oxidative stress and thereby the tissue injury occurs. Similar studies also indicate the failure of cellular antioxidant defense system during hepatotoxicity was recorded 32,33 .
In conclusion, the results of this study demonstrate that the ethanol extract of Polygala chinensis whole plant have a potent hepatoprotective action aqueous CCl 4 induced hepatic damage in rats. Its mode in affording the hepatoprotective activity against CCl 4 induced liver damage may be due to cell membrane stabilization, hepatic cells regeneration and enhancement of antioxidant enzymes such as catalase, superoxide dismutase and glutathione peroxidase production. The hepatoprotective and antioxidant potential of whole plant extract could have been brought about by various phytochemical principles i.e. flavonoids, alkaloids, phenolics and tannins present in P. chinensis whole plant. So results of this study demonstrated that the P. chinensis has significantly protection on CCl 4 induced hepatotoxicity.