Exploiting Marker Genes for Reliable Botanical Authentication of Bacopa monnieri Products

<i>Bacopa monnieri</i>, commonly known as Brahmi, is a perennial herbaceous plant used in Ayurvedic medicine owing to its nootropic properties. The increased demand for bacopa-derived herbal/food products has motivated adulteration practices through plant substitution. This work is aimed at developing a new method for <i>B. monnieri</i> detection and quantification in herbal products. The chloroplast gene encoding the Ycf1 photosystem I assembly protein (<i>Ycf1</i>) and the nuclear gene coding for the flavonoid glucosyltransferase (Flag) were selected as candidate markers to develop a real-time PCR assay with EvaGreen dye for <i>B. monnieri</i> detection. Both markers were specific to the target species, with <i>Ycf1</i> providing the best real-time PCR kinetics and highest sensitivity. Therefore, a new method targeting the <i>Ycf1</i> barcode was developed, exhibiting high specificity and a sensitivity of 1 pg of bacopa DNA. Additionally, a calibration model was proposed using reference mixtures of <i>B. monnieri</i> in <i>Ginkgo biloba</i> with a linear dynamic range of 25-0.1% (<i>w</i>/<i>w</i>). The curve parameters of slope, PCR efficiency and correlation coefficient met the acceptance criteria. The method was successfully validated with blind mixtures and further applied to commercial herbal products, revealing an important level of adulteration in bacopa/Brahmi-labelled products (60%) due to absence of or reduction in bacopa content. In this work, the first quantitative real-time PCR method for the botanical authentication of <i>B. monnieri</i> in herbal products is proposed as a powerful tool, which can be used by quality control laboratories and regulatory authorities to ensure labelling compliance.