Hepatoprotective Effect Of Polyphenols From Phyllanthus Emblica L.against Alcohol-induced Oxidative Damage

In recent years,People's health are threatened by alcoholic liver injury,and the antioxidants that antagonize oxidative stress of alcohol became the focus of prevention and treatment of ALD.Phyllanthus emblica L.?PE?is a good source of polyphenols and has been reported to have significant antioxidant activity,as a traditional medicine food homologous plant.The dry PE fruit were used to treat liver injury.The liver protective activity of polyphenols from PE was also reported in many literatures.In this paper,the polyphenol active components of PE were studied from the following aspects:1.Polyphenols from PE?PPE?were extracted by solvent extraction,the content of polyphenols was determined by the Folin–Ciocalteu method,and total phenolic content was taken as the index.The effect of extraction time,extraction temperature and ethanol concentration on the extraction of polyphenols were determined by single factor experiment,and extracting solution was optimized by orthogonal test.The results showed that the content of gallic acid had a good linear relationship between 1 and 5 mg/m L,and the linear regression equation was y=0.1431x+0.0791?r=0.9997?.The suitable extraction scheme is:the dry powder of PE was extracted with 45%etanol on a rotary shaker at 70°C for 135 min,and the total phenolic content 0.24 mg GAE/mg under the condition.2.HPLC analysis was conducted to quantify the main compounds of PE from different origins,using corilagin,gallic acid and ellagic acid as references standard.The PE extract was chromatographed on the Inertsil ODS C₁₈column?300×7.8 mm?with acetonitrile/0.1%trifluoroacetic acid at 25?for 30 min,with the injection volume of 10?L.The peaks of all compounds were recorded at 254 nm,with the flow rate of 1.0 m L/min.The results showed that the HPLC method has good separation effect,instrument precision,good stability,high repeatability and recovery.The order of the contents of GA,CO and EA in PE was Zhangzhou>Liangshan>Chaoshan>Kunming>Yulin.3.The antioxidant activities of PPE were determined by total reduction force,scavenging assays of DPPH radicals and OH radicals,inhibition activity of spontaneous hepatic lipid oxidation,respectively,and the correlation between antioxidant activities and active substance content was studied.The Kunming sample had the highest reducing power.The Liangshan sample was found to have the strongest antioxidant activities in scavenging DPPH radicals and hydroxyl radicals,with the EC₅₀value of 7.46?g/m L and0.44mg/m L,as well as a significant activity against spontaneous hepatic lipid peroxidation.The markedly correlation coefficients were existed between the phenolic content?GA,CO and EA?of PE and antioxidant activity.Linear regression analysis exhibited a significant correlation between the content of CO and DPPH radical scavenging activity?r=0.964?.The good correlation were also observed between the content of EA and DPPH radical scavenging activity?r=0.933?,and between content of GA and lipid oxidation inhibition activity?r=0.895?.Liangshan sample can be used as the material for further research because of its high content of PPE and prominent antioxidant activity.4.The protective effects of polyphenols from Phyllanthus emblica L.against ethanol-induced oxidative damage in rats liver cell was investigated,and the molecular mechanisms was further revealed by examining the Nrf2/HO-1 pathway.BRL-3A mice were used to evaluate the hepatoprotective effect of PPE on liver injury induced by ethanol exposure.The biochemical parameters?MDA,SOD,ALT,AST and GSH?were measured using commercial kits.HO-1 m RNA level was determined by Q-PCR.Protein levels were measured by western blot.A decreased amount of MDA,ALT and AST release were observed,whereas PPE pretreatment significantly increased the leakage of SOD and GSH in BRL-3A cells.Furthermore,PPE pretreatment apparently up-regulated HO-1 expression,and prevented the overexpression of Nrf2 expressions.These results demonstrate that PPE-mediated up-regulation of HO-1 via Nrf-2 signaling pathway may provide a pivotal mechanism for its hepatoprotective activity.The phenolic compounds including gallic acid,corillagin and ellagic acid present in PPE might be playing a major role against alcohol-induced oxidative damage in rat cells.