DNA-based Simultaneous Identification of Three Terminalia Species Targeting Adulteration

BACKGROUND: Various parts of three Terminalia species, namely, Terminalia arjuna (stem bark), Terminalia bellirica (fruit), and Terminalia chebula (fruit) are widely known for their therapeutic principles and other commercial values. However, stem bark of T. bellirica and T. chebula along with Terminalia tomentosa are reported as adulterants of T. arjuna. Correct botanical identification is very critical for safe and effective herbal drugs. DNA-based identification approaches are advancing the conventional methods and sometime proved more beneficial. OBJECTIVE: The purpose of the study was to develop polymerase chain reaction (PCR) method using internal transcribed spacer (ITS) region to ascertain the identity of T. arjuna herbal material as well as detection of mixing of other three Terminalia species. MATERIALS AND METHODS: DNA from stem barks samples were isolated and subjected to ITS region amplification and sequencing. Sequences were compared for polymorphic nucleotides determination to develop species-specific primers. Final primers were selected on the basis of in silico analysis and experimentally validated. PCR assays for botanical identification of Terminalia species were developed. Sensitivity testing and assay validation were also performed. RESULTS: The PCR assays developed for Terminalia species were resulted in definite amplicons of the corresponding species. No cross-reactivity of the primers was detected. Sensitivity was found enough to amplify as low as 2 ng of DNA. Mixing of DNA in various concentrations for validation also proved the sensitivity of assay to detect original botanicals in the mixture. The developed methods proved very specific and sensitive to authenticate Arjuna bark to develop evidence-based herbal medicines. SUMMARY: Internal transcribed spacer-based species-specific polymerase chain reaction.(PCR) assays were developed to authenticate Terminalia arjuna stem bark and to identify substitution/adulteration of Terminalia bellirica and Terminalia chebula in the genuine starting material. Definite amplicons were obtained specific to particular species and the assay was found of profound sensitivity to amplify as low as 2 ng of DNA. Results of method validation proved that the assay can identify adulterant Terminalia species even when present in lower amounts. The DNA barcodes and PCR methods can also be used to identify Terminalia bellirica and T. chebula related herbal medicinal material. Abbreviations used: ITS: Internal transcribed spacer, BSA: Bovine serum albumin, DMSO: Dimethyl sulfoxide.