A PCR-lateral flow immunochromatographic assay (PCR-LFA) for detecting Aristolochia species, the plants responsible for aristolochic acid nephropathy.

Aristolochic acids (AAs), which are strong carcinogens, have caused dietary supplements with Aristolochia plants to be discontinued worldwide. Therefore, the development of a method to identify these herbs is critical for customer safety. To support the regulation of Aristolochia-free products, a PCR coupled with lateral flow immunochromatographic assay (PCR-LFA) that is specific to the nucleotide signature in plastid rbcL gene region of Aristolochia species was developed to detect Aristolochia plants and related herbal products. Triplex primers (A397F, C357F and R502) were designed based on specific nucleotides observed exclusively in the rbcL sequences of Aristolochia. Positive results for Aristolochia occur when the three pink lines are clearly developed on the developed lateral flow strip and can be seen by the naked eye. In this study, the lateral flow strip has sensitivity for detecting amplicons amplified from genomic DNA at the concentrations as low as 0.01 ng. Various kinds of samples, including purchased crude drugs and polyherbal samples, have been investigated, and the results showed that Aristolochia crude drugs and Aristolochia-containing products are still present in dispensaries. In conclusion, with the goal of protecting consumers from the health risks associated with Aristolochia contamination, PCR-LFA was developed and demonstrated to be efficient for detecting plants belonging to Aristolochia in various kinds of samples.

Combining DNA and HPTLC profiles to differentiate a pain relief herb, Mallotus repandus, from plants sharing the same common name, "Kho-Khlan".

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.

Development of a DNA barcode library of plants in the Thai Herbal Pharmacopoeia and Monographs for authentication of herbal products.

Traditional herbal medicine has long been practiced as a method of health care in many countries worldwide. The usage of herbal products has been increasing and is expected to continue to do so in the future. However, admixture and adulteration are concerns regarding the quality of herbal medicine, including its safety and efficacy. We aimed to develop a reference DNA barcode library of plants listed in the Thai Herbal Pharmacopoeia (THP) and Monographs of Selected Thai Materia Medica (TMM) (n = 101 plant species) using four core barcode regions, namely, the ITS2, matK, rbcL and trnH-psbA intergenic spacer regions, for authentication of the plant origin of raw materials and herbal products. Checking sequences from samples obtained from local markets and the Thai Food and Drug Administration (Thai FDA) against our digital reference DNA barcode system revealed the authenticity of eighteen out of twenty tested samples as claimed on their labels. Two samples, no. 3 and 13, were not Cyanthillium cinereum (L.) H.Rob. and Pueraria candollei Wall. ex Benth. as claimed, respectively. They were recognized as Emilia sonchifolia (L.) DC. and Butea superba (Roxb.), respectively. Hence, it is important for the Thai FDA or regulatory agencies to immediately initiate strict enforcement for the development of pharmacopoeial standards as well as revisions or modifications of available regulatory guidelines and to implement close monitoring for the quality control of herbal products in terms of authentication before they enter the herbal market. The centralized digital reference DNA barcode database developed here could play a very important role in monitoring or checking the authenticity of medicinal plants.

PCR combined with lateral flow immunochromatographic assay to differentiate the narcotic Mitragyna speciosa from related species and detect it in forensic evidence.

Plants in the genus Mitragyna (Rubiaceae) are used in traditional medicine because of their broad therapeutic activity. Four Mitragyna species, M. speciosa (Roxb.) Korth. (MS), M. rotundifolia (Roxb.) Kuntze (MR), M. diversifolia (Wall. ex G. Don) Havil. (MD), and M. hirsuta Havil. (MH), occur in Thailand. M. speciosa, commonly known as 'Kratom' in Thai, is the only narcotic species for which buying, selling, importing or possessing has been prohibited by law in Thailand and some other countries. Mitragynine and 7-hydroxymitragynine, the major psychoactive compounds, are important in the treatment of opioid withdrawal. However, this species is used in traditional medicine to relieve pain and inflammation. Consequently, a rapid and easy technique for differentiating M. speciosa from closely related species is needed for routine forensic analysis. In this study, polymerase chain reaction coupled with lateral flow immunochromatographic assay (PCR-LFA) based on matK was developed for the detection of M. speciosa in forensic specimens. Duplex primers (MS-F-FAM, Ctrl-F-DIG and Ctrl-R-Biotin) were designed based on species-specific nucleotide indels observed exclusively in the matK sequences of M. speciosa. Positive results for M. speciosa are indicated by the clear presence of three black lines on the lateral flow cassette. Forensic samples were investigated, and the three black test lines indicating M. speciosa were observed for seven of eight specimens. PCR-LFA has been proven to be fast, easy and efficient for detecting the narcotic M. speciosa and could be developed as a rapid forensic diagnostic technique for other plants.

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

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.

DNA metabarcoding to unravel plant species composition in selected herbal medicines on the National List of Essential Medicines (NLEM) of Thailand.

Traditional medicines are widely traded across the globe and have received considerable attention in the recent past, with expectations of heightened demand in the future. However, there are increasing global concerns over admixture, which can affect the quality, safety, and efficacy of herbal medicinal products. In this study, we aimed to use DNA metabarcoding to identify 39 Thai herbal products on the Thai National List of Essential Medicines (NLEM) and assess species composition and admixture. Among the products, 24 samples were in-house-prepared formulations, and 15 samples were registered formulations. In our study, DNA metabarcoding analysis using ITS2 and rbcL barcode regions were employed to identify herbal ingredients mentioned in the products. The nuclear region, ITS2, was able to identify herbal ingredients in the products at the genus- and family-levels in 55% and 63% of cases, respectively. The chloroplast gene, rbcL, enabled genus- and family-level identifications in 58% and 73% of cases, respectively. In addition, plant species were detected in larger numbers (Family identified, absolute %) in registered herbal products than in in-house-prepared formulations. The level of fidelity increases concerns about the reliability of the products. This study highlights that DNA metabarcoding is a useful analytical tool when combined with advanced chemical techniques for the identification of plant species in highly processed, multi-ingredient herbal products.

Differentiation of Cyanthillium cinereum, a smoking cessation herb, from its adulterant Emilia sonchifolia using macroscopic and microscopic examination, HPTLC profiles and DNA barcodes.

Cyanthillium cinereum (L.) H.Rob. is one of the most popular herbal smoking cessation aids currently used in Thailand, and its adulteration with Emilia sonchifolia (L.) DC. is often found in the herbal market. Therefore, the quality of the raw material must be considered. This work aimed to integrate macro- and microscopic, chemical and genetic authentication strategies to differentiate C. cinereum raw material from its adulterant. Different morphological features between C. cinereum and E. sonchifolia were simply recognized at the leaf base. For microscopic characteristics, trichome and pappus features were different between the two plants. HPTLC profiles showed a distinct band that could be used to unambiguously differentiate C. cinereum from E. sonchifolia. Four triterpenoid compounds, ß-amyrin, taraxasterol, lupeol, and betulin, were identified from the distinct HPTLC band of C. cinereum. The use of core DNA barcode regions; rbcL, matK, ITS and psbA-trnH provided species-level resolution to differentiate the two plants. Taken together, the integration of macroscopic and microscopic characterization, phytochemical analysis by HPTLC and DNA barcoding distinguished C. cinereum from E. sonchifolia. The signatures of C. cinereum obtained here can help manufacturers to increase the quality control of C. cinereum raw material in commercialized smoking cessation products.

Mitigating the Impact of Admixtures in Thai Herbal Products.

Medicinal plants and their products are extensively used within indigenous healthcare systems in Thailand and several other nations. The international trade of herbal products has a noteworthy impact on the worldwide economy, and the interest in herbal products is expanding in both developing and developed countries. There has been rapid growth in the medicinal plant product market and a broadening consumer base interested in herbal products from Thailand. However, in herbal industries, ingredient substitution and admixture are typical issues wherein species of lower market value are admixed with those of a higher value. The adverse consequences of consuming adulterated drugs are invariably due to the presence of an unintended herb rather than the presence of an intended herb. It has also been argued that admixtures are intentional because of the lack of regulatory policies or centralized tests for product authentication. The consequences of species admixtures can extend from the reduced efficacy of a drug to decreased trade value. This study aims to clarify the nature and extent of species admixtures reported in the Thai herbal trade market and discuss the potential reasons for such adulteration. In the broader context of species admixtures, we strongly propose the establishment of multiple herbal crude drug repositories that can be developed to facilitate the use of comparative identity tests by industry, traders, and researchers to maintain authentic natural health product (NHP) standards and to certify the authenticity of NHPs. The proposition of the establishment of centralized testing (CT) could be a promising initiative in Thailand for the development of science and technology, and the herbal medicines produced as a result of CT could be dispensed as prescription drugs based on disease consideration instead of as health foods or nutraceuticals.

DNA barcoding of species of Bacopa coupled with high-resolution melting analysis.

In Thailand, there are three species of Bacopa, namely, B. monnieri, B. caroliniana, and B. floribunda. Among these species of Bacopa, B. monnieri is the only medicinal species, used for the treatment of cognitive impairment and improvement of cognitive abilities because of its bioactive constituents, bacoside A and B. However, because of the similar characteristics of these species, it is difficult to differentiate among related species, resulting in confusion during identification. For this reason, and to ensure therapeutic quality for consumers, authentication is important. In this study, the three abovementioned species of Bacopa were evaluated using barcoding coupled with high-resolution melting (Bar-HRM) analysis based on primers designed for the trnL-F sequences of the three species. The melting profiles of the trnL-F amplicons of B. caroliniana and B. floribunda were clearly different from the melting profile of the trnL-F amplicon from B. monnieri; thus, the species could be discriminated by Bar-HRM analysis. Bar-HRM was then used to authenticate commercial products in various forms. The melting curves of the six commercial samples indicated that all the tested products contained genuine B. monnieri species. This method provides an efficient and reliable authentication system for future commercial herbal products and offers a reference system for quality control.