Study On Antioxidant Activity Of Xinjiang Grape Bleeding Sap And Structure Identificatuon Of Substances In Kashgar Munage Grape Bleeding Sap

Objective:To study the antioxidant activity and composition of Xinjiang grape bleeding sap,and determine the separation and purification process of antioxidant components in Xinjiang Kashgar munage grape bleeding sap and their structure identification.To investigate the effects of temperature and phenyllactic acid on the stability,antioxidant activity and components of grape bleeding sap.To analysis the polysaccharide in xinjiang grappe bleeding sap.To lay foundation for the basic research of Xinjiang grape bleeding sap.Methods:1 Take Xinjiang 11 kinds of different varieties grape bleeding sap as the research object,Determine their ability of scavenging ABTS⁺·,·OH,DPPH·,O₂^-·,NO₂^-·.free radical.MTT method was used to determine the effect of Xinjiang grape grape bleeding sap on the growth of PC12 cells;and the effect on the oxidative damage model of PC12 cells induced by H₂O₂.2 Qualitative and qualitative experiments were used to identify the saponins,proteins,alkaloids,polysaccharides,flavonoids and water in 6kinds of Xinjiang grape bleeding sap.Using bovine serum albumin as the standard for protein,the content was determined by Coomassie brilliant blue method.Taking Gallic acid as the standard for polyphenol,the content was determined by Folin-Ciocalteu method.Using glucose as the standard for polysaccharide,the content of polysaccharide was determined by anthrone sulfuric acid method.Taking oleanolic acid as standard,the content of saponin was determined by vanillin acetic acid colorimetric method.3Taking·OH,DPPH·free radical scavenging rate and adsorption rate as the evaluation index of 8 different types of macroporous resin:AB-8,D101,HPD400,HPD500,HPD826,LSA-21,HPD100 and NKA-9 to select the most suitable macroporous resin;and determine the separation conditions.Sephadex LH-20 and C18 were used to purify the macroporous resin purification component,the separation conditions were determined,and the necessity of each step purification was investigated by HPLC.Two steps separation conditions of preparative HPLC were prepared.The single component was analyzed by NMR/MS spectrum and structure were determined.4 Water extraction and ethanol precipitation method was used to prepare polysaccharide in Xinjiang Kashgar munage grape bleeding sap.DEAE-52,Sephadex G-150 was used for purification.Gas chromatography was used to determine its monosaccharide composition.And its antioxidant activity was also analysized.5 Study the·OH free radical scavenging rate of Xinjiang Kashgar munage bleeding sap under 4_℃,20_℃,37_℃,47_℃and 57_℃。Observe the physical appearance change changes and quantify the polysaccharide,protein,saponin and polyphenol content.6 Investigate the effect of phenyllactic acid on the·OH clearance rate,composition and physical appearance of bleeding.Results:1.11different kinds Xinjiang grape bleeding sap had scavenging capacity for ABTS⁺·,·OH,DPPH·,O₂^-·,NO₂^-·.free radical to some extend.And·OH scavenging ability was the strongest.The five free radical scavenging methods used to determine the antioxidant activity of the bleeding sap had significant difference with each other.Xinjiang grape bleeding sap could improve the survival rate of PC12 cell.And they could protect oxidative damaged PC12cell induced by H₂O₂,which can improve the activity of GSX-PX,reduce the leakage of LDH.2 6 kinds of grape bleeding sap contain amino acids,proteins,carbohydrates and their glycosides,saponins,and did not contain flavonoids and alkaloids.The solid content of bleeding sap in Xinjiang grape bleeding sap was only about 0.2%,and 99.8%is water.The contents of protein,polysaccharide,saponin and polyphenol were different in grape sap.3 Xinjiang Kashigar munage grape bleeding sap purification conditions of antioxidant component was as follows:HPD400macroporous resin:sample load was 1 BV,elution was 60%ethanol,elution volume was5 BV,sample loading flow rate was 2m L·min^-1,elution speed was 5 m L·min^-1.The purification conditions of C18 were as follows:sample load was≤1m L,sample loading and elution speed were not controlled,elution was 50%methanol,elution volume was3BV.Preparative HPLC separated two steps.After HPLC/NMR/MS analysis,9compounds were identified.They were respectively 16min（2,3-dihydroxypropyl-galactosidase and uracil）,18min（phenyllactic acid,4-hydroxy-5 methyl pyrimidine-2-ketone and glycerol）,19min（4-hydroxyl,2-pyrimidine-α-furacylglucoside）,23min（5-methyl uracil）,30min（phenylalanine）70min（N-α-hydroxy methamphetamine）.3 After DEAE-52 separation,8 polysaccharide fractions from Kashgar munage grape bleeding sap were gained.There were 4 fractions were enough to be purified by Sephadex G150,they were GBSK-1,GBSK-2,GBSK-3,GBSK-5.And they had different degrees of free radical scavenging capacity.The monosaccharide composition of GBSK-3 is relatively simple,mainly composed of xylose;GBSK-2,GBSK-5,were composed of monosaccharide rhamnose,mannose,Arabia sugar,glucose and galactose.GBSK-1 were composed of rhamnose,xylose,mannose,Arabia sugar,glucose,galactose.4 With respect to the original grape bleeding sap,the addition of phenyllactic acid above5 mg·min^-1 can mentanin the high·OH radical scavenging rate of Xinjiang Turpan grape bleeding sap in the experimental time.The addition of phenyllactic acid above 10mg·m L^-1 could mentanin the high·OH radical scavenging rate of Hotan red grape bleeding sap and Kashgar munage grape bleeding sap in the experimental time.At the same time,addition of phenyllactic acid,could prevent the reduction of protein and polysaccharide,polyphenols,saponins component,the precipitation and had positive correlation to the amount of phenyllactic acidaddition.The temperature experiment results showed that 2015 Kashgar munage grape bleeding sap was 20_℃.A higher temperature could keep the polysaccharide components longer.Conclusion:Xinjiang grape bleeding sap had good antioxidant activity.Xinjiang grape bleeding sap contains amino acids,proteins,carbohydrates and their glycosides and saponins,and contains no flavonoids and alkaloids.There is a certain difference in the content of each component in the grape bleeding sap.The solid content of grape bleeding sap is only about 0.2%,and99.8%is water.Separation and purification of Xinjiang Kashgar munage grape bleeding sap gained 9 compounds.There was difference in the monosaccharide composition of grape bleeding polysaccharide components,and had a certain degree antioxidant activity.With respect to the original grape bleeding sap,addition of phenyllactic acid,can prevent the reduction of protein and polysaccharide,polyphenols,saponins component,the precipitation and had positive correlation to the amount of phenyllactic acid addition.The result of temperature experiment showed that at the suitable temperature,the·OH radical scavenging rate could metain high and stable in the experimental time.The addition of phenyllactic acid addition can keep the·OH radical scavenging rate stable for a long time.These experimental results laid a theoretical foundation for further studies on Xinjiang grape bleeding sap.