Comparison of DNA barcoding and metabarcoding approaches for the authentication of herbal infusions in the Portuguese market

The authentication of commercial herbal products such as herbal teas and infusions is essential to ensure consumer safety and product integrity. In this study, 100 commercial herbal infusions, including 94 single-species products and six polyherbal formulations, were analysed using DNA barcoding ( matK and rbcL ) and ITS2 metabarcoding. Moreover, the performance of two custom reference databases (NCBI and BOLD) was compared in ITS2 metabarcoding. DNA extraction was successful for 94 samples, while six single-species products failed to amplify any of the tested barcodes. Among the 88 remaining single-species samples, ITS2 showed the highest amplification success (100 %), outperforming the barcodes rbcL (94 %) and matK (84 %). Sanger sequencing confirmed the labelled species in 69.3 % of cases with rbcL and 48.9 % with matK . While 63 samples would be considered authentic solely based on barcoding (i.e., if either rbcL or matK matched the label), ITS2 metabarcoding revealed that many of these contained additional undeclared species, indicating that barcoding alone overestimated product authenticity. Of the 85 samples successfully analysed by ITS2 metabarcoding, only 27 (32 %) fully matched their label, while 58 (68 %) contained either additional undeclared species or complete substitutions. Several products contained undeclared species in significant proportions, indicating potential economic adulteration. Analysis of mock-mixtures demonstrated that ITS2 metabarcoding can approximate ingredient proportions, though recovery bias may occur. Overall, the results revealed (i) the importance of curated and comprehensive databases, with a higher number of species being identified by NCBI database, (ii) the superior sensitivity of ITS2 metabarcoding, and (iii) the widespread mislabelling in commercial herbal products. • The authenticity of 100 commercial herbal infusions was evaluated. • Results from two approaches, DNA barcoding and metabarcoding, were compared. • Two custom reference databases (NCBI and BOLD) were developed for ITS2 metabarcoding. • ITS2 metabarcoding revealed a high level of adulteration (68 %). • ITS2 allowed the detection of fungal contaminants in some samples.