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.

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.