Extraction And Identification Of Polyphenols In Wuweizi Fresh Fruit And Its Protective Effect On Injured HepG2Cell

Wuweizi (Schisandra chinensis (Turcz.) Baill.) is a berry-like fruit, mainly grown in north-eastern China, including Heilongjiang, Jilin, Liaoning provinces. Wuweizi has been used as a functional ingredient in foods, such as wine, beer, beverages, yoghourt, jam, fruitcake and other products. Wuweizi is also used in Chinese traditional medicine. Recently, some new bioactivities of Wuweizi have been reported, such as hepatoprotection, anti-carcinogenicity, anti-obesity, anti-virus, anti-inflammation and anti-diabetes. Wuweizi has high content of lignan, which mainly exist in seeds which have been considered as the primary constituents for Wuweizi’s antioxidant, hepatoprotective, immuo-modulating, anti-inflammatory and anticancer effects. Compared to the intensive studies on lignan, polyphenols in Wuweizi pulp, especially anthocyanins have not received much attention. There were a few of reports about anthocyanins extracted from Wuweizi.The polyphenol extracted from Wuweizi fresh fruits were studied in this project. Firstly, the extraction optimization and composition analysis of polyphenols in the fresh pulp of Wuweizi have been investigated in this study. The extraction processes of polyphenols from Wuweizi pulp and proanthocyanidins from Wuweizi seed were optimized using Random-Centroid Optimization (RCO) methodology. Six factors for polyphenols and four factors for proanthocyanidins, three extraction targets for polyphenols and proanthocyanidins were considered in the RCO program respectively. Three sets of optimum proposed factor values were obtained corresponding to three extraction targets respectively. The set of optimum proposed factor values for polyphenol extraction given was chosen in further experiments as following:liquid and solid ratio (v/w)8, ethanol67.3%(v/v), initial pH1.75, temperature55℃for4h and extraction repeated for4times. The Wuweizi polyphenol extract (WPE) was obtained with a yield of16.37mg/g and composition of polyphenols1.847mg/g, anthocyanins0.179mg/g, sugar9.573mg/g and protein0.327mg/g. The WPE demonstrated high scavenging activities against DPPH radicals of88.9%. The set of optimum proposed factor values for proanthocyanidins extraction given was chosen in further experiments as following:liquid and solid ratio (v/w)9, ethanol65%(v/v), initial pH5.21and the extraction time is4h. The Wuweizi seed extract (WSE) was obtained with a yield of167.3mg/g and composition of proanthocyanidins7.12mg/g and the scavenging activities against DPPH radicals of82.9%.Secondly, the stability of Wuweizi polyphenols in Wuweizi has been studied. The study investigated the effect of organic acids including citric acid, oxalic acid, acetic acid, malic acid, succinic acid and erythorbic acid in comparison with HC1on the stability of Wuweizi anthocyanins crude extacts. Higher concentration (0.250M and0.125M) of malic acid, succinic acid increased the absorbance at520nm of anthocyanins significantly. Erythorbic acid has no obvious hypochromic effect on the WPE. In the extraction processing,0.125M of hydrochloric acid and oxalate could obviously increase the polyphenol content of extracts compared with the control without acid added; 0.125M of hydrochloric acid, oxalic acid, malic acid could increase the scavenging activities against DPPH radicals of WPE-S, WPE-SC and WPE, coarse solution, producing clearance rate is significantly higher than acid free blank group, have higher antioxidant ability. The extraction efficiency was evaluated with three parameters, including anthocyanin content, polyphenol content and DPPH radical scavenging activity. All three parameters of the anthocyanin extracts were increased significantly, with oxalic, malic and succinic acid, but not with citric and acetic acid.Thirdly, Wuweizi anthocyanin crude extract was purified by XAD-7resin, and the profile and the structure of Wuweizi anthocyanin has been confirmed using HPLC-ESI-MS and ’H-13C NMR spectroscopy. Two types of anthocyanins were detected and identified as cyanidin-3-O-glucoside (11%of total anthocyanin content) and cyanidin-3-O-xylosylrutinoside (89%of total anthocyanin content).Fourthly, the polyphenol extracts were prepared from Wuweizi fresh fruits, including the polyphenol extract (WPE), anthocyanin extract (WAE), and lignin extract (WLE), and their protective effects were investigated on injured HepG2cells by EtOH, CC14and AAPH, respectively. Cell proliferation was tested by the WST-1assay, which showed that the WPE, WAE and WLE treatments increased the cell numbers of HepG2cells injured by EtOH, CCl4in a dose-dependent manner. At the protective concentrations of0.1mg/mL of WPE and WAE, and0.01mg/mL of WLE, cell numbers were significantly increased when compared to the no-treatment control (p<0.05). The AnnexinV-propidium iodide assay showed Wuweizi extract treatments, could also decrease the apoptosis rate and necrosis rate. After injured HepG2cell treated by different Wuweizi extracts, the inhibition expression of apoptosis related gene families (TIMPs, Caspase-8, Caspase-9and FADD) were presented, but showed the high expression on oxidative stress gene families (SOD, GPX1and CAT) and SATA3gene signal. These results indicated that protective effects of Wuweizi polyphenol extracts were generalizable to HepG2cell.