茶叶中甲基化儿茶素的分离、纯化和高效液相色谱法分析

Isolation of Methylated Catechins from Tea and Their Aanalysis by High Performance Liquid Chromatography

以日本Benifuji绿茶为原料,用50%乙腈提取茶叶有效成分,提取液经氯仿脱咖啡碱和色素、乙酸乙酯和HP-20大孔树脂富集儿茶素,得多酚含量高于80%的茶多酚粗品,再经ToyopearHW-40S柱层析分离纯化,得EGC、ECG、EGCG、EC及两种未知成分。经1H-NMR、MS和HPLC等分析,两种未知成分为（-）-表没食子儿茶素3-O（3-O-甲基）没食子酸酯（EGCG3″Me）和（-）-3-O-甲基-表儿茶素没食子酸酯（ECG3′Me）。采用TSKgelODS-100Z色谱柱和二极管阵列检测器（DAD）,建立了一种快速、灵敏分析茶叶中儿茶素和甲基化儿茶素的高效液相色谱法。色谱条件：TSKgelODS-100Z色谱柱,流动相为KH2PO4（pH2.5）-甲醇系统,采用梯度洗脱,流速1.0mL/min,柱温40℃,进样量20μL。结果表明,6种儿茶素在一定范围内线性良好,相关系数为0.9996~1.0000,平均加标回收率在90.8%-105.9%之间,相对标准偏差均小于3.5%。

The extract was obtained using 50% acetonitrile to extract Japanese Benifuji green tea. The extract was treated by chloroform, ethyl acetate, and Diaion HP-20 resin to afford a crude preparation with high content of polyphenol （ 〉 80% ）. The solution of crude tea polyphenol was loaded onto a column of Toyopearl HW-40 s, and the column was eluted with ethanol/water （80：20） to yield six fractions： EGC, ECG, EGCG, EC, and two unknown compounds. Through the analysis of ^1H NMR, MS, and HPLC, the two unknown compounds were elucidated as EGCG3Me and ECG3Me. Futhermore, a high performance liquid chromatographic method with DAD as detector and TSKgel ODS-100Z as column was developed for the simultaneous determination of catechins and methylated catechins in tea. It was fast （ less analysis time） and sensitive. Chromatographic conditions were as follows ： TSKgel ODS-100Z column, gradient eluant of KH2PO4 （ pH 2.5 ） and methanol, flow rate 1.0 mL/min, temperature of column 40℃, DAD detection, sample amount 20 μL. As results, the correlation coefficients were 0. 9996 - 1. 000 in the linear range. The average recoveries of catechins ranged from 90.8% to 105.9%, and the relative standard deviations were 3.5%.