Complementary authentication of Chinese herbal products to treat endometriosis using DNA metabarcoding and HPTLC shows a high level of variability

Abstract

Traditional Chinese Medicine (TCM) is popular for the treatment of endometriosis, a complex gynecological disease that affects 10% of women globally. The growing market for TCMs has yielded a significant incentive for product adulteration, and although emerging technologies show promise to improve their quality control, many challenges remain. We tested the authenticity of two traditional Chinese herbal formulae used in women’s healthcare for the treatment of endometriosis, known as Gui Zhi Fu Ling Wan (FL) and Ge Xia Zhu Yu Tang (GX). Dual-locus DNA metabarcoding analysis coupled with high-performance thin-layer chromatography (HPTLC) were used to authenticate 19 FL and six GX commercial herbal products, as well as three ad hoc prepared artificial mixtures. HPTLC was able to detect most of the expected ingredients via comparative component analysis. DNA metabarcoding was able to detect an unexpected species diversity in the products, including 38 unexpected taxa. Chromatography has a resolution for all species indirectly through the identification of marker compounds for the different species ingredients. Metabarcoding on the other hand yields an overview of species diversity in each sample, but interpretation of the results can be challenging. Detected species might not be present in quantities that matter, and without validated quantification, some detected species can be hard to interpret. Comparative analysis of the two analytical approaches also reveals that DNA for species might be absent or too fragmented to amplify as the relevant chemical marker compounds can be detected but no amplicons are assigned to the same species. Our study emphasizes that integrating DNA metabarcoding with phytochemical analysis brings valuable data for the comprehensive authentication of Traditional Chinese Medicines ensuring their quality and safe use.

Three-tiered authentication of herbal traditional Chinese medicine ingredients used in women’s health provides progressive qualitative and quantitative insight

“…Each band in the fingerprints was given a score from zero to five, based on the intensity perceived by the naked eye, compared to the standard, where zero is “not detectable”, and five is the highest intensity. BISs with scales 0-5 refer to the quality of the ingredients’ entire fingerprint with respect to the visibility and positions of bands for the pharmacopoeial reference marker compounds and botanical references in each chromatogram ( Mück et al, 2023 ).…”

“…DNA extraction, PCR, normalization and pooling of amplicons, such as library preparation and sequencing was conducted according to Mück et al (2023) . The samples were sequenced and analyzed alongside the sample set presented in Mück et al (2023) .…”

“…DNA extraction, PCR, normalization and pooling of amplicons, such as library preparation and sequencing was conducted according to Mück et al (2023) . The samples were sequenced and analyzed alongside the sample set presented in Mück et al (2023) . We used dual index fusion primers for amplicon libraries of the internal transcribed spacers nrITS1 and nrITS2, based on 18S-ITS1F and 58S-ITS1R ( Omelchenko et al, 2019 ), and ITS2F and ITSp4 primers ( Timpano et al, 2020 ).…”

“…Bioinformatic processes related to the metabarcoding analysis were done as described in ( Mück et al, 2023 ). After applying strict filtering controls to delete any false positive detections for each sample, the taxonomic assignment step was conducted by selecting top-scored species as the target species (search tool: BLAST) ( Yao et al, 2022 ).…”

“…Interestingly, TCM ingredients are described with herbal drug names defining a plant’s genus and the part of the plant used. However, the ingredient name is seldom coherent with scientific taxonomy and ingredient names often refer to more than one plant species ( Mück et al, 2023 ). The Chinese Pharmacopoeia states which species are accepted under the ingredient name with scientific taxonomy ( Chinese Pharmacopoeia Committee, 2020 ).…”