新鲜丹参中酚酸成分的含量测定及其多酚氧化酶对含量的影响研究

Content determination of phenolic acids in fresh Salvia miltiorrhiza and effect of polyphenol oxidase on content

新鲜丹参中酚酸成分的含量及其积累与转化,以及含量测定方法尚未形成共识。为进一步探明新鲜丹参中酚酸成分的真实含量,该实验采用多种处理方法制备供试品溶液,测定新鲜丹参中酚酸成分的含量,并考察烘干、阴干过程中不同脱水率的丹参样品中酚酸成分的含量变化;以硫酸铵沉淀、透析提取纯化丹参多酚氧化酶(PPO),进行部分酶学性质研究。采用UPLC测定丹参中迷迭香酸、紫草酸、丹酚酸B的含量,结果显示用1 mol·L^(-1)的HCl溶液或1 mol·L^(-1)的HCl甲醇溶液匀浆,测得新鲜丹参中酚酸成分的含量最高,不同脱水率丹参中酚酸成分的含量无明显差异,表明酚酸含量与脱水率无相关性。丹参PPO的最适pH为7.6,最适温度为40℃,以邻苯二酚为底物,丹参PPO的酶促褐变反应符合米氏方程,米氏常数Km为0.12 mol·L^(-1),Vmax为588.23 U·min^(-1);4种抑制剂柠檬酸、乙二胺四乙酸二钠、抗坏血酸和亚硫酸钠对丹参PPO的抑制效果均随抑制剂浓度的增大而增加,其中亚硫酸钠的抑制作用最强。该研究证明新鲜丹参中已存在大量的酚酸成分,酚酸是丹参生长过程中原始积累的次生代谢产物,而非采后干燥脱水胁迫的诱导产物;对丹参栽培、采收加工等具有参考价值和借鉴意义。

There is no consensus on the content,accumulation,transformation and content determination methods of phenolic acids in fresh Salvia miltiorrhiza.In order to find out the true content of phenolic acids in fresh S.miltiorrhiza,a variety of treatment me-thods were used in this study to prepare sample solution.The content changes of phenolic acids in S.miltiorrhiza samples with different dehydration rates were investigated during drying and shade drying processes.Polyphenol oxidase(PPO)of S.miltiorrhiza was extracted and purified by ammonium sulfate precipitation and dialysis to investigate the enzymatic properties.The content of rosmarinic acid,lithosperic acid and S.nolic acid B in S.miltiorrhiza was determined by UPLC.The results showed that the content of phenolic acids in fresh S.miltiorrhiza was highest when it was homogenized with 1 mol·L^(-1)HCl solution or 1 mol·L^(-1)HCl methanol solution.There was no significant difference in the content of phenolic acids in S.miltiorrhiza with different dehydration rates,indicating that there was no correlation between phenolic acid content and dehydration rate.The optimum pH of S.miltiorrhiza PPO was 7.6 and the optimum temperature was 40℃.With catechol as substrate,S.miltiorrhiza PPO had the enzymatic browning reaction which was in compliance with Michaelis equation,with Michaelis constant Km of 0.12 mol·L^(-1)and Vmax of 588.23 U·min^(-1).The inhibitory effect of citric acid,disodium ethylenediamine tetraacetate,ascorbic acid and sodium sulfite on S.miltiorrhiza PPO increased with the increase of inhibitor concentration,and sodium sulfite showed the strongest inhibitory effect.The present study proved that there were a large number of phenolic acids in fresh S.miltiorrhiza,which were the secondary metabolite of primitive accumulation during the growth of S.miltiorrhiza,rather than the induced product of postharvest drying and dehydration stress.This study has reference value and significance for the cultivation,harvest and processing of S.miltiorrhiza.