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Background: Milk thistle is one of the most popular hepatoprotectants, and is often sold in combination with other ingredients. Botanical supplements are known to be vulnerable to contamination and adulteration, and emerging technologies show promise to improve their quality control. Methods: Untargeted and semi-targeted metabolomics based on UHPLC-QTOF-ESI+MS techniques, UV spectrometry, and DNA metabarcoding using Illumina MiSeq were used to authenticate eighteen milk thistle botanical formulations (teas, capsules, tablets, emulsion). Results: Untargeted metabolomics separated 217 molecules and by multivariate analysis the discrimination between the different preparations was established. The semi-targeted metabolomics focused on 63 phytochemicals, mainly silymarin flavonolignans and flavonoids, that may be considered as putative biomarkers of authenticity. All formulations contained molecules from silymarin complexes at different levels. The quantitative evaluation of silybins was done using in parallel UV spectrometry and UHPLC-QTOF-ESI+MS and their correlations were compared. DNA metabarcoding detected milk thistle in eleven out of sixteen retained preparations, whereas two others had incomplete evidence of milk thistle despite metabolomics validating specific metabolites, e.g., silymarin complex, identified and quantified in all samples. Meanwhile, the DNA metabarcoding provided insights into the total species composition allowing the interpretation of the results in a broad context. Conclusion: Our study emphasizes that combining spectroscopic, chromatographic, and genetic techniques bring complementary information to guarantee the quality of the botanical formulations. © 2023, The Author(s).

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BackgroundMilk thistle is one of the most popular hepatoprotectants, and is often sold in combination with other ingredients. Botanical supplements are known to be vulnerable to contamination and adulteration, and emerging technologies show promise to improve their quality control.MethodsUntargeted and semi-targeted metabolomics based on UHPLC-QTOF-ESI+MS techniques, UV spectrometry, and DNA metabarcoding using Illumina MiSeq were used to authenticate eighteen milk thistle botanical formulations (teas, capsules, tablets, emulsion).ResultsUntargeted metabolomics separated 217 molecules and by multivariate analysis the discrimination between the different preparations was established. The semi-targeted metabolomics focused on 63 phytochemicals, mainly silymarin flavonolignans and flavonoids, that may be considered as putative biomarkers of authenticity. All formulations contained molecules from silymarin complexes at different levels. The quantitative evaluation of silybins was done using in parallel UV spectrometry and UHPLC-QTOF-ESI+MS and their correlations were compared. DNA metabarcoding detected milk thistle in eleven out of sixteen retained preparations, whereas two others had incomplete evidence of milk thistle despite metabolomics validating specific metabolites, e.g., silymarin complex, identified and quantified in all samples. Meanwhile, the DNA metabarcoding provided insights into the total species composition allowing the interpretation of the results in a broad context.ConclusionOur study emphasizes that combining spectroscopic, chromatographic, and genetic techniques bring complementary information to guarantee the quality of the botanical formulations.

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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.

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Milk thistle (Silybum marianum (L.) Gaertn.) is among the top-selling botanicals used as a supportive treatment for liver diseases. Silymarin, a mixture of unique flavonolignan metabolites, is the main bioactive component of milk thistle. The biological activities of silymarin have been well described in the literature, and its use is considered safe and well-tolerated in appropriate doses. However, commercial preparations do not always contain the recommended concentrations of silymarin, failing to provide the expected therapeutic effect. While the poor quality of raw material may explain the low concentrations of silymarin, its deliberate removal is suspected to be an adulteration. Toxic contaminants and foreign matters were also detected in milk thistle preparations, raising serious health concerns. Standard methods for determination of silymarin components include thin-layer chromatography (TLC), high-performance thin-layer chromatography (HPTLC), and high-performance liquid chromatography (HPLC) with various detectors, but nuclear magnetic resonance (NMR) and ultra-high-performance liquid chromatography (UHPLC) have also been applied. This review surveys the extraction techniques of main milk thistle metabolites and the quality, efficacy, and safety of the derived food supplements. Advanced analytical authentication approaches are discussed with a focus on DNA barcoding and metabarcoding to complement orthogonal chemical characterization and fingerprinting of herbal products.

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Herbal medicines and preparations are widely used in healthcare systems globally, but concerns remain about their quality and safety. New herbal products are constantly being introduced to the market under varying regulatory frameworks, with no global consensus on their definition or characterization. These biologically active mixtures are sold through complex globalized value chains, which create concerns around contamination and profit-driven adulteration. Industry, academia, and regulatory bodies must collaborate to develop innovative strategies for the identification and authentication of botanicals and their preparations to ensure quality control. High-throughput sequencing (HTS) has significantly improved our understanding of the total species diversity within DNA mixtures. The standard concept of DNA barcoding has evolved over the last two decades to encompass genomic data more broadly. Recent research in DNA metabarcoding has focused on developing methods for quantifying herbal product ingredients, yielding meaningful results in a regulatory framework. Techniques, such as loop-mediated isothermal amplification (LAMP), DNA barcode-based Recombinase Polymerase Amplification (BAR-RPA), DNA barcoding coupled with High-Resolution Melting (Bar-HRM), and microfluidics-based methods, offer more affordable tests for the detection of target species. While target capture sequencing and genome skimming are considerably increasing the species identification resolution in challenging plant clades, ddPCR enables the quantification of DNA in samples and could be used to detect intended and unwanted ingredients in herbal medicines. Here, we explore the latest advances in emerging DNA-based technologies and the opportunities they provide as taxa detection tools for evaluating the safety and quality of dietary supplements and herbal medicines.

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Sea buckthorn (Hippophae rhamnoides L.) (SB) is increasingly consumed worldwide as a food and food supplement. The remarkable richness in biologically active phytochemicals (polyphenols, carotenoids, sterols, vitamins) is responsible for its purported nutritional and health-promoting effects. Despite the considerable interest and high market demand for SB-based supplements, a limited number of studies report on the authentication of such commercially available products. Herein, untargeted metabolomics based on ultra-high-performance liquid chromatography coupled with quadrupole-time of flight mass spectrometry (UHPLC-QTOF-ESI+MS) were able to compare the phytochemical fingerprint of leaves, berries, and various categories of SB-berry herbal supplements (teas, capsules, tablets, liquids). By untargeted metabolomics, a multivariate discrimination analysis and a univariate approach (t-test and ANOVA) showed some putative authentication biomarkers for berries, e.g., xylitol, violaxanthin, tryptophan, quinic acid, quercetin-3-rutinoside. Significant dominant molecules were found for leaves: luteolin-5-glucoside, arginine, isorhamnetin 3-rutinoside, serotonin, and tocopherol. The univariate analysis showed discriminations between the different classes of food supplements using similar algorithms. Finally, eight molecules were selected and considered significant putative authentication biomarkers. Further studies will be focused on quantitative evaluation.

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Botanicals and derived herbal products have been long used in meeting primary healthcare needs around the world. They have gained increasing popularity in industrialized countries as complementary and alternative therapies to synthetic pharmaceuticals. Botanical preparations are however susceptible to contamination, adulteration, and substitution, that raise serious quality and public health safety concerns. The deliberate or accidental use of undeclared ingredients may occur throughout the entire value chain of the botanical product, from the cultivation or wild harvesting of the raw botanical material to the final packaged and marketed product. The current identification and quality inspection use tests to establish the identity, purity, and constituents of the herbal products. These tests most often utilize sensory and/or phytochemical inspection to detect species-specific characteristics or compounds, along with assays for toxic constituents such as heavy metals and toxins. However, marketed herbal products are very often highly processed and sold as complex mixtures. Classical analytical methods are often limited in their ability to identify the target plant species that should be present, and it is even more difficult to detect non-targeted species using these methods. This chapter presents a review of DNA barcoding and metabarcoding approaches to assess species diversity in herbal products and the use of these approaches in their quality control. © 2022 Elsevier Inc. All rights reserved.

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Herbs and spices are some of the most vulnerable products in terms of fraud and adulteration in the food sector. Although standard analytical methods are accurate for quality control of specific lead or marker compounds, they cannot accurately assess the entire species composition of many marketed products. Complementary analytical approaches are thus often used for comprehensive screening of herbs and spices. In this study we evaluate DNA metabarcoding for the identification and authentication of 62 products, containing basil, oregano, and paprika collected from different retailers and importers in Norway. Our results show varying degrees of discrepancy between the constituent species and those listed on the product labels, despite high product authenticity. We suggest the false positives result from the sensitivity of DNA metabarcoding and filtering thresholds should be integrated into protocols to reduce false positives. Our results highlight how integrating DNA metabarcoding into the toolbox of analytical methods for quality control of fresh and/or processed plant-based food can improve product quality.