Research Unit of DNA Barcoding of Thai Medicinal Plants, Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, 10330, Thailand.
Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok, 10330, Thailand.
Sci Rep. 2021 Mar 24;11(1):6738. doi: 10.1038/s41598-021-86228-9.
Mitragyna speciosa (Korth.) Havil. [MS], or "kratom" in Thai, is the only narcotic species among the four species of Mitragyna in Thailand, which also include Mitragyna diversifolia (Wall. ex G. Don) Havil. [MD], Mitragyna hirsuta Havil. [MH], and Mitragyna rotundifolia (Roxb.) O. Kuntze [MR]. M. speciosa is a tropical tree belonging to the Rubiaceae family and has been prohibited by law in Thailand. However, it has been extensively covered in national and international news, as its abuse has become more popular. M. speciosa is a narcotic plant and has been used as an opium substitute and traditionally used for the treatment of chronic pain and various illnesses. Due to morphological disparities in the genus, the identification of plants in various forms, including fresh leaves, dried leaf powder, and finished products, is difficult. In this study, DNA barcoding combined with high-resolution melting (Bar-HRM) analysis was performed to differentiate M. speciosa from allied Mitragyna and to assess the capability of Bar-HRM assays to identify M. speciosa in suspected kratom or M. speciosa-containing samples. Bar-HRM analysis of PCR amplicons was based on the ITS2, rbcL, trnH-psbA, and matK DNA barcode regions. The melting profiles of ITS2 amplicons were clearly distinct, which enabled the authentication and differentiation of Mitragyna species from allied species. This study reveals that DNA barcoding coupled with HRM is an efficient tool with which to identify M. speciosa and M. speciosa-containing samples and ensure the safety and quality of traditional Thai herbal medicines.
泰国的四种钩藤属植物（Mitragyna）中，只有美托占（Mitragyna speciosa）（Korth.）Havil. [MS]，即泰语中的“咔特”，属于麻醉品种。其他三种分别为宽果钩藤（Mitragyna diversifolia）（Wall. ex G. Don）Havil. [MD]、纤细钩藤（Mitragyna hirsuta Havil.）[MH]和圆叶钩藤（Mitragyna rotundifolia）（Roxb.）O. Kuntze [MR]。美托占是一种热带树种，属于茜草科（Rubiaceae），在泰国被法律禁止。然而，由于其滥用情况越来越普遍，它在国内外新闻中被广泛报道。美托占是一种麻醉植物，曾被用作鸦片替代品，传统上用于治疗慢性疼痛和各种疾病。由于该属植物在形态上存在差异，因此识别各种形式的植物，包括新鲜叶片、干叶粉和成品，都具有一定难度。在这项研究中，采用 DNA 条形码结合高分辨率熔解（Bar-HRM）分析，以区分美托占与其近缘种，并评估 Bar-HRM 分析在鉴定疑似咔特或含有美托占的样品中的能力。PCR 扩增子的 Bar-HRM 分析基于 ITS2、rbcL、trnH-psbA 和 matK DNA 条形码区。ITS2 扩增子的熔解曲线图谱清晰可辨，可用于鉴定钩藤属物种及其近缘种。本研究表明，DNA 条形码结合 HRM 是一种有效的工具，可用于鉴定美托占及其含美托占的样品，以确保泰国传统草药的安全性和质量。
