三叶青叶化学成分鉴定及其总黄酮含量测定研究  

Identification of chemical components and determination of flavonoids in Tetrastigma hemsleyanum leaves

作　　者：范世明[1] 谢心月 曾繁天 周小红[1] 蔡碧雅 许文[1,2] 谢治森 黄心平 

机构地区：[1]福建中医药大学药学院,福州350122 [2]福建中医药大学生物医药研发中心,福州350122 [3]福州集珍园生物科技有限公司,福州350002

出　　处：《药物分析杂志》2017年第8期1481-1488,共8页Chinese Journal of Pharmaceutical Analysis

基　　金：国家公益性行业科研专项(201407002-3);福建省科技厅引导性项目(2016N0015);康复技术协同创新中心课题(X2015002-协同);福建中医药大学校管课题(X2014134-学科);福建省大学生创新创业训练计划项目(201610393084;201510393014)

摘　　要：目的:建立超快速液相色谱-串联四极杆飞行时间质谱法(RRLC-Q-TOF-MS法)快速鉴定三叶青叶化学成分,根据鉴定的黄酮成分建立相适应的紫外-可见分光光度法(UV-Vis法),测定三叶青叶中总黄酮的含量。方法:RRLC-Q-TOF-MS法采用CORTECS C_(18)色谱柱(2.1 mm×100 mm,1.6μm),流动相为乙腈(A)-0.1%甲酸水溶液(B),梯度洗脱,流速0.25 m L·min^(-1),柱温45℃;飞行时间质谱采用负离子全扫描模式,数据采集范围m/z 50~1 000,通过一级质谱和二级质谱、相关文献及对照品推测鉴定化学成分。根据鉴定的黄酮进行3种总黄酮显色方法比较,确定三乙胺法显色,以UV-Vis法在400 nm波长处测定三叶青叶总黄酮含量。结果:采用RRLC-Q-TOF-MS共鉴定了三叶青叶中异荭草苷、荭草苷、异荭草素鼠李糖苷、荭草素鼠李糖苷、牡荆苷、异牡荆苷、牡荆素鼠李糖苷、异牡荆素鼠李糖苷共8个主要黄酮类成分;采用UV-Vis法测定三叶青叶总黄酮含量,以牡荆苷计,质量浓度在1.956~9.780μg·m L^(-1)范围内与吸光度值呈良好线性关系(r=0.999 5),平均加样回收率99.8%(RSD=3.4%),三叶青叶总黄酮含量范围为13.38~28.67 mg·g^(-1)。结论:本文建立RRLC-Q-TOF-MS联用技术,为鉴定三叶青叶的黄酮成分提供了一种快速、高效的定性分析方法;建立以三乙胺显色的UV-Vis法定量分析三叶青叶总黄酮方法准确,为综合评价三叶青叶的质量提供参考。Objective： To develop a qualitative analytical method by rapid resolution liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry （ RRLC-Q-TOF-MS ） for identification of chemical components and an UV-Vis analytical method for determination of total flavonoids in Tetrastigma hemsleyanum Diels et Gilg leaves. Methods： The separation was performed on a CORTECSCIs（ 2. 1 mm x 100 mm, 1.6 μm ） column with the mobile phase consisting of acetonitrile （ A ） and water containing 0. 1% formic acid （ B ） in gradient mode at a flow rate of 0. 25 mL·min^-1. The column temperature was set at 45 ℃. TOF-MS was used in negative scan mode. The data acquisition ranged from 50 to 1 000 and compounds were identified by MS spectrum, MS/ MS spectrum, references and reference substances. Then triethylamine developer was chosen by comparing three UV specific visualisation methods of the identified flavonoids. With vitexin as the standard, triethylamine as the developer, UV-Vis method was applied to determination of total flavonoids at 400 nm. Results： 16 compounds including 8 organic acids （ neochlorogenic, chlorogenic, cryptochlorogenic acid, caffeic acid, caffeoylquinic acid, feruloylquinic acid and two coumaroylquinie acids ）and 8 flavonoids （ isoorientin, orienfin, isoorienfin-2＂ -O-rhamnoside, orientin-2＂ -O-rhamnoside, vitexin, isovitexin, vitexin-2＂ -O-rhamnoside and isovitexin-2＂ -O-rhamnoside ） in Tetrastigma hemsleyanum leaves were identified by RRLC-Q-TOF-MS. There was a good linear relationship between the concentration and absorbance of vitexin within the range of 1. 956-9. 780 μg·mL^-1 （ r=0. 999 5 ）. The average recovery was 99. 8% with RSD of 3.4%. The total flavonoids in the Tetrastigma hemsleyanum leaves ranged from 13.38 to 28.67 mg· g^-1. Conclusion： The results indicate that the new approach conbining RRLC- Q-TOF-MS and UV-Vis is applicable in qualitative and quantitative quality control of medicinal leaves from Tetrastigma-hemsleyanum.

关 键 词：三叶青 新绿原酸 绿原酸 隐绿原酸 荭草苷 异荭草苷 荭草素鼠李糖苷 牡荆苷 异牡荆苷 牡荆素鼠李糖苷 总黄酮 C-苷黄酮 三乙胺显色剂 超快速液相色谱-串联四极杆飞行时间质谱 紫外-可见分光光度法 

分 类 号：R917[医药卫生—药物分析学]