Differentiation of Centella asiatica (L.) Urb. from Hydrocotyle umbellata L. using the trnH-psbA region, species-specific bands and anisocytic stomata as markers for quality control of raw materials and their products

Centella asiatica (L.) Urb. has been confused with Hydrocotyle umbellata L. in Thailand. The plants grow in the same habitat and look alike, making H. umbellata a potential adulterant species for C. asiatica. This study aimed to (i) establish reference markers of authentic C. asiatica and H. umbellata using DNA barcoding, thin-layer chromatography (TLC) and microscopic methods and (ii) determine whether H. umbellata adulterants are present in C. asiatica products in Thailand. Ribulose-bisphosphate carboxylase (rbcL), maturase K (matK), internal transcribed spacer (ITS) and chloroplast intergenic spacer (trnH-psbA) were identified as core DNA barcode regions that can be used to differentiate C. asiatica and H. umbellata. The trnH-psbA region showed the highest nucleotide variation between C. asiatica and H. umbellata and had distinct amplicon sizes of approximately 506 and 398 bp, respectively. TLC analysis revealed three distinct bands at Rf 0.20, 0.30 and 0.32 that could be used to differentiate C. asiatica and H. umbellata. Anisocytic stomata were present only in C. asiatica and were purposed as a microscopic marker. The trnH-psbA amplicon, TLC species-specific markers and anisocytic stomata were utilized to examine six Bua Bok products traded in Thailand. Agarose gel electrophoresis showed PCR amplicons at approximately 500 bp, TLC showed species-specific bands at Rf 0.20, 0.30 and 0.32, and anisocytic stomata were found in all products, confirming that all tested products were C. asiatica. These markers will benefit the herbal industry and other end users by allowing the detection of the adulterant species H. umbellata in C. asiatica raw materials and products.