Studies On The Purification And Antioxidant Activity Of The Polysaccharide From Citrus Reticulata ’chachi’ Peel Extract

The Citrus reticulata ‘chachi’ peel is the dry and ripe peel，is one with long history ofmedicinal and edible，and it is the source of Tangerine peel. Previous research group haveoptimized the extraction process of Citrus reticulata ‘chachi’ peel with muti-vitroantioxidant response surface methodology. It is confirmed that there are polysaccharide andflavones in the extract. It is found that the extract have significant effects on D-Galactoseinduced aging rats. The extract could reduce the MDA contents. It could also increase theSOD and the GSH-Px activity. Testing antioxidant activity of the six polymethoxylatedflavones through establishing the cells injury model of vitro oxidation stress damage.This study adopts the method to extract the polysaccharides with hot water，throughstep by step ethanol concentration gradients from Citrus reticulata ‘chachi’ peel extract. Weget the purification of polysaccharide fractions though separation and purification steps.Determining of the molecular weight of polysaccharide fractions by high performance GelPermeation Chromatography and studying on the vitro cells antioxidant activities of Citrusreticulata ‘chachi’ peel extract polysaccharide. Establishing a HPLC method to determinatethe flavones in it in order to control the quality of Citrus reticulata ‘chachi’ peel extract.The main study contents were summarized as follow:1. The Citrus reticulata ‘chachi’ peel extract polysaccharide was extracted by hotwater with1:10solid-liquid ratio，extract time was3hours with80℃water bath for3times.Added the ethanol in the solution and kept the ethanol volume fraction to be50%、70%and90%. Collected and freeze-dried the deposit that to be crude polysaccharide of CCP50、CCP70and CCP90. The CCP50was the highest rate of yield but with lowest content ofpolysaccharide. The CCP90was the lowest rate of yield but with highest content of polysaccharide.2. Investigated the effects of resin type and decolorization conditions to the crudepolysaccharide by step to step. Compared of the different types of macro porous adsorptionresin and anion exchange resin. D-101resin was berrer than other macro porous resin.Thebest decoloration condition was8%D-101resin，with30℃and4hour. The decolorationrate of CCP50and polysaccharide retention were79.56%and73.27%，CCP70were78.20%and68.09%，CCP90were81.68%and68.39%. The protein removal rate were69.70%、74.88%and73.19%with the Sevag method. Finally，CP50、CP70and CP90weremade after dialysising and freeze drying.3. CP50、CP70and CP90were separated and purified through DEAE-Sepharose FastFlow and Sepahdex G-100column chromatograph，got the G-1^G-3、G-4^G-5and G-6.HPGPC determination the molecular weight and purity. The result showed that themolecular weight of high volume fraction ethanol precipitating polysaccharide was largerthan low volume fraction ethanol precipitating polysaccharide. The molecular weight ofG-1、G-3、G-4and G-6were72059u、109931u、47433u and18743u and they had highpurity. The molecular weight of G-2and G-5were88219u and31869u，with lower purity.4. Established the100μmol/L H₂O₂damage model of PC12cells and HUVEC cells.Focused on the protective effect of Citrus reticulata ‘chachi’ peel extract polysaccharide onPC12cells and HUVEC cells from oxidant damage. The result showed that CCP50、CCP70and CCP90had protective effect in PC12cells and HUVEC cells with H₂O₂damage. D50、D70and D90remained antioxidant activity but weaker than CCP50、CCP70and CCP90.CP50、CP70and CP90had no antioxidant activity.Compared with normal control group，PC12cells damaged by H₂O₂increased thecontent of MDA，decreased SOD and GSH-Px activity. Compared with model group，CCP50、CCP70and CCP90decreased the content of MDA，increased SOD and GSH-Pxactivity. The more polysaccharide molecular weight，the stronger cells antioxidant activity.5. Established the HPLC method for the determination of flavone from the Citrusreticulata ‘chachi’ peel extract，used C18column、acetonitrile-0.2%acetic acid as the mobilephase for gradient elution，283nm and330nm as the detection wavelength、1.0mL/min as the flow velocity. Hesperidin、Nobiletin and Tangeretin could reach good separation effectfor the Citrus reticulata ‘chachi’ peel extract.