Study Of The Antioxidative Activities And The Separation And Purification Of Chemical Composition In Bleeding Sap Of Xinjiang Hotan Red Grape Tree

Objective:1.The Xinjiang Hotan red GBS as the research object and the vitro antioxidant activity as evaluation means,The purification and separation of the Hotan red GBS the chemical composition by HPD400,SephadexLH-20,reversed-phase C₁₈column.2.The analysis,separation and purification,structure identification of the chemical composition of Xinjiang Hotan red GBS by using HPLC,Semi-Preparative HPLC,NMR,UPLC-MS,IR.Methods:1.With Vc as positive control,the antioxidant activities of Xinjiang Hotan red GBS were determined by ABTS·method,·OH kit method,DPPH·method and NO₂^-·method.Using gallic acid as the standard,the optimized conditions for determining content of total polyphenol and the change of antioxidant activity for Xinjiang Hotan red GBS of different storage years by Folin-Ciocalteu Colorimetry.2.The work of purification chose Sephadex LH-20 to 60%ethanol elution by macroporous adsorption resin to get parts for further purified,and focuses on the three factors:sample amout,dosage of eluent,eluent concentration,then did the orthogonal tests.Using the reversed-phase C₁₈ column to purify the SephadexLH-20 purification part.Using HPLC to determine six kinds of free phenol content of Hotan red GBS.Determining the best detection wavelength of Semi-Preparative HPLC with DAD detector.Analysis of liquid phase mode directly amplified to Semi-Preparative of liquid,the relatively high content of compound was separated of gradient elution.The monomer compounds were separated from NMR(¹H-NMR、¹³C-NMR、H-HCOSY、HSQC、HMBC)and TOF-MS spectrum analysis to Structural identification.Results:1.Xinjiang Hotan red GBS cleared different free radicals with different clearance ability.,and the strongest was to·OH,which was stronger than the positive control（0.2mg/mL Vc）.The order of radical clearance rate was·OH>DPPH·>ABTS·>NO₂^-.Through determining content of total polyphenol and the change of antioxidant activity for Xinjiang Hotan red GBS of different storage years,the total phenolic content and antioxidant activity as the storage time extension all showed a trend of decline.2.The best purification Sephadex LH-20 is determined through the single factor,orthogonal test and DPPH·clearance as evaluation index,which was sample amout with 2%CBV,dosage of eluent with 5CBV,eluent concentration with 70%methanol,flow rate with 1d/s.The 20%-70%methanol part was eluented by using the reversed-phase C₁₈ column.The 60%methanol eluente part was separated for two part（Ⅰ,Ⅱ）by SephadexLH-20 column.The gradient elution conditions were as follows:0³0 min,90⁸5B%;30⁶0min,85⁹0B%（A–B:methanol-water）.The results show that the retention time of 10-30min separation effect is best with partⅡ,but partⅠwasn’t separated before 10min and under the condition of the purification effect is not good,so the separation and purification of subsequent mainly concentrated in partⅡ.DAD detector determining the best detection wavelength of Semi-Preparative of liquid were 260nm and 280nm,Under this two wavelength,the peak signal strong and high purity.Xinjiang Hotan red GBS contain Gallic acid,catechin and epicatechin,and low content.3.The Six compounds to be separated from Semi-Preparative of liquid,and combining the data of NMR,TOF-MS to analysis the relatively high content of three compound.Got NO.0 monomer compounds is2-hydroxy-3phenyl lactic acid.Conclusion:1.The Xinjiang Hotan red GBS have scavenging activity,and the strongest was to·OH.First using HPD400,Sephadex LH-20 and reversed-phase C₁₈ column in turn to purify Xinjiang Hotan red GBS.The Six compounds to be separated from Semi-Preparative of liquid.On the basis of the HPD400 macroporous resin separation,the separation effect of Sephadex LH-20 is remarkable.Using the reversed-phase C₁₈ column have two main functions is that further purification for partⅡto remove the sample easy death adsorption material,and to protect the chromatographic column of Semi-Preparative.Combining with IR,NMR,TOF-MS and other means to analysis the relatively high content of NO.0,NO.1 and NO.5 compounds.