Faculty of Pharmaceutical Sciences Chulalongkorn University, Department of Pharmacognosy and Pharmaceutical Botany, Center of Excellence in DNA Barcoding of Thai Medicinal Plants, Bangkok, Thailand.
PLoS One. 2022 Jun 9;17(6):e0268680. doi: 10.1371/journal.pone.0268680. eCollection 2022.
The pain relief formula "Ya Pa Som Kho-Khlan (YPSKK)" or "ยาผสมโคคลาน" in Thai is officially recorded in the Natural List of Essential Medicines (NLEM) of Thailand. The main component is Mallotus repandus (Willd.) Müll. Arg.; however, Anamirta cocculus (L.) Wight & Arn and Croton caudatus Gleiseler share the same common name: "Kho-Khlan". Confused usage of A. cocculus or C. caudatus can have effects via toxicity or unsuccessful treatment. This study aimed to combine a high-performance thin-layer chromatography (HPTLC) technique and DNA barcoding coupled with high-resolution melting (Bar-HRM) to differentiate M. repandus from the other two species. The M. repandus extract exhibited a distinct HPTLC profile that could be used to differentiate it from the others. DNA barcodes of the rbcL, matK, ITS and psbA-trnH intergenic spacer regions of all the plants were established to assist HPTLC analysis. The rbcL region was selected for Bar-HRM analysis. PCR amplification was performed to obtain 102 bp amplicons encompassing nine polymorphic nucleotides. The amplicons were subjected to HRM analysis to obtain melting curve profiles. The melting temperatures (Tm) of authentic A. cocculus (A), C. caudatus (C) and M. repandus (M) were separated at 82.03±0.09°C, 80.93±0.04°C and 80.05±0.07°C, respectively. The protocol was applied to test crude drugs (CD1-6). The HPTLC profiles of CD2-6 showed distinct bands of M. repandus, while CD1 showed unclear band results. The Bar-HRM method was applied to assist the HPTLC and indicated that CD1 was C. caudatus. While ambiguous melting curves from the laboratory-made formulae were obtained, HPTLC analysis helped reveal distinct patterns for the identification of the plant species. The combination of HPTLC and Bar-HRM analysis could be a tool for confirming the identities of plant species sharing the same name, especially for those whose sources are multiple and difficult to identify by either chemical or DNA techniques.
泰国官方将一种名为“Ya Pa Som Kho-Khlan（YPSKK）”或“ยาผสมโคคลาน”的止痛配方列入泰国基本药物自然清单（NLEM）。其主要成分是 Mallotus repandus（Willd.）Müll. Arg.；然而，Anamirta cocculus（L.）Wight & Arn 和 Croton caudatus Gleiseler 有相同的通用名：“Kho-Khlan”。A. cocculus 或 C. caudatus 的混淆使用可能会导致毒性或治疗失败。本研究旨在结合高效薄层色谱（HPTLC）技术和 DNA 条形码与高分辨率熔解（Bar-HRM）技术，从其他两种物种中区分 Mallotus repandus。M. repandus 提取物表现出独特的 HPTLC 图谱，可用于将其与其他物种区分开来。所有植物的 rbcL、matK、ITS 和 psbA-trnH 基因间隔区的 DNA 条形码已建立，以辅助 HPTLC 分析。选择 rbcL 区域进行 Bar-HRM 分析。进行 PCR 扩增以获得包含九个多态核苷酸的 102 bp 扩增子。将扩增子进行 HRM 分析以获得熔解曲线图谱。鉴定的 A. cocculus（A）、C. caudatus（C）和 M. repandus（M）的扩增子 Tm 分别在 82.03±0.09°C、80.93±0.04°C 和 80.05±0.07°C 处分离。该方案应用于测试原料药（CD1-6）。CD2-6 的 HPTLC 图谱显示出 Mallotus repandus 的独特带，而 CD1 的带结果不清晰。Bar-HRM 方法用于辅助 HPTLC，并表明 CD1 为 C. caudatus。虽然从实验室配方中获得了模糊的熔解曲线，但 HPTLC 分析有助于揭示植物物种鉴定的独特模式。HPTLC 和 Bar-HRM 分析的结合可以成为确认同名植物物种身份的工具，特别是对于那些来源多样且难以通过化学或 DNA 技术识别的物种。
