Antioxidant and hepatoprotective effects of Lithocarpus polystachyus against carbon tetrachloride-induced injuries in rat

The present study aims to investigate the hepatoprotective and antioxidant effects of the total flavonoid of Lithocarpus polystachyus Rehd.(LP-F) in vitro and in vivo . The in vitro antioxidant property of total flavonoids was investigated by employing various established systems. Rats with carbon tetrachloride-induced liver injury were used to assess the hepatoprotective and antioxidant effect of total flavonoids in vivo . The level of activity of glutamate pyruvate transaminase, aspartate aminotransferase, alkaline phosphatase and gamma-glutamyl transpeptidase, total bilirubin, total cholesterol, triglycerides total protein and albumin contents in the serum and malondialdehyde, superoxide dismutase, catalase, and glutathione in the liver and kidney of the rats were assayed using standard procedures. The results showed the total flavonoids of L. polystachyus has strong hepatoprotective and antioxidant activity in vitro and in vivo . These data were supplemented with histopathological studies of rat liver sections. This suggests that the hepatoprotective activity of formulation is possibly attributed to its free radical scavenging properties.

L. polystachyus of family Fagaceae, is a shrub distributed widely in the mountainous area in southern China (Institute of Botany, 1972). Its tender leaves have been a traditional Chinese herb and been taken as a sweet tonic drink for several hundreds of years (China Pharmacopoeia Committee, 1999). In the course of seeking the sweet components in the leaves, the sweet compounds were identified phlorizin, phloretin, querce -tin, dihydrochalcone-2'--D-glucopyranoside, luteolin, and quercitrin , and their content in the leaves was up to around 10.6% (Li et al., 2008), L. polystachyus is claimed to possess multiple bioactivities, including hepatoprotective, anti-hypertensive, antioxidant, vasodilative, cerebrovascular, cardiovascular, hypoglycemic, and antiobesity activities (Dong et al., 2012;Hou et al., 2012;Liao et al., 1994;Yang et al., 2007). According to the Dictionary of Chinese Traditional Drugs, hepatoprotective activity is the major pharmacological effect of L. polystachyus but no scientific data has been reported for the hepatoprotective effect of this medicinal plant so far.
The present study was carried out to investigate the hepatoprotective effect and a possible underlying antioxidant activity of indifferent extracts of the leaves of L. polystachyus by assaying various marker enzymes, antioxidant enzymes in carbon tetrachloride-induced rats.

Materials and Methods
Plant materials: L. polystachyus was collected from the Huaihua county of the western region of Huna province, China in March, 2010 and authenticated by Prof. Xianjin Wu, Department of Bioscience, Huaihua University, where a voucher specimen has been deposited. The plant material was air dried indoor at room temperature before extraction.
Experimental animal: Male Wistar rats (220 ± 4.5 g) purchased from the animal house of Xiangya School of Medicine of Central South University. They were kept in departmental animal house in an environmenttally control room (25 ± 2°C), relative humidity 50 ± 5% RH and with 12 hours light and 12 hours darkness cycle. All the animals were free to standard laboratory feed and tap water before experiment. All the studies were performed in accordance with the guidelines for the care and use of laboratory animals, as adopted and promulgated by the Institutional Animal Care Committee.
Determination and preparation of total flavonoids of L. polystachyus: The extract from L. polystachyus was obtain -ed by ultrasound-assisted extraction with 70%ethanol for 45 min (Li et al., 2008), and then filtrated through filter paper The filtrate was evaporated till dryness under reduced pressure weighed, the yield of the extract 15.2%. The extract was solved with hot water, added into column loading-treated AB-8 macroreticular resin for adsorption for 12 h, and then washed with 20% ethanol respectively for getting rid of impurity. LP-F in the column was eluted with 60%ethanol, eluting solution was evaporated till dryness under reduced pressure and weighed, the yield of total flavonoids of L. polystachyus 8.74%.
Preparation of standard curve: A calibration rutin solution containing 200.8 µg/mL rutin was prepared with 70%ethanol. In all 0, 1, 2, 3, 4, 5 and 6 mL of calibration rutin solution was respectively added to seven 25 mL volumetric flasks; to each was added 6 mL of 70%ethanol and 1 mL 5%NaNO2. Each flask was shaken fully, mixed and then stand still for 6 min; each flask was added 1 mL 10%AlCl3 solution, shake them fully again, mixed and stand still 6 min subsequently, 10 mL 10%NaOH solution was added into each volumetric flask and dilution to the mark with 70% ethanol, shaken fully, mixed and then let it rest for 15 min, Its absorbency (A) was measured at 500 nm, 70% ethanol as a blank control. 1 mL of above the extraction was transferred to a 25 mL volumetric flask, to measure its absorbance (A). Using the methodology described above, we finally calculated its total flavones content according to a regression equation

In vitro antioxidant activity
Scavenging activity on DPPH radical: The radical scavenging activity of total flavonoids of L. polystachyus was determined according to our previous procedure (He et al., 2012). In brief, 3.0 mL of 0.1 mM DPPH in methanol solution was added to 1.0 mL of total flavonoids of L. polystachyus preparative solution at different concentrations. The mixture was stood for 20 min in the dark before the absorbance was measured with the visible spectrometer at 517 nm. The DPPH scavenging effect was calculated as follows: DPPH scavenging activity (%) = [1 − (Absorbance of sample− Absorbance of background)/Absorbance of blank] × 100.
Scavenging activity on ABTS radical: The ABTS radical cation scavenging activity of DDP was identified using a method (Re et al, 1999) with some modifications. ABTS was produced from the reaction of 7 mM of ABTS solution with 2.45 mM of potassium persulfate. The mixture was then kept in the dark at room temperature for 16 hours. At the time of use, the ABTS solution was diluted with ethanol to yield an absorbance of 0.70 ± 0.02 at 734 nm. Samples (0.2 mL) of various concentrations (0.2 to 1.0 mg/mL) were mixed with ABTS solutions (2 mL). After reaction at room temperature for 6 min, the absorbance at 734 nm was recorded. The ABTS scavenging effect was calculated based on the following formula: ABTS scavenging activity (%) = [1 − (Absorbance of sample− Absorbance of background)/ Absorbance of blank] × 100.
Scavenging activity on hydroxyl radical: The HO -scavenging activity of LP-F was measured by an improved Fenton-type reaction . In brief, 1.0 mL of LP-F, at different concentrations, was spiked with 1.0 mL of 6 mM FeSO4, 1.0 mL of 6 mM salicylic acid-ethanol and 1.0 mL of 6 mM H2O2 and incubated at 37°C for 1 h. BHT was used as positive control. The absorbance at 510 nm was measured with a visible spectrometer. The percentage of HO --scavenging activi ty of total flavonoids of L. polystachyus was calculated according to the following formula: Scavenging activity against HO -(%) = [1 − ( Absorbance of sample − Absor -bance of blank)/ Absorbance of control] × 100.
Scavenging activity on superoxide radical: The scavenging activity on O2 •of total flavonoids of L. polystachyus was determined as described in our previous study . 1.0 mL of Mm nitroblue tetrazolium, 1.0 mL of mM nicotinamide adenine diuncleotide hydrogen, 1.0 mL of different concentration sample solution and 0.4 mL of 3 mM phenazine methosulfate were successively added into test tube, the reaction mixture was incubated at ambient temperature for 5 min and the absorbance of the mixture solution was determined at 560 nm with a visible spectrometer. O2 •scavenging capability of total flavonoids of L. polystachyus was calculated according to the following formula:

In vivo studies
Carbon tetrachloride-induced hepatotoxicity: Rats (n=60) were divided into six groups consisting of 10 animals in each group. Group I served as normal group and received distilled water with 0.3%sodium carboxymethylcellulose (CMC-Na)(1 mL/kg body weight, p.o.) for 5 days, and olive oil (1 mL/kg bodyweight, s.c.) on days 2 and 3 (Jain et al., 2008). Group II served as control group and received 0.3% CMC-Na (1 mL/kg body weight, p.o.) for 5 days, and CCl4-olive oil (1:1, 2 mL/kg body weight, s.c.) on days 2 and 3. Group III was treated with the standard drug silymarin (100 mg/ kg body weight, p.o.) daily for 5 days, and also received CCl4-olive oil (1:1, 2 mL/kg body weight, s.c.) on days 2 and 3, 30 min after administration of silymarin. Groups IV-VI (test group animals) were administered orally a dose of 100, 200, and 400 mg/kg body weight of total flavonoids of L. polystachyus (p.o.), respectively, for 5 days; in addition, they received CCl4-olive oil (1:1, 2 mL/kg, s.c.) on days 2 and 3, 30 min after administration of total flavonoids of L. polystachyus. On day 6, animals were anesthetized, blood was collected and allowed to clot, and serum was separated for assessment of biochemical parameters. The rats were then sacrificed by bleeding, the liver and kidney were carefully dissected and cleaned of extraneous tissue, and part of the liver tissue was immediately transferred into 10%formalin for histopathological investigation.
Serum analysis for liver marker enzymes: Serum samples were assayed for alkaline phosphatase, glutamate pyruvate transaminase, aspartate aminotransferase, gammaglutamyl transpeptidase by assay kits which were obtained from the Institute of Biological Engineering of Nanjing Jiancheng (Nanjing, China).
Serum analysis for biochemical studies: Total bilirubin, total cholesterol, triglycerides total protein and albumin were estimated by assay kits which were obtained from the Institute of Biological Engineering of Nanjing Jiancheng (Nanjing, China).

Determinations of antioxidant activity in vivo:
The liver and kidney of superoxide dismutase, catalase, malondialdehyde and glutathion were determined by assay kits which were obtained from the Institute of Biological Engineering of Nanjing Jiancheng (Nanjing, China).
Histopathological studies: Liver tissues were fixed in 10% formalin for at least 24 h, embedded in paraffin, and cut into 5 µm thick sections in a rotary microtome. The sections were stained with hematoxylin-eosin dye and observed under a microscope (IX51, Olympus, Japan) to detect histopathological changes in the liver.
Statistical analysis: Data obtained from this work were analyzed statistically using Students' t-test and ANOVA (One-or Two-way) followed by a post test (Tukey-Kramer multiple comparison test). Differences between means will be considered significant at 5% level of significance (p<0.05).

Results
Total flavonoids of L. polystachyus was extracted from L. polystachyus with 70%ethanol and purified with AB-8 macroreticular resin, dried under reduced pressure and weighed, the yield of 8.7%. Regression equation: A= 10.014C−0.007, r = 0.9998, C (concentration), with a better linear correlation between 7.9 and 54.4 μg/mL. Quantitative analysis indicated that the content of total flavonoids of L. polystachyus was up to 76.8%.

As depicted in
The levels of serum total bilirubin, total cholesterol, triglycerides, total protein and albumin were estimated and the results were given in Table II. Administration of CCl4 in rats caused a significant increase (p<0.05) in serum total bilirubin, total cholesterol, triglycerides and albumin levels, ranging from 0.6 ± 0.9 mg/dL , 68.6 ± 5.6 mg/dL , 32.6 ± 1.0 mg/dL, 8.2 ± 0.7 and 1.0 ± 0.1 g/ dL for the normal control rats to 1.9 ± 0.2 mg/dL, 113.6 ± 6.6 mg/dL, 79.4 ± 2.2 mg/dL, 4.9 ± 0.1 and 2.7 ± 0.0 g/ dL, while caused a significant decrease (p<0.05) in serum total protein ranging from 8.2 ± 0.7 g/dL for the normal rats to 4.9 ± 0.1 g/dL respectively. However, the protective administration of indifferent dose of LP-F decreased the CCl4-elevated levels of serum total bilirubin, total cholesterol, triglycerides and albumin (p<0.05), and silymarin as positive control also exhibited a similar effect. While increased the CCl4elevated levels of serum total protein.
Determinations of the in vivo antioxidant activities of total flavonoids of L. polystachyus in liver and kidney are presented in Figure 2. Compared with the normal group, the CCl4-intoxicated rats exhibited a significant decrease in total superoxide dismutase, catalase and glutathione (liver and kidney) (p<0.001), significant increase in the level of malondialdehyde (in liver and kidney)(p<0.001).These changes were significantly reversed after treatment with LP-F and silymarin.
Histopathological observations of liver sections from the control group showed normal cellular architecture with distinct hepatic cells, sinusoidal spaces and a Values are expressed as mean ± SEM, n=10; a p<0.001 considered statistically significant as compared to normal control group; b p<0.001, c p<0.05 considered statistically significant as compared to carbon tetrachloride treated group Values are expressed as mean ± SEM, n=10; a p<0.001 considered statistically significant as compared to normal control group; b p<0.001, c p<0.05 considered statistically significant as compared to carbon tetrachloride treated group central vein (Figure 3a). In contrast, the CCl4 group revealed the most severe damage of any of the groups; the liver sections showed massive fatty changes, necrosis, ballooning degeneration, broad infiltration of lymphocytes, and the loss of cellular boundaries (Figure 3b). The liver sections of the rats treated with total flavonoids of L. polystachyus (Figure 3e-f) showed a more or less normal lobular pattern with a mild degree of fatty change, necrosis, and lymphocyte infiltration almost comparable to the control (Figure 3a) and the silymarin-treated group (Figure 3c).

Discussion
The assays for scavenging DPPH, ABTS, HOand O2 • -showed that total flavonoids of L. polystachyus had a concentration-dependent antioxidant effect in vitro, and this effect was further verified by testing the antioxidant potential of total flavonoids of L. polystachyus in an animal model with experimental CCl4 -induced oxidative stress.
In this study, elevated levels of liver marker enzymes such as glutamate pyruvate transaminase, aspartate aminotransferase, alkaline phosphatase and gammaglutamyl transpeptidase in serum are obtained with CCl4 treatment indicating the pathophysiology of liver. Generally, CCl4 is metabolized by the liver in to highly reactive metabolites which either directly or indirectly cause lipid peroxidation of the hepatocytes. Different cytosolic liver marker enzymes would then leaked out Values are expressed as mean ± SEM; n=10; a p<0.001 considered statistically significant as compared to normal control group; b p<0.001, c p<0.05 considered statistically significant as compared to carbon tetrachloride treated group; Group I: Normal, Group II: control, Group III: silymarin 100 (mg/kg), Group IV: total flavonoid of L. polystachyus 100 (mg/kg), Group V: total flavonoid of L. polystachyus 200 (mg/kg), Group VI: total flavonoid of L. polystachyus 400 (mg/kg).
from these swollen and necrotic hepatocytes in to blood circulation and evidently elevated levels are obtained that is associated with the massive centrilobular necrosis, ballooning, degeneration and cellular infiltration of the liver (Singh et al., 2008). Treatment of rats by both CCl4 and the LP-F/silymarin reversed the increase in liver marker enzymes towards the control group and hepatic lesions are also minimized ( Table I).
It is well known that the liver play a pivotal role in the regulation of various chemicals. Administration of CCl4 causes hepathopathy as indicated by elevation in serum level of total bilirubin, total cholesterol, triglycerides and albumin whereas decreases total protein in rat. These pathological changes signify the potential damage in hepatic induced with CCl4 treatment. Treatment of rat with CCl4 and with either LP-F or silymarin ameliorated the toxic effects of CCl4 and restored the level of above biochemicals towards the normal group in accordance with other findings (Lin et al., 2008).
Carbon tetrachloride is accumulated in hepatic parenchyma cells and metabolized to the CCl3 • (Recknagel, 1983). CCl3 • radical reacts very rapidly with oxygen to yield a highly reactive CCl3OO • . These radicals react with proteins and lipids. They remove hydrogen atoms from unsaturated lipids thus initiating lipid peroxidation, which causes loss of integrity of cell membranes and damage to hepatic tissue . The enzymes superoxide dismutase, catalase and glutathione are key antioxidant enzymes that protect against oxidative stress and tissue damage (Halliwell and Gutteridge, 1990).
These enzymes are critical for defence mechanisms against the harmful effects of reactive oxygen species (ROS) and free radicals in biological systems. The superoxide dismutase converts superoxide radicals into H2O2 and O2, thus participating with other antioxidant enzymes, in the enzymatic defense against oxygen toxicity. Catalase is a key component of the antioxidant defense system. Inhibition of this protective mechanism results in enhanced sensitivity to free radical induced cellular damage. The decrease of catalase may result in a lot of deleterious effects due to the accumulation of superoxide radicals and hydrogen peroxide (Srinivasan et al., 2007). glutathione content was another important parameter that revealed oxidative damage in both liver and kidney. glutathione constitutes the first line of defense against free radicals. Reduction in liver glutathione activity in carbon tetrachloride-treated rats as observed in this study indicates the damage to the hepatic cells. malondialdehyde is a cytotoxic product that is a hallmark of lipid peroxidation. The fact that LP -F treatment reduced elevated malondialdehyde and increased levels of superoxide dismutase, catalase and glutathione, indicated that it prevent the peroxidation of lipids by carbon tetrachloride.
In conclusion, L. polystachyus possessed strong hepatoprotective and antioxidant activity in a rat model of CCl4-induced. The hepatoprotective activity may be due to its free radical-scavenging and antioxidant activity, resulting from the presence of some flavonoids in the extracts.

Contribution
First two authors contributed equally in this study.