Multiplex Bar-HRM for differentiating Centella asiatica (L.) Urb. from possible substituent species

Global market demand for plant-derived products has increased significantly due to their extensive use in the treatment and prevention of various diseases. However, mislabeling of commercial herbal products poses a major safety concern and affects their efficacy. Differentiating species among Centella asiatica (L.) Urb., Hydrocotyle umbellata L., Bacopa monnieri L., and Bacopa caroliniana (Walter) B.L.Rob, which share morphological features and common local names is challenging. To address this issue, DNA barcoding coupled with high-resolution melting (Bar-HRM) analysis was performed using ITS, matK, and rbcL loci. Suitable primer sets were designed based on nucleotide diversity analysis from multiple alignments of each barcode. Our findings showed that multiplex Bar-HRM (mBar-HRM) using matK1 and rbcL1 successfully discriminated C. asiatica from the other species and enabled contamination detection of C. asiatica and species specificity of various medicinal plants. Analytical sensitivity determined the minimal DNA concentration to generate melting patterns at 0.01 ng per reaction. mBar-HRM was also tested for adulteration detection by mixing DNA from C. asiatica with H. umbellata or B. monnieri at more than 75%. Results demonstrated the potential of mBar-HRM as a reliable method for species authentication in C. asiatica-derived commercial products.