Department of Forensic Medicine, Juntendo University School of Medicine, 2-1-1, Hongo, Bunkyo-Ku, Tokyo, 113-8421, Japan.
Department of Forensic Medicine, Saitama Medical University, 38 Morohongo, Moroyama, 350-0495, Saitama, Japan.
J Nat Med. 2020 Jan;74(1):275-281. doi: 10.1007/s11418-019-01360-1. Epub 2019 Sep 6.
We evaluated whether the origins of crude drugs derived from arthropods and annelids could be identified using molecular biological techniques. DNA was extracted from 20 crude drugs prepared from different animals using a commercial kit with added phenol treatment. The target regions used to identify origin were the mitochondrial 16S ribosomal RNA (rRNA), 12S rRNA, and cytochrome oxidase subunit I (COI) gene regions. Extracted DNA was amplified by polymerase chain reaction, and then sequenced by the Sanger method. The aligned sequences were compared with all available sequences using BLAST to estimate the origins of the crude drugs. The origin of crude drugs used in this study could be estimated using this method. The COI region was the best for identifying origin among three regions examined, based on the success rate of PCR amplification and analysis. Moreover, the 12S rRNA region was also useful for origin identification, with the exception of the earthworm. However, the origin of some crude drugs could not be strictly identified due to matches to various species in all three regions. One likely cause was that the species of origin of a crude drug has not been registered in DNA databases. We found that even the same crude drug from the same pharmaceutical company had different origins by production lot or import source country. Therefore, this method is useful not only for DNA-based origin identification but also quality control of production lots.
我们评估了使用分子生物学技术是否可以鉴定来源于节肢动物和环节动物的生药的起源。使用添加了苯酚处理的商业试剂盒从 20 种不同动物制备的生药中提取 DNA。用于鉴定起源的目标区域是线粒体 16S 核糖体 RNA(rRNA)、12S rRNA 和细胞色素氧化酶亚基 I(COI)基因区域。提取的 DNA 通过聚合酶链反应扩增，然后通过 Sanger 法测序。使用 BLAST 将对齐的序列与所有可用序列进行比较，以估计生药的起源。通过这种方法可以估计本研究中使用的生药的起源。根据 PCR 扩增和分析的成功率，COI 区域是三个检查区域中鉴定起源的最佳区域。此外，除了蚯蚓之外，12S rRNA 区域也可用于鉴定起源，但某些生药的起源不能严格鉴定，因为在所有三个区域中都与各种物种匹配。一个可能的原因是生药的起源物种尚未在 DNA 数据库中注册。我们发现，即使是来自同一制药公司的同种生药，由于生产批次或进口来源国的不同，其起源也不同。因此，该方法不仅可用于基于 DNA 的起源鉴定，还可用于生产批次的质量控制。
