Hepatoprotective studies on methanolic extract of whole plant of Lindernia ciliata

25 C; 35-60% of relative humidity). The animals standard rat pellet diet water libutum Abstract Lindernia ciliata is traditionally used in the treatment of jaundice. In the present study, the methanolic extract of L. ciliata was evaluated for its hepatoprotective activity in paracetamol, ethanol and D-galactosamine intoxicated rats. The activity of the extract was assessed on the basis of improvement in the altered level of various serum biochemical parameters and in the changes occurred in the histology of liver of the rats. The extract was also investigated for its antioxidant potential by employing different in vitro methods. Among the three test doses of extract, 200 mg/kg was found to be the most effective dose. The significant (p<0.01) hepatoprotective and antioxidant activities exhibited by the extract, in different in vivo models and in vitro studies respectively may be attributed to the flavonoids and phenolic compounds present in the extract.

Acute toxicity study: Acute toxicity study was carried out for methanolic extract of L.ciliata according to the Organization for Economic Co-operation and Development (OECD) 420 guidelines (OECD, 2001). All animals were observed for toxic symptoms and mortality for 72 hours.
Total phenolic content: The total phenolic content of the extract was determined using the Folin-Ciocalteu colorimetric method as described elsewhere (Marinova et al., 2005). The extract (100-1,000 µg/mL) or standard solution of gallic acid (10-100 µg/mL) was added to 25 mL volumetric flask containing 9 mL of distilled deionised water. A reagent blank was prepared using distilled water instead of sample. One milliliter of Folin -Ciocalteus phenol reagent was added to the mixture and shaken. After 5 min, 10 mL of 7% aqueous sodium carbonate was added to the mixture. The solution was diluted to 25 mL with dd H2O and mixed. After incubition for 90 min at room temperature, the absorption against prepared reagent blank was determined at 760 nm using UV-Visible spectrophotometer. Quantification was done with respect to the standard gallic acid and expressed as gallic acid equivalents (GAE) in mg per gram of extract.
Total flavonoid content: The total flavonoid content of the extract was measured using the aluminum chloride colorimetric method as described in the literature (Marinova et al., 2005). The extract (100-1000 µg/mL) or standard solution of rutin (10-100 µg/mL) was added to 10 mL volumetric flask, containing 4 mL of double distilled H2O. To the flask 0.3 mL of 5%sodium nitrite solution was added. After 5 min, 0.3 mL of 10% aluminum chloride solution was added. At 6 th minute 2 mL of 1M NaOH was added and the total volume was made up to 10 mL with dd H2O. The solution was mixed well and the absorbance was measured against prepared reagent blank at 510 nm. The total flavonoid content was expressed as rutin equivalents in mg per gram of extract.

Paracetamol-induced hepatotoxicity in rats:
The experiment was performed according to the method given in the literature with minor modifications (Gini et al., 2010). The rats were divided into six groups comprising six in each. 2%gum acacia was used as vehicle for suspending the standard drug and the extract. Group I was kept as control received single daily dose of vehicle (2% gum acacia 1 mL/kg) for seven days. Groups II, III, IV, V and VI were given orally daily dose of vehicle (2%gum acacia 1 mL/kg), silymarin (100 mg/kg), extract (100 mg/kg; 200 mg/kg and 400 mg/kg) once a day for seven days respectively. On 8 th day a dose of paracetamol (3 g/kg) was administered to the animals of all groups leaving Group I. Then blood and liver samples were collected from the animals of all groups 24 hours after administration of paracetamol for estimation of various biochemical parameters and histopathological studies respectively.

Ethanol-induced hepatotoxicity in rats:
The protective effect of extract against ethanol induced liver damage was done according to the procedure given in the literature with minor modifications (Zhen-Ming Lu et al., 2007). The rats were divided into six groups and pretreated for ten days with single daily dose of vehicle, silymarin, and the extract in different doses as described in paracetamol induced hepatotoxicity experiment. On 10 th day one hour after the daily treatment, the animals of all the groups except group I, intoxicated with an acute oral dose of ethanol (5 g/kg) diluted with distilled water (6:4 v/v). The blood and liver samples were collected 18hr after administration of ethanol under ether anesthesia, for quantification of various biochemical parameters and histopathological studies respectively.

Assessment of antihepatotoxic activity of effective dose of extract against D-GaIN induced hepatotoxicity in rats:
The antihepatotoxic activity of extract against D-GaIN induced hepatotoxicity was carried out according to the procedure given in the literature with minor modifications (Karan et al., 1999). The rats were randomly divided into four groups of six animals each. Group I served as normal and received the vehicle (1 mL/kg orally of 2% gum acacia in water) for 3 days. On the first day, D-GaIN (400 mg/kg i.p) was given to groups II, III and IV. Vehicle (2%gum acacia 1 mL/kg), silymarin (100 mg/kg) and EXTRACT (200 mg/kg) were given to the animals of Groups II, III and IV respectively for three times at the time point of 2 hours, 24 hours, 48 hours after the administration of D-GaIN. The blood and liver samples were collected from the animals 1 hour after the last treatment for estimation of various biochemical parameters and histopathological studies respectively. (Yanling et al., 2008):

Determination of prothrombin time
The prothrombin time was determined by collecting blood in normal capillary tubes and breaking it into pieces until a thread is observed. Time was noted between the collections of blood to the appearance of thread.
Histological studies: Histological studies were done by staining the fine section of liver isolates and examining under the microscope. The liver samples collected from the rats of the study were washed with normal saline (0.9%). Then, 2-3 pieces of approximately 6 cm size were cut and fixed in phosphate buffered 10% formaldehyde solution. After embedding in paraffin wax, thin sections of 5 mm thickness of liver tissue were cut and stained with hemotoxylin-eosin stain.
In vitro antioxidant studies on extract DPPH radical scavenging assay: This method was performed as described in the literature with minor modifications (Blios et al., 1958). 1 mL of 0.1 mM solution of DPPH in methanol was added to 2.5 mL of the test extract in methanol (10-100 µg/mL). The reaction mixture was then allowed to stand at room temperature in a dark chamber for 30 min. After 30 min, absorbance was measured at 517 nm using UV-Visible spectrophotometer. Rutin was used as a standard. The scavenging activity of DPPH radical (%) was calculated from the following equation: Lower absorbance of the reaction mixture indicates higher free radical scavenging activity.
Superoxide scavenging activity: The procedure was done according to the method described in the literature with slight modification (Nishikimi et al., 1972) Nitric oxide scavenging activity: The assay was carried out according to the reported method (Garratt et al., 1964). 1.0 mL each of 10 mM sodium nitroprusside, phosphate buffered saline (pH 7.4) and various concentrations (100-1,000 μg/mL) of the test extracts were mixed. The mixture was incubated for 150 min at 25°C. To 0.5 mL of the incubated solution, 1 mL sulfanilamide (0.33% in 20% glacial acetic acid) was added and allowed to stand for 5 min. Then 1 mL of napthylethylenediamine dihydrochloride (NED) (0.1% w/v) was added and the mixture was incubated for 30 min at 25°C. The pink chromophore generated was measured spectrophotometrically at 540 nm against a blank sample. Ascorbic acid was used as standard.
Reducing power assay: The assay was performed according to the reported method (Oyazu et al., 1986). Stock solutions were prepared by dissolving 10 mg of the extract in 1 mL of DMSO and working concentrations (10-100 µg/mL) were prepared in PBS (0.2 M). 2.5 mL of the test extract at different concentrations was mixed with 2.5 mL of 1%potassium ferricyanide. After incubiting the mixture at 50ºC for 20 min, 2.5 mL of 10%TCA was added and centrifuged at 1,036 rpm for 10 min. Then 2.5 mL of upper layer of reaction mixture was mixed with 2.5 mL of distilled water and 0.5 mL of 0.1% freshly prepared FeCl3 solution. The absorbance of the reaction mixture was measured at 700 nm. Ascorbic acid was used as positive control. Increased absorbance of the reaction mixture indicated increased reducing power.

Statistical analysis:
The data obtained were analyzed by one-way of variance (ANOVA) followed by Durnett all Vs control for the significant interrelation between the various groups using Graph pad prism-3 computer software. p<0.05 and p<0.01 were considered to be significant.

Results
The total flavonoid content in the methanolic extract of L.ciliata was found to be 75.5 ± 0.9 mg of rutin equivalents per gram of extract and the total phenollic content was 31.3 ± 1.9 mg of gallic acid equivalents/g of extract.
Paracetamol intoxication in normal rats significantly (p<0.01) elevated the level of hepatospecific enzymes (AST, ALT, ALP), TB, DB, LDH and decreased the level of TP, ALB in serum ( Table I). It indicates acute hepatocellular damage and biliary obstruction which was endorced by the histopathological examination of the liver sections of rats showing cetrilobular necrosis, dilatation of sinusoidal spaces, and bleeding in hepatic lobes (Figure 1). The rats treated with extract and silymarin showed a significant (p<0.01) protection, against paracetamol-induced hepatic damage by normalizing serum biochemical parameters and by minimizing the histopathological abnormalities. Among the test doses, percentage protection shown by extract at 200 mg/kg was well comparable to that of silymarin (100 mg/kg).
Ethanol intoxication in normal rats also caused significant (p<0.01) alteration in the level of the serum biochemical parameters (Table II) and changes in the histology of the liver of the rats showing kupffer cell proliferation, dilatation of sinusoidal space and bleeding in hepatic lobes (Figure 2). Treatment with standard and all test doses showed a significant (p<0.01) recovery from the ethanol induced hepatic damage which was evident from the reversal of alterations in the level of serum biochemical parameters and changes in histology of liver. The extract, at all test doses exhibited a phenomenal hepatoprotective activity. Among the test doses, percentage protection shown by extract at 200 mg/kg was well comparable to that of silymarin 100 mg/kg.
The results of the study are presented in Table III and histopathological photographs of liver section of .7 ± 1.9 a 100.7 ± 3.2 a 378.5 ± 8.9 a 0.3 ± 0.0 a 0.1 ± 0.0 a 8.2 ± 0.3 a 3.3 ± 0.1 a 183.7 ± 4.9 a 24.1 ± 5.5 a Extract 100 mg/kg 94.9 ± 2.2 a 153.3 ± 5.5 a 444.7 ± 5.1 a 0.5 ± 0.0 a 0.4 ± 0.0 a 6.7 ± 0.2 a 2.7 ± 0.0 a 224.1 ± 3.2 a 54.1 ± 2.5 a Extract 200 mg/kg 87 ± 2.6 a 128.8 ± 3.7 a 403 ± 3.9 a 0.5 ± 0.0 a 0.2 ± 0.0 a 7.5 ± 0.3 a 2.9 ± 0.2 a 198.6 ± 5.6 a 39.5 ± 4.5 a Extract 400 mg/kg 92.3 ± 2.1 a 139.4 ± 4.8 a 410.3 ± 5.9 a 0.5 ± 0.0 a 0.3 ± 0.0 a 7.3 ± 0.2 a 2.8 ± 0.1 a 207.9 ± 6.7 a 51.6 ± 6.1 a Data expressed as mean ± SD; n=6; values in parenthesis indicate percentage recovery. P value-Paracetamol Vs vehicle; P value Paracetamol Vs treatmentsa <0.01 rats of the study are shown in Figure 3. Extract at 200 mg/kg orally was evaluated in the study since the percentage protection offered by it was well comparable to that of reference drug, silymarin 100 mg/kg in hepatoprotective studies. D-GaIN intoxication in normal rats significantly (p<0.01) increased The test extract has shown a concentration dependent in vitro free radical scavenging activity. The IC50 of the extract and the standard are shown in Table  IV. The extract also showed a concentration dependent reducing power. The reducing power of the extract is expressed in terms of ascorbic acid equivalents and was found to be 44.7 ± 1.7 mg.

Discussion
In the present study, extract was investigated for its hepatoprotective potential against three mechanistically devised models (paracetamol, ethanol and Dgalactosamine) hepatotoxicity in rats. It is well known that overdoses of paracetamol are hepatotoxic. Drug induced liver injuries are wide spread and account for one half of the cases of acute liver fai -lure and mimics all forms of acute and chronic liver diseases (Kaplowitz et al., 2001). Similarly, alcohol abuse is another major health problem worldwide.
Administration of paracetamol or ethanol increased the levels of AST, ALT, ALP and LDH in serum indicating damage to the liver cell plasma membrane as these enzymes are normally present in cytoplasm. Treatment with extract at 100, 200 and 400 mg/kg significantly reduced the elevated level of these enzymes in paracetamol or ethanol treated rats. This reflects that the extract have some functions in preserving structural integrity of hepatocellular membrane thus prevented enzymes leakage into the blood circulation. Besides various normal functions, liver excretes the breakdown product of hemoglobin namely bilirubin into bile. Administration of the necrotizing agents like paracetamol or ethanol produ -ces sufficient injury to hepatic parenchyma to cause large increase in bilirubin content (Dufour et al., 2000). Pretreatment with extract at all the test doses significantly (p<0.01) decreased their levels and are comparable with that of silymarin (100 mg/kg). In paracetamol or ethanol-induced hepatotoxicity, a decrease in TP and albumin occurs due to the disruption and dissociation of polyribosomes on endoplasmic reticulum leading to defective protein 15.6 ± 2.5 199.9 ± 4.4 263.1 ± 6.2 1282.6 ± 11.6 2.7 ± 0.2 1.8 ± 0.1 4.2 ± 0.3 1.4 ± 0.1 373.2 ± 6.3 157.1 ± 5.4 Standard 64.3 ± 3.5 a 81.5 ± 2.0 a 87.8 ± 2.1 a 537.8 ± 13.1 a 0.6 ± 0.0 a 0.2 ± 0.0 a 8.0 ± 0.2 a 3.3 ± 0.2 a 227.0 ± 5.6 a 29.8 ± 4.4 a LC 200 50.3 ± 1.5 a 88.6 ± 3.6 a 96.9 ± 7.2 a 704.5 ± 21.5 a 0.8 ± 0.0 a 0.1 ± 0.0 a 8.0 ± 0.3 a 3.2 ± 0.2 a 242.5 ± 4.2 a 40.1 ± 4.4 a Data expressed as mean ± SD, n=6, values in parenthesis indicate percentage recovery. P value -D-galactosamine Vs vehicle; P value Dgalactosamine Vs treatmentsa <0.01 biosynthesis. extract at 100, 200, 400 mg/kg increaseed the serum TP and ALB levels with varying degree of significance. This may be due to promotion of the assembly of ribosomes on endoplasmic reticulum to facilitate uninterrupted protein biosynthesis. Prothrombin or factor II is one of the clotting factors made by the liver. In paracetamol or ethanol intoxicated rats the PT was drastically increased which may be attributed to decreased synthesis of clotting factors. Recovery of PT after administration of test extracts provides an index of recovery of synthetic capacity of the damaged liver.
The hepatoprotective activity of extract was further verified by histopathalogical observations. It is well documented that over doses of paracetamol or ethanol leads to drastic alterations in histoarchitecture of liver showing centrilobular necrosis, fatty changes, dilation of sinusoidal spaces, kupffer cell proliferation, ballooning degeneration and bleeding area in hepatic lobes. The extract at all test doses showed a definite sign of protection and recovery against injury. Of the three test doses, extract at 200 mg/kg exhibited significant liver protection against the toxicants as evident from the reversal of changes in biochemical parameters and histoarchitecture of liver.
The hepatotoxicity induced by D-galactosamine resembles that of human viral hepatitis both in metabolic and morphological aberrations that always caused periportal necro inflammation and hepatocyte apoptosis (Keppler et al., 1968, Decker et al., 1974, Katunnama et al., 2006. D-GaIN has been known to cause hepatotoxicity by the accumulation of UDP-GaIN derivatives in the liver followed by a depletion of hepatic UTP, resulting in the inhibition of mRNA and proteins (Aniya et al., 2005). Treatment with extract 200 mg/kg body weight significantly decreased the serum ALT, AST, LDH, ALP, TB and DB levels suggesting that L. ciliata might scavenge reactive oxygen species generated from D-GaIN intoxication and hence prevent hepatic cellular enzymes and bilirubin from leaking into the blood (Dufour et al., 2000). The extract also showed a remarkable recovery in PT and serum level of TP and ALB. Treatment with extract 200 mg/kg exhibited significant liver protection, by normalizing the histological architecture of liver of the rats, which was almost similar to that of silymarin (100 mg/kg).
Oxidative stress can be increased either by increase in free radical production in the body or by decrease in endogenous antioxidants. As so many free radicals can contribute to oxidative stress, individual assessment of susceptibility becomes important. Oxidative stress plays an important role in the pathogenesis of many liver diseases (Tanikawa and Torimura, 2006). In view of that, extract was systematically evaluated for its antioxidant ability using different in vitro methods. DPPH is a stable free radical, when antioxidant reacts with DPPH• the electron is paired off and the DPPH solution is decolorized (Bhagat, 2011). Nitric oxide plays a vital role in various inflammatory processes. Higher levels of these radical are toxic to tissue and contribute to the vascular collapse (Rajan et al., 2011). The hydroxyl radical is an extremely reactive free radical formed in biological systems and has been implicated as a highly damaging species in free radical pathology, capable of damaging almost every molecule found in living cells viz., DNA, lipids and proteins (Hochestein, 1988). Superoxide anion is also very harmful to cellular components and produced from molecular oxygen due to oxidative enzyme of body as well as via non-enzymatic reaction such as autoxidation by catecholamine's (Naskar et al., 2010).
The methanolic extract of L. ciliata has shown a concentration dependent DPPH, nitric oxide, hydroxyl and superoxide radical scavenging activities. The extract also exhibited significant reducing power which ultimately adds to its antioxidant potential. The antioxidant activity may be attributed to the phenolic and flavonoidal compounds present in the extract.
In conclusion, the overall results of the study indicated that the three activities exhibited by the extract are complementing each other in giving protection to the liver either as prophylactic or curative and substantiated the traditional claim of the plant, L. ciliata in the treatment of jaundice.