Authentication of milk thistle commercial products using UHPLC-QTOF-ESI + MS metabolomics and DNA metabarcoding

Abstract

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.

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

“…In conjunction with chemical fingerprinting, researchers have gradually turned their focus to DNA based molecular methods, which are recognized as techniques to identify edible and medicinal plant species, and also to detect their substitutes and adulterants in crude or processed products ( Raclariu et al, 2017 ; Raclariu-Manolică et al, 2023b ). These methods are independent of species life stage, tissue type, or the physiological conditions of constituents ( Techen et al, 2014 ; Lo and Shaw, 2018b ; Hao and Xiao, 2020 ) and can discriminate botanicals at species level ( Besse et al, 2021 ; Mishra et al, 2016 ).…”

“… Xin et al (2018) performed shotgun sequencing of CHMs to obtain the barcode regions ITS2, psbA-trnH, and matK. Barcoding techniques can further be used in conjunction with metabolomics, transcriptomics or proteomics ( Mishra et al, 2016 ; Raclariu-Manolică et al, 2023b ). The possibility to process data via multivariate analysis, pattern recognition and metabolomics then gives a broader scope for applications in medicinal plant analysis and the spectrum of compounds found within medicinal plants ( Feng et al, 2018 ; Nöst et al, 2019 ; Kaigongi et al, 2020 ; Raclariu-Manolică et al, 2023b ).…”

“…Barcoding techniques can further be used in conjunction with metabolomics, transcriptomics or proteomics ( Mishra et al, 2016 ; Raclariu-Manolică et al, 2023b ). The possibility to process data via multivariate analysis, pattern recognition and metabolomics then gives a broader scope for applications in medicinal plant analysis and the spectrum of compounds found within medicinal plants ( Feng et al, 2018 ; Nöst et al, 2019 ; Kaigongi et al, 2020 ; Raclariu-Manolică et al, 2023b ). Modern analytical technologies combined with chemometrics are increasingly used for quality monitoring of medicinal plant matrices, but it is still challenging to choose the adequate type of analysis and statistical method as this is highly dependent on the specific authenticity issue ( Liu 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 ). PCRs were run using indexing primers as in Raclariu-Manolică et al (2023b) with applying the indexing strategy of Fadrosh et al (2014) .…”

“…DNA barcoding techniques, as described in the British Pharmacopoeia ( British Pharmacopoeia Commission, 2018 ), are a valuable screening tool for raw, single species botanical materials. Metabarcoding techniques have been successfully used for ingredient profiling of commercial herbal products, which are composed of varying, processed botanical materials, such as CHMs and provide information about unknown ingredients ( Coghlan et al, 2012 ; Arulandhu et al, 2017 ; Seethapathy et al, 2019 ; Anthoons et al, 2021 ; Zhu et al, 2022 ; Raclariu-Manolică, 2023a ; Raclariu-Manolicăet al, 2023b ). Chromatographic authentication gives high resolutions for the detection of target compounds of known ingredients and are basic authentication tools for herbal remedies ( Liang et al, 2010 ; Booker et al, 2016 ; Heinrich et al, 2022 ).…”