进口药材“大、小广天仙子”的鉴别研究

Identification of the Semen Hygrophilae Megalanthae in Two Size from Imported Materials

目的多种方法联合鉴别进口药材“大、小广天仙子”。方法参照《儿茶等43种进口药材质量标准》中性状和显微鉴别方法,并根据2020年版中国药典中药材DNA条形码分子鉴定法指导原则分析相关ITS1、ITS2、pshA-trnH、matK、rbcL基因序列信息,利用关键差异位点探索建立聚合酶链式反应法-限制性片段长度多态性分析(PCR-RFLP)鉴别方法。结果进口“大、小广天仙子”在显微特征和5个DNA条形码基因上均具有明显差异。“大广天仙子”符合进口药材标准规定;“小广天仙子”来源于另一个物种,为混伪品。利用ITS2上能被限制性内切酶NdeⅠ识别的关键差异位点可建立新的PCR-RFLP鉴别法。结论采用显微鉴别和PCR-RFLP法可有效鉴别广天仙子真伪。

OBJECTIVE To identify the semen hygrophilae megalanthae in two size from imported materials by various methods.METHODS The traditional identification methods were used according to the Regulatory Specification of 43 Imported Medicinal Materials such as Catechu.The PCR-RFLP identification method was established on sequencing the ITS1、ITS2、pshA-trnH、matK、rbcL with DNA bacording technique from the Chinese pharmacopoeia 2020.RESULTS There were obvious difference in microscopic characteristics,and some variation sites in the 5 DNA bacording genes with the semen hygrophilae megalanthae in two size.The larger one was in compliance with the imported regulations,while the smaller adulterants was a separate species.The PCR-RFLP method with recognizing the criticalsite in ITS2 and digestingthe amplicifition product of semen hygrophilae megalanthae by restriction enzyme NdeⅠwas established.CONCLUSION The microscopic identification and PCR-RFLP method can effectively identify semen hygrophilae megalanthae and its adulterants.