蒙成药安神补心六味丸多维鉴定研究

Multidimensional Identification of Inner Mongolian Medicine Anshen Buxin Liuwei Pills(安神补心六味丸)

目的 建立蒙成药安神补心六味丸组成药材基原系统化的鉴定方法。方法 通过高通量测序技术对蒙成药安神补心六味丸进行总DNA的提取,并对其COI及ITS2序列进行PCR扩增,双端测序后进行序列的整理和聚类分析,并与组成安神补心六味丸中牛心的COI序列,木香、丁香、广枣、肉豆蔻药材的ITS2序列进行测序的结果利用邻接法构建系统树。利用薄层色谱方法以正己烷-石油醚(60~90℃)-乙酸乙酯-冰醋酸(6∶2∶3∶0.2)为展开剂。展开后烘箱烘干,置紫外光灯(254 nm)下检视。最后对蒙成药安神补心六味丸进行光学显微鉴定观察,确定其显微特征,并将显微特征与组成中药显微特征进行对应。结果 基于对蒙成药安神补心六味丸的ITS2序列进行高通量测序后得到高质量序列共138 633条,经过比对与安神补心六味丸组成药味相关的序列共584条,分别为木香(Aucklandia lappa)、南酸枣(Choerospondias axillaris)、丁香(Eugenia caryophyllata),均为木香、广枣、丁香药材的正品植物来源。在新鲜牛心中成功提取到COI序列,但未提取到安神补心六味丸成药及干燥牛心的COI序列,木香、丁香、广枣、肉豆蔻的ITS2序列成功扩增,扩增的ITS2序列与高通量测序比对后获得的OTU代表序列在系统树与木香、丁香、广枣分别聚为一支。安神补心六味丸与枫香脂及对照药材在相同条件下,薄层色谱同一处出现斑点。在对成药进行显微鉴定后发现与组成药味一致的显微特征。结论 结果表明以ITS2序列作为条形码,利用高通量测序的ITS2序列技术为主要鉴别手段,结合薄层色谱及对蒙成药显微特征观察,可以对安神补心六味丸中6种中药的基原进行准确的鉴别。

Objective To establish a systematic identification method for the basic material of Inner Mongolian medicine Anshen Buxin Liuwei Pills(安神补心六味丸).Methods High-throughput sequencing technology was used to extract total DNA from Anshen Buxin Liuwei Pills,and PCR amplification was performed on its COI and ITS2 sequences.After double-ended sequencing,sequence arrangement and cluster analysis were carried out,and the COI sequences of cow heart in Anshen Buxin Liuwei Pills were analyzed.The ITS2 sequence was sequenced for the medicinal materials of Muxiang(Aucklandiae Radix),Dingxiang(Caryophylli Flos),Guangzao(Choerospondiatis Fructus),Roudoukou(Myristicae Semen).The system tree was constructed by adjacency method.N-hexan-petroleum ether(60-90℃)-ethyl acetate-glacial acetic acid(6∶2∶3∶0.2)was developed by thin layer chromatography.After unfolding,dry in oven and place under ultraviolet lamp(254 nm)for inspection.Finally,the optical microscopic identification and observation of Anshen Buxin Liuwei Pills were carried out to determine its microscopic characteristics,and the microscopic characteristics were corresponding to the composition of traditional Chinese medicine.Results The COI sequence of Anshen Buxin Liuwei Pills was not extracted.Based on high-throughput sequencing of ITS2 sequence of Anshen Buxin Liuwei Pills,a total of 138633 high-quality ITS2 sequences were obtained,and 584 sequences related to the composition of Anshen Buxin Liuwei Pills were compared.Aucklandialappa,Choerospondiasaxillaris and Eugeniacaryophyllata were the plant sources of the medicinal materials of Muxiang(Aucklandiae Radix),Guangzao(Choerospondiatis Fructus),Dingxiang(Caryophylli Flos),respectively,which were stipulated in the 2020 edition of Chinese Pharmacopoeia of the People's Republic of China.COI sequences were successfully extracted from fresh cattle,but not from dry cattle.ITS2 sequences of Muxiang(Aucklandiae Radix),Dingxiang(Caryophylli Flos),Guangzao(Choerospondiatis Fructus),Roudoukou(Myristicae Semen)were amplified successfully.The amplified ITS2 sequences were compared with the OTU representative sequences obtained by high-throughput sequencing,and the ITS2 of Muxiang(Aucklandiae Radix),Dingxiang(Caryophylli Flos),Guangzao(Choerospondiatis fructus)were clustered into one branch in the system tree,respectively.The spots of Anshen Buxin Liuwei Pills and maple Fengxiangzhi(Liquidambaris Resina)and control herbs appeared in the same spot of thin layer chromatography under the same conditions.The microscopic characteristics were found to be consistent with the composition of the drug.Conclusion The results showed that ITS2 sequence was used as bar code,high-throughput sequencing technology was used as the main identification means,combined with thin layer chromatography and observation of the microscopic characteristics of Inner Mongolian medicine,the six traditional Chinese medicine bases in Anshen Buxin Liuwei Pills could be accurately identified.