Comprehensive analysis using DNA metabarcoding, SCAR marker based PCR assay, and HPLC unveils the adulteration in Brahmi herbal products

Background Brahmi is one of the important nootropic botanicals, widely sold in the market, with the name “Brahmi’’ being used to describe both Bacopa monnieri and Centella asiatica species. The Brahmi herbal products market is expanding; hence, economically motivated adulteration is highly prevalent. Methods and results This study aimed to develop DNA-based methods, including SCAR marker-based PCR and metabarcoding, to authenticate Brahmi herbal products and compare these methods with HPLC. These methods have been validated using mock controls ( in-house blended formulations). All targeted plant species in mock controls were detected successfully with all three methods, whereas, in market samples, only 22.2%, 55.6%, and 50.0% were found positive for Brahmi by PCR assay, DNA metabarcoding, and HPLC, respectively. Metabarcoding can detect the presence of non-labeled plants together with targeted species, which is an advantage over PCR assay or HPLC. Conclusion SCAR marker-based PCR is a rapid and cost-effective method for detecting the presence of B. monnieri and C. asiatica . However, in this study, the success rate of PCR amplification was relatively low because the primers targeted either RAPD or ITS-based SCAR markers. HPLC assay, although an alternative, was unable to detect the presence of other botanicals, just like the SCAR marker-based PCR assay. On the other hand, metabarcoding can be utilized to identify the target plants, even in very small quantities, while also providing simulated identification of other botanicals. This study successfully addressed the need for quality control of Brahmi herbal products and provided the first-time report of DNA metabarcoding for such products.