Antioxidant and hepatoprotective activity of ethanolic and aqueous extracts of Limonia elephantum leaves

Limonia elephantum (wood apple) has been widely used in an Indian folk medi-cine system. In the present study, the acute toxicity studies were carried out to determine the safety of the compounds in liver diseases. The antioxidant and the hepatoprotective properties of the L. elephantum are evaluated against paracetamol induced hepatic damage in rats. Liver superoxide dismutase, lipid peroxidation, glutathione peroxidase, catalase levels and serum biochemical profile such as serum glutamate oxalate transaminase, serum glutamate pyruvate transaminase, alkaline phosphatase, bilirubin and total protein were examined for the antioxidant and hepatoprotective effects of these treatments. The results of the present studies strongly indicate that the higher dose (400 mg/kg) of ethanolic extract of L. elephantum proved against hepatoprotective effects and also the antioxidant properties.

using Soxhlet apparatus with methanol as a solvent. The methanolic extract were obtained through hot percolation process were subjected to solvent evaporation through vacuum distillation method to obtain dried methanolic extract.
The aqueous extract of leaves of L. elephantum was obtained by simple maceration process. In this process the marc obtained through hot continuous extraction process were utilized for the experimental purpose. The marc was weighed and placed into the maceration flask and added with sufficient quantity of water and macerate for 24 hours, during the process the first six hours the flask were subjected to gentle agitation and set aside for remaining 18 hours. After 24 hours the solvent was filtered and clarified to get clear filtrate. The clear filtrate was evaporating to obtain the dried extract. Both extracts were stored in desiccators, which was used for further experimental purpose.
Experimental animals: Male albino rats of Wistar strain weighing about 180 and 220 g were housed individually in polypropylene cages and fed on standard pellet diet (Hindustan liver). The animals were obtained from the animal house, Perundurai Medical College, Erode, Tamil Nadu, India. Water was given at libitum. The animals were maintained at standard laboratory conditions (temperature 24±2°C, relative humidity 30-70% and 1:1 dark and light cycle). Ethical clearances for the handling of experimental animals were obtained from Institutional Animal Ethical Committee (NCP/ IAEC/Ph.D/2010-001) for the present investigation (Wagh and Gangadhar, 2010).
Acute toxicity studies: Healthy adult male albino mice (18 -22 g) were subjected to acute toxicity studies as per guidelines (AOT 425) subjected by the organization for economic co-operation and development (OECD-2001). The selected animals were used to determine the dose. The animals were divided into seven groups of six in each. The animals were fasted over night prior to the acute experimental procedure. The Karber's method (Ghosh and Kale, 2005) was used to determine the dose; gum acacia (2% w/v) was used as vehicle to suspend the extracts and administered intraperitoneally. The control group was received 2 mL/kg of the vehicle intra peritoneally. The other group received the extract as test drug in one of the following doses-100, 200, 400, 800, 1,000, 2,000 and 3,000 mg/kg in a similar manner. Immediately after dosing, the animals were observed continuously for first four for behavior changes and for mortality at the end of 24 hours, 48 hours and 72 hours respectively. The toxicity studies shows that the ethanolic and aqueous extract of drug at minimum dose of 200 mg/kg onwards shows the reaction in experimental animals. However, no mortality was reported even after 72 hours. This indicates that the alcoholic extract was safe up to a single dose of 3 g/kg body weight.

Experimental design:
Paracetamol-induced hepatotoxicity in rats (acute model): Animals were randomized and divided into seven groups of six animals in each group. Group I served as untreated control and fed orally with normal saline 5 mL/kg body weight daily for seven days. Group II rats were similarly treated as Group I. Group III and IV were treated with 200 mg and 400 mg/kg body weight of the aqueous extract of L. elephantum and Group V and VI were treated with the ethanolic extract of L. elephantum orally daily for seven days respectively (Hiroshini et al., 1987). Animals of Group VII were fed with standard drug silymarin 25 mg/kg (Veereshwarayya and Thiruvengadam, 2004) p.o daily for seven days. On the seventh day, paracetamol suspension was given by oral route, in a dose of 750 mg/kg body weight to all rats except the rats in Group I. The biochemical parameters were estimated after an 18 hours fast following the last dose.
Biochemical studies: The blood was obtained from all animals by puncturing retro-orbital plexus. The blood samples were allowed to clot for 45 min at room temperature. Serum was separated by centrifugation at 2,500 rpm at 30°C for 15 min and utilized for the estimation of various biochemical parameters namely SGOT, SGPT (Retimen and Frankel, 1957) SALP (King and Armstrong, 1934), serum bilirubin (Malloy and Evelyn, 1937) and total protein (Gornall et al., 1949).
After collection of blood samples the rats in different groups were sacrificed and their livers were excised immediately and washed in ice cold normal saline, followed by 0.15 M Tris-HCl (pH 7.4) blotted dry and weighed. A 10% w/v of homogenate was prepared in 0.15 M Tris-HCl buffer and processed for the estimation of lipid peroxidation (Ohkawa et al., 1979). A part of homogenate after precipitating proteins with trichloroacetic acid was used for estimation of glutathione (Ellman, 1959). The rest of the homogenate was centrifuged at 1,500 rpm for 15 min at 40°C. The supernatant thus obtained was used for estimation of SOD and CAT activities (Kakkar et al., 1984;Aebi, 1974).

Serum hepatospecific markers:
Activities of serum glutamate oxaloacetate transaminase (SGOT) and serum glutamate pyruvate transaminase (SGPT) were estimated by the method of Retimen and Frankel (1957). 0.05 mL of serum with 0.25 mL of substrate (aspartate and α -ketoglutarate for SGOT; alanine and α-keto glutarate for SGPT, in phosphate buffer pH 7.4) was incubated for an one hour in case of SGOT and 30 min for SGPT. 0.25 mL of DNPH solution was added to arrest the reaction and kept for 20 min in room temperature. After incubation one mL of 0.4N NaOH was added and absorbance was read at 505 nm in uv-vis spectrophotometer. Activities were expressed as IU/L.
Based on the method of King and Armstrong (1934) alkaline phosphatase activity was assayed by using disodium phenyl phosphate as substrate. The color was developed and read at 510 nm in uv-vis spectrophotometer after 10 min. Activities of ALP was expressed as IU/L. Serum total bilirubin level was estimated based on the method of Malloy and Evelyn (1937). Diazotised sulphonilic acid (0.25 mL) reacts with bilirubin in diluted serum (0.1 mL serum + 0.9 mL distilled water) and forms purple colored azobilirubin, which was measured at 540 nm in uv-vis spectrophotometer. Activities of total bilirubin were expressed as mg/dL. Serum total protein level was estimated based on the method of Gornall et al. (1949). Biuret reagent (1.0 mL) reacts with serum (10 μL) and the color was developed and read at 578 nm in uv-vis spectrophotometer. Activities of total protein were expressed as mg/dL.

Assay of superoxide dismutase (SOD):
Superoxide dismutase (SOD) activity was determined by the method of Kakkar et al. (1984). The assay mixture were contained 0.1mL of sample, 1.2 mL of sodium pyrophosphate buffer (pH 8.3, 0.052M), 0.1 mL of phenazine methosulfate (186 μm), 0.3 mL of nitro blue tetrazolium (300 μm), 0.2 mL of NADH (750 μm). Reaction was started by addition of NADH. After incubation at 30°C for 90 sec, the reaction was stopped by the addition of 0.1 mL of glacial acetic acid. The reaction mixture was stirred vigorously with 4.0 mL of n-butanol. The mixture was allowed to stand for 10 min, centrifuged and butanol layer was separated. The color intensity of the chromogen in butanol layer was measured at 560 nm against nbutanol and concentration of SOD was expressed as units/g of liver tissue. Absorbance values were compared with a standard curve generated from known SOD.
Assay of catalase (CAT): Catalase was assayed according to the method of Aebi (1974). The estimation was done spectrophotometrically following the decrease in absorbance at 230 nm. The liver tissue was homogenized in M/150 phosphate buffer (pH 7.0) at 1-40°C and centrifuged at 5,000 rpm. The reaction mixture contained 0.01 M phosphate buffer (pH 7.0), 2 mM H2O2 and the enzyme extract. The specific activity of catalase was expressed in terms of units/gram of liver tissue. Absorbance values were compared with a standard curve generated from known CAT.

Assay of glutathione peroxidases (GPX):
Glutathione peroxidases activities (total and Se-dependent) were measured at 340 nm following NADPH oxidation in the presence of excess glutathione reductase, reduced glutathione and corresponding peroxide (Lawrence and Burk, 1976). Cumene hydroperoxide and H2O2 were used as substrates for the determination of total and Sedependent peroxidase activity. The difference in the absorbance per unit of time (the rate of blank reaction was subtracted from the total rate) was taken as the measure of GPx activity (C =−6.22 mM − 1 cm − 1 ). The GPx activities in the gills and mantle of B. azoricus are expressed as µmol/min/mg of total protein concentrations.
Lipid peroxidation: The method was used and designed to evaluate malondialdehyde (MDA) and 4-hydroxyalkenals (4-HNE) concentrations upon the decomposetion by polyunsaturated fatty acid peroxides (Erdelmeier et al., 1998). This procedure is based on the reaction of two moles of N-methyl-2-phenylindole, a chromogenic reagent, with one mole of either MDA or 4 -HNE at 45°C for 60 min to yield a stable chromophore that has a maximum absorbance at 586 nm. Therefore, 10 µL of 0.5M butylated hydroxytoluene, 650 µL of a mixture of 6 mL of methanol with 18 mL of 10.3 mM Nmethyl-2-phenylindole and 150 µL of 15.4M methanesulfonic acid were added to 200 µL of the first cytosolic fraction of the homogenate of the gills or mantle of B. azoricus and incubated at 45°C for 60 min.MDA + 4-HNE concentrations were estimated by measuring the maximum absorbance of the formed chromophore at 586 nm using malondialdehyde bis-tetrametoxypropan (SIGMA) as a standard. Lipid peroxidation is expressed as nmoles of MDA and 4-HNE g − 1 of total protein concentrations.
Statistical analysis: All the results were expressed as mean ± SEM. Statistical analysis was performed using GraphPad Prism version 5. The data were statistically analyzed by one way analysis of variance (ANOVA) between plant extract treated groups and disease control group. P values ˂0.05 were considered to be significant.

Results
The effects of L. elephantum on serum transaminase, alkaline phosphatase, bilirubin and total protein levels in paracetamol-induced liver damage in rats summaryzed in Table I. Administration of paracetamol (750 mg/ kg; body weight), after 18 hours of intoxication resulted a significant (p<0.05) elevation of hepatospecific serum markers SGOT, SGPT, SALP, bilirubin and total protein in paracetamol-treated group, in comparison with the normal control group. On administration of L. elephantum (Group III to VI) and silymarin at the dose of 25 mg/kg (Group VII) the level of these enzymes were found retrieve towards normal control.
The levels of SOD and CAT recorded a significant decline and the level of lipid peroxidation and GPx were increased in paracetamol treated animals when compared with control group of animals (Table I and II), while in L. elephantum treated rats the activities of these enzymes attained a near-normal control (p<0.05).

Discussion
The pretreatment with aqueous and ethanolic extract of L. elephantum, both at the dose of 200 mg/kg and 400 mg/kg, significantly attenuated the elevated levels of the serum markers. The normalization of serum markers by aqueous and ethanolic extract of L. elephantum suggests that they are able to condition the hepatocytes so as to protect the membrane integrity against paracetamol induced leakage of marker enzymes into the circulation. The above changes can be considered as an expression of the functional improvement of hepatocytes, which may be caused by an accelerated regeneration of parenchyma cells. Serum ALP and bilirubin levels, on the other hand are related to hepatic cell damage. Increase in serum level of ALP is due to increased synthesis in presence of increasing billiary pressure (Moss and Butterworth, 1974). Effective control of bilirubin level and alkaline phosphatase activity points towards an early improvement in the secretory mechanism of the hepatic cell.
Lipid peroxidation has been postulated to the destructive process of liver injury due to acetaminophen administration. In the present study, the elevations in the levels of end products of lipid peroxidation in the liver of rat treated with paracetamol were observed. The increase in malondialdehyde (MDA) levels in liver suggests enhanced lipid peroxidation leading to tissue damage and failure of antioxidant defense mechanisms to prevent formation of excessive free radicals.
Treatment with aqueous and ethanolic extract of L. elephantum significantly reversed these changes. Hence it may be possible that the mechanism of hepatoprotection by aqueous and ethanolic extract of L. elephantum is due to its antioxidant effect.
The non-enzymic antioxidant, glutathione is one of the most abundant tripeptides present in the liver. Its functions are mainly concerned with the removal of free radical species such as hydrogen peroxide, superoxide radicals, alkoxy radicals and maintenance of membrane protein thiols and as a substrate for glutathione peroxidase and GST (Prakash et al., 2001). In the present study the decreased level of GSH has been associated with an enhanced lipid peroxidation in paracetamol-treated rats. Administration of aqueous and ethanolic extract of L. elephantum significantly increased the level of glutathione in a dose-dependent manner.
The enzymatic antioxidant defense system is the nature protector against lipid peroxidation. SOD, CAT and GPx enzymes are important scavengers of superoxide ion and hydrogen peroxide. These enzymes prevent generation of hydroxyl radical and protect the cellular constituents from oxidative damage (Scott et al.,1991). In the present study, it was observed that the aqueous and ethanolic extract of L. elephantum significantly increased the hepatic SOD activity in paracetamol induced liver damage in rats. This shows that the aqueous and ethanolic extract of L. elephantum can reduce reactive free radicals that might lessen the oxidative stress to the tissues and improves the activity of hepatic antioxidant enzyme.
Catalase (CAT) is an enzymatic antioxidant widely distributed in all animal tissues and the highest activity is found in the red cells and in the liver. CAT decomposes hydrogen peroxide and protects the tissue from highly reactive hydroxyl radicals (Chance et al., 1952). Therefore the reduction in the activity of these enzymes may result in a number of deleterious effects due to the accumulation of superoxide radicals and hydrogen peroxide. Administration of aqueous and ethanolic extract of L. elephantum increased the activities of CAT in paracetamol-induced liver damage in rats to prevent the accumulation of excessive free radicals and protected the liver from paracetamol intoxication.
The observations in paracetamol-treated rats showed severe necrosis. This could be due to the formation of highly reactive radicals because of oxidative threat caused by paracetamol. All these changes were very much reduced in rats treated with aqueous and ethanolic extract of L. elephantum. Based on the above results, it could be concluded that aqueous and ethanolic extract of L. elephantum exerts significant hepatoprotection against paracetamol-induced toxicity.