【作者】 姚育翠；

【导师】 李向荣；

【作者基本信息】 浙江大学 ， 药物分析学， 2008， 硕士

【摘要】 玫瑰花是我国传统药材之一,《本草纲目拾遗》载:“玫瑰花活血、行血、健脾降火、理气调经、滋补养颜”,其养颜美容的功效被广泛接受。原花青素是具有极强抗氧化能力的多酚类物质,药理作用广泛,目前国外已制成多种药品、保健品、化妆品上市,是一种很有开发前途的天然抗氧化剂。本文主要研究玫瑰花中原花青素的提取及纯化工艺,并建立玫瑰花原花青素中单体和二聚体的HPLC分析方法,最后对玫瑰花原花青素的抗氧化能力进行评价。本论文首先采用微波法从玫瑰花中提取原花青素,通过单因素实验考察了微波反应时间、反应次数和料液比对提取率的影响,最终确定提取条件为:60%乙醇为溶剂,料液比1:20,微波功率5.00V,微波反应时间20min,反应次数为1次,原花青素的提取率达到1.59%。并将微波法与传统溶剂浸提法进行了对比,结果表明微波法下原花青素的提取率高于溶剂浸提法。继而采用大孔吸附树脂柱进一步纯化初提液,首先通过静态实验从五种树脂（D101、D1300、NKA,AB-8和NKA-Ⅱ）中筛选出适合纯化原花青素的树脂型号,以饱和吸附量和解吸率为指标综合考虑,结果显示:D101型树脂综合性能最好,其静态饱和吸附量为9.3mg/g湿树脂,解吸率为77.20%。论文进一步通过正交实验考察了纯化工艺参数,包括上样浓度、上样pH、洗脱液浓度、洗脱流速四个因素,以原花青素纯度和回收率为指标,极差分析和方差分析结果表明:上样pH是影响最显著的因素,最优纯化工艺条件为:上样浓度为1.25mg/ml,上样pH为2,洗脱液浓度为70%,洗脱流速为2ml/min。本文还对树脂纯化过程的动力学特征进行了初步探索,绘制了渗漏及洗脱曲线,并用数学方法将渗漏曲线拟合为⁽y=1.18(e^0.31x-e^-0.79x)),由此方程积分可得出树脂的动态饱和吸附量即最大上样浓度。论文进一步建立了玫瑰花原花青素中单体及二聚体的HPLC分析方法,具体色谱条件为:分析柱:Eslipse XDB-C18,4.6mm×150mm,5μm;流动相:A:乙腈,B:1.0%冰醋酸（pH=2.71）;洗脱梯度:0-10min:5-10%A;10-100min:10-25%A;流速:1.0ml/min;检测波长:280nm;柱温:30℃;进样量:20μl。在此条件下,四种物质（儿茶素、表儿茶素、表儿茶素没食子酸酯和原花青素B2）的分离度、方法的线性、精密度和加样回收率均符合要求。定量分析结果显示:原花青素B2的含量最高,达2.54%,表儿茶素含量为0.612%,表儿茶素没食子酸酯含量为0.257%。论文最后将纯化得到的玫瑰花原花青素产品进行抗氧化活性研究,采用DPPH法测定其抗氧化能力,以维生素C为对照,实验测得原花青素对[DPPH·]的半抑制量EC50=0.053mg/ml,维生素C对[DPPH·]的半抑制量EC50=0.066mg/ml,结果表明:原花青素的抗氧化能力强于维生素C。更多还原

【Abstract】 Flos Rosae Rugosae is a kind of chinese traditional medicine material.It is recorded in ’Compendium of Materia Medica’ that it has great effectiveness on keeping young,and this is recepted extensively.Procyanidins are kinds of polyphenol with great antioxidant activity and then extensive pharmacological activity. Procyanidins are natural antioxidants which have prospects of development and hasve been marketed by a lot of countries as medicine,health food and cosmetics.This paper studied on the extraction and isolation of procyanidins from Flos Rosae Rugosae,developed a HPLC analysis method for determination of them and evaluated the antioxidant activity of procyanidins comparing with that of VC.Procyanidins were extracted from Flos Rosae Rugosae with microwave assisted. The effection of microwave reaction time,times and ratio of solid/solvent on the percentage of procyanidins extraction was investigated by the single-factor experiment.The best extracting conditions were as follows:60%ethanol as the extracting solvent,ratio of solid/solvent 1:20,microwave power 5.00V,microwave reaction time 20min,one times,percentage of procyanidins extraction can achieve to 1.59%.Compared with solvent extraction only,extraction with microwave assisted had better performance.The extracted crude procyanidins powder was purified by macroporous adsorbent resin.In the beginning,a suitable type of macroporous adsorbent resin had been selected from five types including D101,D1300,NKA,AB-8 and NKA-Ⅱ.Evaluated by static saturated adsorption ability and elution percentage,D101 was thought to be the most suitable type of macroporous adsorbent resin for the purification of procyanidins.Its static saturated adsorption ability was 9.3mg/g wet resin and elution percentage 7.2%.Evaluated by product purity and recovery,the influence of concentration of sample,pH of sample,concentration of eluant and flow rate of elution had been investigated by orthogonal experimental design.Analysis of range and analysis of variance were used to determine the optimal parameters of the purification process,the results were:1.25mg/ml as the concentration of sample,2 as the pH value of sample,70%ethanol aqueous solution as the eluant and 2ml/min as the flow rate of elution.Kinetics about process of purification had also been studied.The curves of dynamic adsorption-elution process had been plotted,and the kinetic equation was ⁽Y = 1.18(e^0.31x -e^-0.79x))A HPLC method was developed for analysing monomers and dimers of procyanidins from Flos Rosae Rugosae.The column was Eslipse XDB-C18, 4.6mm×150mm,5μm,and thermostated at 30℃.The elution conditions were as follows:flow rate,1 ml/min;solvent A,1.0%AcOH in H₂O;solvent B,CH₃CN; elution starting with 5%B and continuing with a linear gradient from 5 to 10%B in 10min,then 10%to 25%in 100min.The method was validated with respect to resolution,linearity,precision and recovery.Epicatechin,epicatechingallate and procyanidins B2 had been quantitatively analysised,and their contents respectivly were 0.612%,0.257%,2.54%.The antioxidant activity of procyanidins was evaluated by the amount of antioxidant necessary to decrease by 50%the initial DPPH concentration.The study showed that EC50 for procyanidins was 0.052mg/ml while for VC was 0.066 mg/ml, the conclusion can be drawn that the antioxidant activity of procyanidins is strong than that of VC.更多还原