Study On Separation, Purification And Antioxidant Activities Of Rosmarinic Acid And Flavonoid Aglycones From Yam Leaves

As a byproduct of yam（Dioscorea alata）cultivation,yam leaves are abandoned directly in the field.To our best known,there is no relevant report about bioactive compouds from yam leaves except for our group.Preliminary study found that yam leaves rich in rosmarinic acid and flavonoid aglycones and show a high value of application.If these effective components were extracted,not only it can turn waste into treasure and improve the comprehensive utilization of yam,but also develop new plant resources.Therefore,this study selected rosmarinic acid（RA）and flavonoid aglycones as the research object on the basis of clear polyphenol composition.Experimental conditions were optimized for isolation and purification of RA and flavonoid aglycones from yam leaves and the antioxidant activity of them were studied.Yam phenolic compounds were analyzed and identified by UHPLC-DAD-ESI-MS in the positive/negative ion monitoring mode.Based on retention time,ultraviolet absorption,relative molecular weight,MS² fragmentation pattern in LC-MS chromatography,twenty-nine phenolic compounds were found in different parts of yam.In addition to 5-O-caffeoylquinic acid,feruloylquinic acid,rosmarinic acid,rutin and quercetin existed in roots and leaves,other eleven phenolic compounds were identified from root of yam including sinapic acid,sinapic acid-glucosides,anthocyanins,and other thirteen phenolic compounds were found in yam leaves such as caffeoylquinic acid methyl ester,caffeoyl shikimic acid,p-coumaroylquinic acid,coumaroylquinic acid methyl ester,kaempferol-3-O-rutinoside,etc.Quantitative and cluster analysis of the main polyphenols in 9 kinds of yam leaves were conducted.Results showed the most prominents are leaves with high content of RA and leaves with lower RA but high rutin content.Microwave-assisted extraction process was optimized to separate RA from yam leaves with high content of RA,and then the extract was purified with polyamide column.After adsorption separation,the content of RA in the extracts raised from8.57%to 29.30%.Semi-preparative HPLC was used for further puridication and the the content of RA in the product was 98.15%.Simutaneous microwave-assisted extraction and hydrolysis of flavonoid glycosides was optimized to prepare flavonoid aglycones from yam leaves with high rutin content,and then the crude extract was purified with D101 macroporous resin column.After adsorption separation,the content of flavonoid aglycones in the product raised from 5.26%to 65.30%.The process dynamics model for flavonoid aglycones from yam leaves was deduced which belongs to an apparent first order kinetic equationAntioxidant properties of RA and flavonoid aglycones enrichments were assessed by scavenging free radical action and protective effects on H₂O₂-induced oxidative stress.Free radical scavenging experimental results showed that the antioxidant activity of RA and flavonoid aglycones enrichments from yam leaves was significantly increased.And cell experimental results showed that yam（Dioscorea alata）leaves purified rosmarinic acid extract（DLP）dose-dependently inhibited the increase of ROS,MDA,TNF-αand IL-6,and the decrease of cell viability and SOD activity in H₂O₂-exposed HUVECs.Weston blotting analyses indicated that protective effects of DLP on H₂O₂-exposed HUVECs was accompanied by up-regulation of the expression of Bcl-2 and Nrf2,and down-regulation of Bax,NFκB-p65 and p-NFκB-p65.These findings confirm that phenolic compounds of yam leaves can protect endothelial cells against H₂O₂-induced alterations acting as antioxidative agents and cell signaling modulators.