HPLC and DNA barcoding profiles for identification of the selected twelve Mucuna species and its application for detecting prohibited aphrodisiac Mucuna products.

Aphrodisiac herbal products originated from various plants including Mucuna species. In Thai folklore, Mucuna macrocarpa Wall. and M. pruriens (L.) DC. have long been consumed and utilized for their aphrodisiac properties. Consumption of these plants can lead to serious adverse effects caused by l-dopa. The plants have been legally banned for use as foods, dietary supplements, or nutraceuticals by the FDA of several countries. To protect consumers, methods for the identification of illicit plants or herbal products are needed. This study aimed to identify the selected twelve Mucuna species and examine the aphrodisiac herbal products containing M. macrocarpa and M. pruriens by using HPLC analysis of l-dopa coupled with DNA barcoding profiles of ITS, matK, rbcL, and trnH-psbA. The results showed that l-dopa could be found not only in the seeds of M. macrocarpa and M. pruriens but also in associated allied Mucuna species. Then, a DNA barcode was introduced to support in HPLC profiling to identify the plants. DNA barcodes of twelve Mucuna species found in Thailand were established and used to reconstruct a phylogenetic tree. In this study, ITS2 sequences showed the highest interspecific variability and could be used to differentiate all Mucuna species. The results of ITS2 sequence coupled with HPLC analysis revealed that all the purchased aphrodisiac products originated from M. pruriens only. Therefore, the integration of HPLC analysis and DNA barcoding profile was an efficient method for the identification of prohibited Mucuna species for safety monitoring of herbal supplements and protecting customer safety. Regulatory agencies should raise awareness and restrain the use of these commercial products.

The Identification and Cytotoxic Evaluation of Nutmeg (Myristica fragrans Houtt.) and Its Substituents.

The aril and seed of nutmeg, Myristica fragrans Houtt. (Myristicaceae), hold significant value in various industries globally. Our preliminary research found two morphological variations: a globose shape and an oval shape. Due to these different characteristics, the safety of consumers is of primary concern. Thus, authentication and comparative pharmacological and toxicity analyses are necessary. In this study, pharmacognostic and advanced phytochemical analyses, DNA barcoding, cytotoxicity, and the anti-nitric oxide production of commercial Thai nutmeg were examined. Via morphologic examinations and TLC fingerprinting, all the sampled aril and seed were categorized into globose and oval-shaped groups. The results of HPLC, GC-MS, and LC-MS/MS experiments revealed distinct differences between these groups. The DNA barcoding of the trnH-psbA region using the BLAST method and neighbor-joining tree analyses confirmed the globose nutmeg as M. fragrans and the oval-shaped variant as M. argentea. A comparison was then carried out between the potential toxicity and anti-inflammatory capabilities of M. fragrans and M. argentea. Cytotoxicity tests on HaCaT, 3T3-L1, Caco-2, HEK293, and RAW264.7 were performed using both methanolic extracts and volatile oil from the arils and seeds of both species. This study concludes that blending or substituting these two species maintains their therapeutic integrity without posing safety concerns.

A Comprehensive Review of Andrographis paniculata (Burm. f.) Nees and Its Constituents as Potential Lead Compounds for COVID-19 Drug Discovery.

The COVID-19 pandemic has intensively disrupted global health, economics, and well-being. Andrographis paniculata (Burm. f.) Nees has been used as a complementary treatment for COVID-19 in several Asian countries. This review aimed to summarize the information available regarding A. paniculata and its constituents, to provide critical points relating to its pharmacological properties, safety, and efficacy, revealing its potential to serve as a source of lead compounds for COVID-19 drug discovery. A. paniculata and its active compounds possess favorable antiviral, anti-inflammatory, immunomodulatory, and antipyretic activities that could be beneficial for COVID-19 treatment. Interestingly, recent in silico and in vitro studies have revealed that the active ingredients in A. paniculata showed promising activities against 3CLpro and its virus-specific target protein, human hACE2 protein; they also inhibit infectious virion production. Moreover, existing publications regarding randomized controlled trials demonstrated that the use of A. paniculata alone or in combination was superior to the placebo in reducing the severity of upper respiratory tract infection (URTI) manifestations, especially as part of early treatment, without serious side effects. Taken together, its chemical and biological properties, especially its antiviral activities against SARS-CoV-2, clinical trials on URTI, and the safety of A. paniculata, as discussed in this review, support the argument that A. paniculata is a promising natural source for drug discovery regarding COVID-19 post-infectious treatment, rather than prophylaxis.

Conventional and molecular pharmacognostic characters integrated with chemical profiles of five Piper plants in the Thai herbal pharmacopoeia and their admixture/adulteration/substitution situations in Thailand.

The morphological and microscopy were combined with DNA-barcoding, together with rapid TLC for the characterization of Piper betle (PB), P. nigrum (PN), P. retrofractum (PR), P. sarmentosum (PS), and P. wallichii (PW), five medicinal Piper plants announced in the Thai Herbal Pharmacopoeia (THP). The authentic plants collected from various locations and voucher Piper products bought from commercial sites in Thailand were studied. The reproductive parts of authentic plants were subjected to ensure their morphological characters. Using sequencing analysis and genetic divergence for analyzing discriminatory performance, ITS2 was selected from eight candidate DNA markers to authenticate the origin of Piper crude drugs together with microscopic and TLC profiles for examining their characters, admixtures, adulterants, and substituents. PB and PR exhibited unique characters of the species, with no admixture, adulteration, and substitution. PN showed no variable characters of morphology and genetics. However, the microscopy could illustrate some commercial products of PN sold in Thailand have been adulterated with rice starch and roasted rice. In the herbal trade, PS has been sold in the form of mixed leaf, root, and stem more than the isolated part, but there is no variable character of the species. PW has shown more than one character of species explained by microscopic, chemical components, and genetic data. In conclusion, the conventional and molecular pharmacognostic data combined with chemical profile of authentic five Piper plants could be applied to indicate the plant origin and clarify the situations of admixture, adulteration, and substitution of the commercial Piper products launched in Thailand.

Specification and DNA Barcoding of Thai Traditional Remedy for Chronic Kidney Disease: Pikad Tri-phol-sa-mut-than.

The Pikad Tri-phol-sa-mut-than (TS) remedy, a Thai traditional medicine, is officially recorded in Tamra Paetsart Sonkrau Chabub Anurak for its capabilities in treating kidney deficiency. TS remedy is composed of three fruit species-Aegle marmelos (L.) Corrêa., Coriandrum sativum L., and Morinda citrifolia L.-in an equal part by weight. The quality of the raw material is one of the essential factors that can affect the effectiveness and safety of treatment by herbal remedy. The pharmacognostic evaluation and DNA barcode of the three fruit species and TS remedy were performed in this study to authenticate them from contamination, and to provide the scientific database for further uses. Macroscopic and microscopic examination, chemical profile by TLC, and DNA barcoding were employed to positively identify the raw materials bought from the herbal market, especially the powder form. Consequently, the outcomes of this investigation can be used to develop an essential and effective tool for the authentication of crude drugs and herbal remedies.

Verification of Thai ethnobotanical medicine "Kamlang Suea Khrong" driven by multiplex PCR and powerful TLC techniques.

Kamlang Suea Khrong (KSK) crude drug, a traditional Thai medicine used for oral tonic and analgesic purposes, is obtained from three origins: the inner stem bark of Betula alnoides (BA) or the stems of Strychnos axillaris (SA) or Ziziphus attopensis (ZA). According to the previous reports, SA contains strychnine-type alkaloids that probably cause poisoning; however, only organoleptic approaches are insufficient to differentiate SA from the other plant materials. To ensure the botanical origin of KSK crude drug, powerful and reliable tools are desperately needed. Therefore, molecular and chemical identification methods, DNA barcoding and thin-layer chromatography (TLC), were investigated. Reference databases, i.e., the ITS region and phytochemical profile of the authentic plant species, were conducted. In case of molecular analysis, multiplex polymerase chain reaction (PCR) based on species-specific primers was applied. Regarding species-specific primers designation, the suitability of three candidate barcode regions (ITS, ITS1, and ITS2) was evaluated by genetic distance using K2P model. ITS2 presented the highest interspecific variability was verified its discrimination power by tree topology. Accordingly, ITS2 was used to create primers that successfully specified plant species of authentic samples. For chemical analysis, TLC with toluene:ethyl acetate:ammonia (1:9:0.025) and hierarchical clustering were operated to identify the authentic crude drugs. The developed multiplex PCR and TLC methods were then applied to identify five commercial KSK crude drugs (CK1-CK5). Both methods correspondingly indicated that CK1-CK2 and CK3-CK5 were originated from BA and ZA, respectively. Molecular and chemical approaches are convenient and effective identification methods that can be performed for the routine quality-control of the KSK crude drugs for consumer reliance. According to chemical analysis, the results indicated BA, SA, and ZA have distinct chemical profiles, leading to differences in pharmacological activities. Consequently, further scientific investigations are required to ensure the quality and safety of Thai ethnobotanical medicine known as KSK.

Evaluation of white Kwao Krua (Pueraria candollei Grah. ex Benth.) products sold in Thailand by molecular, chemical, and microscopic analyses.

The tuberous roots of Pueraria candollei Grah. ex Benth. (Fabaceae), commonly known as white Kwao Krua, are used to relieve menopausal symptoms in Thai traditional medicine because they contain phytoestrogens. Black and red Kwao Krua crude drugs exist as well, but they have different botanical origins and pharmacological activities. There is a high demand for white Kwao Krua products, but because of the limited availability of the plant material, it is suspected that the adulteration and misidentification of white Kwao Krua crude drugs and products occur. In this study, we authenticated white Kwao Krua products collected from Thai herbal markets by molecular, chemical, and microscopic analyses. The nucleotide sequences in the internal transcribed spacer (ITS) and trnH-psbA regions of 23 samples of authentic P. candollei were analyzed, and both regions were found to have intraspecific DNA polymorphisms. Based on the single nucleotide polymorphisms in the ITS1 region, species-specific primer sets of P. candollei were designed to authenticate white Kwao Krua and differentiate it from red and black Kwao Krua. Only the PCR products of KWP02 were not amplified by the primer sets. Isoflavonoid contents and microscopic features were used to support the results of molecular analysis to clarify the botanical origin of white Kwao Krua. Molecular, chemical and microscopic methods confirmed that all the Thai Kwao Krua products examined in this study contained authentic "white Kwao Krua" as claimed on their labels.

The combination of ITS2 and psbA-trnH region is powerful DNA barcode markers for authentication of medicinal Terminalia plants from Thailand.

The dried fruits of Terminalia plant (Combretaceae) called "Samo" have been used as herbal medicine in Thai traditional medicine. Four "Samo" crude drugs, namely, Samo thai, Samo thed, Samo dee-ngu, and Samo phiphek, are used as the main ingredients in Triphala and Trisamo recipes. Their commercial products are available in processed and powdered form, but are difficult to authenticate by conventional methods. In this study, we aimed to discriminate species of genus Terminalia for the identification of their crude drugs by a DNA barcoding technique. A total of 208 closely related nucleotide sequences were obtained from nine Terminalia species collected from Thailand and the DDBJ/EMBL/GenBank database. An effective DNA barcode marker was selected from six DNA loci (matK, rbcL, psbA-trnH, ITS, ITS1, and ITS2) and their two-locus combination. All sequences were analyzed by three major methods: (1) BLAST search; (2) the genetic divergence method using Kimura 2-parameter (K2P) distance matrices; and (3) tree topology analysis based on the neighbor-joining method. Comparison of the six candidate DNA loci indicated that ITS identified Terminalia with 100% accuracy at the species and genus levels in the BLAST1 method. ITS2 showed the highest K2P variability. The data from the single markers and the two-locus combinations revealed that only the two-locus combinations, namely, the combinations of rbcL, ITS, ITS1, and ITS2 with psbA-trnH, clearly discriminated all the species. From the results of DNA sequence analysis and the three methods, ITS2 is recommended for the identification of Terminalia species to supplement psbA-trnH.

Molecular Analysis of Terminalia spp. Distributed in Thailand and Authentication of Crude Drugs from Terminalia Plants.

Terminalia, a large genus of Combretaceae, is distributed in Tropical Asia, Africa, and America. Some Terminalia plants are used in folk medicine because they possess powerful medicinal properties. Dried fruits of Terminalia bellirica and Terminalia chebula are used as the main ingredient in Triphala, a famous polyherbal formulation in Ayurvedic medicine and Thai folk medicine, because of their laxative, detoxifying, and rejuvenating effects. To clarify the phylogenetic relationships of medicinal Terminalia species (T. bellirica, T. chebula, and T. catappa) and authenticate their crude drugs, "Samo" and Triphala, nucleotide sequencing alignments in the internal transcribed spacer one-two (ITS 1-2) regions of Terminalia plants collected in Thailand were performed. The amplified fragments of Terminalia species were approximately 800 bp in length. To compare these sequences and DDBJ registered data, a molecular phylogenetic tree was constructed. Phylogenetic analysis clearly separated the sequences into two groups: Asian Terminalia and African Terminalia with some exceptions. In the analyzed sequences, the length of the ITS1-5.8S-ITS2 region was 674 bp in T. chebula, and 677 bp in T. bellirica and T. catappa. Eighty-one single nucleotide polymorphisms (SNPs) and nine insertion-deletions (indels) were observed, and the nucleotide sequences of this region showed species-specific sequences. Based on these differences, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and amplification refractory mutation system (ARMS) were applied to identify medicinal Terminalia species. Moreover, the ARMS method was chosen for fingerprinting analysis of Samo crude drugs and Triphala formulations because it was a fast, cost-effective, and reproducible approach.