哈蟆油基原考证及DNA条形码物种鉴定研究  

Herbalogical studies of Ranae oviductus(Hamayou) and its species identification using DNA barcoding method

作　　者：石林春[1,4] 刘金欣 唐先明 刘建辉[2] 姚辉[1] 张立秋[3] 向丽[4] 张琴[1] 谢彩香[1] 宋经元[1] SHI LinChun;LIU JinXin;TANG XianMing;LIU JianHui;YAO Hui;ZHANG LiQiu;XIANG Li;ZHANG Qin;XIE CaiXiang;SONG JingYuan(Engineering Research Center of Traditional Chinese Medicine Resource,Ministry of Education,Key Lab of Chinese Medicine Resources Conservation,State Administration of Traditional Chinese Medicine of China,Institute of Medicinal Plant Development,Chinese Academy of Medical Sciences&Peking Union Medical College,Beijing 100193,China;Harbin Food and Drug Inspection Center,Harbin 150525,China;College of Pharmaceutical and Food Science,Tonghua Normal University,Tonghua 134002,China;Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China)

机构地区：[1]中国医学科学院北京协和医学院药用植物研究所,国家中医药管理局中药资源保护重点研究室,中药资源教育部工程研究中心,北京100193 [2]哈尔滨市食品药品检验检测中心,哈尔滨150525 [3]通化师范学院制药与食品科学学院,通化134002 [4]中国中医科学院中药研究所,北京100700

出　　处：《中国科学：生命科学》2019年第8期1032-1040,共9页Scientia Sinica(Vitae)

基　　金：国家自然科学基金(批准号:81703659);中国博士后科学基金(批准号:2017M610815);公益性行业科研专项经费项目(批准号:201507002-4-1-2);四川省科技计划项目重点研发项目(批准号:2019YFS0017)资助

摘　　要：哈蟆油为我国珍稀名贵中药材,存在混伪品多、基原有争议、系统分类历史复杂、鉴定困难等难题.本研究首先通过本草考证和现代研究资料整理,结合基于线粒体全基因组的分子系统发育关系分析,以及哈蟆油传统产区与原动物地理分布叠加比较,证实中国林蛙、东北林蛙和黑龙江林蛙均为有效种,中国林蛙仅限于华北和华东分布,拉丁名应为“R.chensinensis David,1875”,《中国药典》采用“Rana temporariachensinensis David”为中国林蛙拉丁名不妥,哈蟆油的基原应为东北林蛙R.dybowskii Günther,1876.然后设计新的PCR引物以解决蛤蟆油COI序列的非特异性扩增难题,建立包含哈蟆油及其混伪品的DNA条形码物种鉴定数据库.最后,基于市售药材验证所建立的哈蟆油DNA条形码物种鉴定方法,发现市场中存在青蛙油、牛蛙油等混伪品,DNA条形码技术可将哈蟆油及其混伪品准确鉴定到物种水平.本研究将为保障哈蟆油的临床用药安全和消费者的合法权益提供技术支持.Ranae oviductus is a rare and precious Chinese medicine;however,there are many challenges connected with identifying this medicine,such as presence of many adulterants,the controversy over its origin,and the complexity of its classification history.First,we performed textual criticism of herbal literature and modern research data,constructed a phylogenetic relationship using whole mitochondrial genomes,and created a spatial overlay for the traditional production areas of Ranae oviductus and animal geographical distribution.The results obtained confirm that Rana chensinensis,R.dybowskii,and R.amurensis can be considered as a valid species for Ranae oviductus and that the Chinese Pharmacopoeia’s use of Rana temporaria chensinensis David.as the scientific name for Chinese brown frog is inappropriate.We further believe that the original species for Ranae oviductus could be R.dybowskii.Second,a debonding solution was prepared and used to pretreat medicinal materials to overcome the harmful effects of high expansion properties of Ranae oviductus during its DNA extraction.A new polymerase chain reaction primer pair was designed to resolve nonspecific amplification of cytochrome c oxidase subunit I(COI)sequences of Ranae oviductus using universal primers.A DNA barcoding reference library was established that contained 259 COI barcodes for 43 species covering Ranae oviductus and most of its adulterants.Finally,the DNA barcoding procedure for the identification of Ranae oviductus was verified based on simulation of real identification practices using commercially available medicinal materials.The results indicate that there are several adulterants,such as frog oil(Pelophylax nigromaculatus)and bullfrog oil(R.catesbeiana),in the herbal market.This study shows that DNA barcoding can successfully distinguish Ranae oviductus and its adulterants.In addition,the procedure established in this study can provide strong support for the safety of Ranae oviductus and protect the interests of consumers.

关 键 词：哈蟆油 基原物种 DNA条形码 混伪品 鉴定 

分 类 号：R28[医药卫生—中药学]