银杏类黄酮O-甲基转移酶活性的高效液相色谱分析

首次明确了银杏叶片类黄酮生物代谢关键酶O-甲基转移酶(flavonoid O-methyltransferase,FOMT)活性的HPLC测定技术与方法。采集扬州大学银杏种质资源圃银杏雄株成熟叶片,提取粗酶液,根据FOMT催化反应生成S-腺苷-L-高半胱氨酸(SAH)的原理,以山奈酚、槲皮素和异鼠李素等3种黄酮苷元为反应底物,采用优化的HPLC体系检测样品中FOMT催化反应生成的SAH峰面积,通过标准曲线求得不同反应底物的FOMT相对活性。FOMT活性反应生成物SAH在1.5～20μg/mL内呈良好的线性关系,RSD为2.1%(n=5),建立了FOMT活性测定优化体系。不同反应底物的FOMT活性表现不同,山奈酚与异鼠李素显著高于槲皮素。FOMT活性HPLC测定方法准确、快速、可靠、灵敏度高。

Analysis on flavonoid O-methyltransferase activity in Ginkgo by high performance liquid chromatography

To research and get a technique and method for determination on the activity of flavonoid O-methyltransferase(FOMT),a key-enzyme of flavonoid biosynthesis in Ginkgo leaves,by RP-HPLC for the first time.The adult leaves of male plants,were obtained from Ginkgo germplasm resources garden of Yangzhou university,then that were used to extract the crude enzyme solution.Based on the principle of S-adenosyl-L-homocysteine(SAH)derived from FOMT catalytic reaction and the correlationship between content and peak area of SAH,the relative activities of FOMT on the different substrates such as kaempferol,quercetin and isorhamnetin,were determined,the standard curve detected by optimized high performance liquid chromatography(HPLC)system in which there was a significant linear relationship within the range of 1.5 ~ 20μg/mL,RSD=2.1%(n=5),so the optimized system for assaying FOMT activity had been established.FOMT activities of the different substrates showed different,kaempferol and isorhamnetin significiantly higher than quercetin.This HPLC system for determination on FOMT activity could be considered as a method which was accuracy,fastness,reliability and high sensitivity.