Integrated microscopic, HPTLC, and HRM analyses for differentiating Centella asiatica (L.) Urb., Bacopa monnieri (L.) Wettst, and Hydrocotyle umbellata L.

In the herbal market, Bacopa monnieri and Centella asiatica are often used interchangeably, which can lead to mislabelling, especially since both are known as "Brahmi" in some regions of India. Similarly, in Thailand, C. asiatica is frequently confused with Hydrocotyle umbellata due to their similar appearance. Both countries are recognized as raw herbal material markets. This study employed a comparative approach using cell morphology, chemical fingerprinting, and DNA profiling to differentiate these herbs. Authentic samples were first analysed to establish species-specific identifiers which were then applied to commercial herbal products obtained online. Microscopic analysis revealed that B. monnieri had glandular trichomes and wavy epidermal cells, whereas C. asiatica featured unicellular trichomes and rosette aggregate crystals. H. umbellata resembled C. asiatica but lacked rosette aggregate crystals and had distinct stomata. Chemical fingerprinting via HPTLC and DNA analysis using high-resolution melt (HRM) further distinguished the herbs. The study confirmed that six samples were correctly labelled, while one sample identified as C. asiatica was actually H. umbellata, and another was C. asiatica contaminated with B. monnieri. This study underscores the importance of combining microscopic, chemical, and DNA analyses for accurate herbal product identification to ensure authenticity, safety, and sustainable use.