绞股蓝皂苷酶促反应规律研究及其转化产物的鉴定
Enzymatic reaction pattern of saponins in Gynostemma pentaphyllum and identification of their transformed products
杨紫晴 1李敏 2徐书杨 1杨丹 2黄琴 3金德珍 3马云璞 3金佩芬 4朱晶晶 2刘守金 5王智民2
作者信息
- 1. 安徽中医药大学,安徽合肥 230012;中国中医科学院中药研究所,北京 100700
- 2. 中国中医科学院中药研究所,北京 100700
- 3. 平利县茶叶和绞股蓝发展中心,陕西 安康 725500
- 4. 嘉兴市食品药品与产品质量检验检测院,浙江 嘉兴 314050
- 5. 安徽中医药大学,安徽合肥 230012
- 折叠
摘要
稀有皂苷活性普遍好于其原形皂苷,不同干燥方式绞股蓝中稀有皂苷/原形皂苷的组成比例及药效差异显著,可能与葡萄糖苷酶的酶促反应有关.为探究绞股蓝加工过程中葡萄糖苷酶的酶促转化反应及其规律,同时也为绿色生产稀有皂苷提供技术支撑.该实验从新鲜绞股蓝叶片中提取葡萄糖苷酶,构建模拟反应体系与原型绞股蓝皂苷提取物进行体外模拟反应,并采用超高效液相色谱-四极杆飞行时间质谱跟踪检测反应前后的底物、中间产物、终产物,进一步采用中药化学手段纯化并鉴定部分底物和产物,探讨其酶促反应规律.研究结果证实了绞股蓝皂苷-β-葡萄糖苷酶的酶促转化作用,指认了该转化过程中4组共15个皂苷类成分,包括PPD型中性皂苷和酸性皂苷(乙酰基皂苷、丙二酰基皂苷等).纯化鉴定了其中4个底物与产物,并明确了绞股蓝皂苷-β-葡萄糖苷酶具有一定的选择性,酶解反应规律如下:①仅水解中性皂苷,乙酰基和丙二酰基等酸性皂苷不易被水解;②水解位点均为皂苷C-3位的葡萄糖基,C-20位的葡萄糖基不易被水解.利用绞股蓝的酶促反应,可实现高效转化生成稀有皂苷,研究结果有助于扩大和提升绞股蓝的利用价值.
Abstract
The activity of rare saponins is generally better than that of their prototypes.There are significant differences in the pro-portions and pharmacological effects of rare saponins/prototype saponins in Gynostemma pentaphyllum samples dried with different methods,which may be related to the reaction catalyzed by glucosidase.To explore the pattern of the enzymatic reaction catalyzed by glucosidase during the processing of G.pentaphyllum and provide technical support for the green production of rare saponins,this ex-periment extracted glucosidase from fresh leaves of G.pentaphyllum,constructed a simulated reaction system,and conducted in vitro simulation reactions with the prototype saponins extract of G.pentaphyllum.Ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry was employed to detect the substrates,intermediate products,and final products of the reaction.Fur-thermore,traditional Chinese medicine chemical methods were used to purify and identify some substrates and products,on the basis of which the enzymatic reaction pattern was explored.The results confirmed the role of gypenoside-β-glucosidase in the enzymatic reaction and identified a total of 15 saponins,including PPD neutral saponins and acidic saponins(acetyl saponins,malonyl saponins,etc.,)in 4 groups during the reaction process.Four substrates and products were purified and identified,and it was clarified that gypenoside-β-glucosidase had selectivity.The enzymatic reaction pattern was summarized as follows.①The enzyme only hydrolyzed neutral sapo-nins,while acidic saponins such as acetyl and malonyl saponins were not easily hydrolyzed.②The hydrolysis occurred at the glucosyl group at the C-3 position,while the glucosyl group at the C-20 position was not easily hydrolyzed.The enzymatic reaction of G.pen-taphyllum can be employed to generate rare saponins,and the findings of this study can help expand and enhance the utilization value of G.pentaphyllum.
关键词
绞股蓝/皂苷/酶促转化/分离鉴定Key words
Gynostemma pentaphyllum/saponins/enzymatic conversion/separation and identification引用本文复制引用
基金项目
中国中医科学院科技创新工程项目(CI2021A04408)
中国中医科学院科技创新工程项目(CI2023E001TS03)
北京市自然科学基金项目(7242248)
国家自然科学基金项目(81573600)
出版年
2024
NETL
NSTL
万方数据