Differentiation of Mitragyna speciosa, a narcotic plant, from allied Mitragyna species using DNA barcoding-high-resolution melting (Bar-HRM) analysis

Chayapol Tungphatthong,Thongchai Sooksawate,Supita Awachai,Suchada Sukrong,Santhosh Kumar J Urumarudappa

doi:10.1038/s41598-021-86228-9

Chayapol Tungphatthong, Thongchai Sooksawate + Show 3 more

Open Access

https://doi.org/10.1038/s41598-021-86228-9

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Journal: Scientific Reports	Publication Date: Mar 24, 2021
Citations: 12	License type: open-access

Affiliation: Chulalongkorn University

Abstract

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.

Highlights

In Thailand, Mitragyna speciosa (Korth.) Havil. [MS], Mitragyna diversifolia
This study showed that the ITS2 amplicons had the highest GC content (63.27%), the trnH-psbA intergenic spacer amplicons had the lowest GC content (35.35%) (Table 2), and nucleotide variations in these regions exhibited the largest difference in Tm among the four Mitragyna species (Table 3)
To the best of our knowledge, this is the first study of DNA barcoding coupled with HRM analysis for the differentiation of M. speciosa, a narcotic species, from closely related species and for the investigation of suspected kratom samples

Summary

Introduction

In Thailand, Mitragyna speciosa (Korth.) Havil. [MS], Mitragyna diversifolia Kuntze [MR], species belonging to the Rubiaceae family with specific medicinal importance, are commonly distributed in the central to southern regions[1,2,3] Among these Mitragyna species, M. speciosa is a narcotic plant, and recently, it has been used as an opium substitute and for the treatment of addiction to m orphine[4]. M. speciosa is substituted with other plant species, including Mitragyna species, or adulterated. Even species within the same genus may exhibit differences in pharmacological action This inadvertent substitution and adulteration can cause intoxication and even death[20,22]. Several studies have shown that very closely related medicinal plant species can be distinguished accurately by using DNA barcoding combined with high-resolution melting (Bar-HRM) a nalysis[21,25,26,27]. The generated Bar-HRM analysis profiles enable verification of the authenticity of narcotic plant species for law enforcement

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