Journal of Applied Botany and Food Quality 92, 33 - 38 (2019), DOI:10.5073/JABFQ.2019.092.005 1 Department of Biotechnology and Biosciences, University of Milano-Bicocca, Milano, Italy 2 FEM2 Ambiente Srl, Milano, Italy DNA barcoding to trace Medicinal and Aromatic Plants from the field to the food supplement Jessica Frigerio1, 2, Tommaso Gorini1, Andrea Galimberti1, Ilaria Bruni1, Nicola Tommasi1, Valerio Mezzasalma2, Massimo Labra1* (Submitted: October 8, 2018; Accepted: January 3, 2019) * Corresponding author Summary The global market of food supplements is growing, along with con- sumers demand for high-quality herbal products. Nevertheless, substitution fraud, and adulteration cases remain a common safety problem of global concern. In the last years, the DNA barcoding ap- proach has been proposed as a valid identification method and it is now commonly used in the authentication of herbal and food pro- ducts. The objective of this study was to evaluate whether DNA barcoding can be applied to trace the plant species from the start- ing raw material to the finished commercial products. We selected a panel of 28 phytoextracts obtained through three different extraction methods (i.e., maceration, percolation and sonication) with different solvents (i.e., ethanol, deionized water and glycerol). Furthermore, we chose six plant species for which we collected and analysed all the intermediates of the industrial production. We sequenced and analyzed the sequence variability at DNA barcoding (psbA-trnH, ITS) and minibarcoding (rbcL 1-B) marker regions. Phytoextracts obtained through hydroalcoholic treatment, with the lower per- centage of ethanol (<40%), and aqueous processing, at the lowest temperature, had major rate of sequencing and identification success. This study proves that DNA barcoding is a useful tool for Medicinal and Aromatic Plants (MAPs) traceability, which would provide con- sumers with safe and high-quality herbal products. Key Words: Herbal products, ITS, MAPs, Minibarcode, psbA-trnH, Phytoextracts, rbcL Introduction Medicinal and Aromatic Plants (MAPs) and their preparations are products used in medicine, cosmetics and food industry, belonging to plants, fungi, algae or lichens (Efsa, 2009). Such products are prepared using plants or their parts to exploit their therapeutic and healthy properties (e.g., antioxidant, anti-inflammatory), as well as their flavor or scent (Who, 1999). According to a report published by the Persistence Market Research, the global market of herbal supplements had a value of USD 40 billion in 2017 and is expected to reach a market valuation in excess of USD 65 billion by 2025 (PErsistEncE MarkEt rEsEarch, 2017). In the last years, the in- creasing consumption of natural food supplements and the growing awareness of consumers concerning the healthy benefits of these products have been progressively enhancing the market of MAPs (Efsa, 2009). Although most of the herbal products used as food in- gredients have been available to consumers since decades, the regu- lation of these products differs greatly among jurisdictions. While some countries consider MAPs as reliable ingredients for food pro- duction, others regulate them as healthy products or medicines. For example, in the European Union (EU), most products containing Medicinal and Aromatic Plants are sold as food supplements and re- gulated under the food law (silano et al., 2011); in Australia dietary supplements are considered medicinal products and in Canada they are subject to the complex regulation of the Natural Health Pro- ducts Directorate (NNHPD) of Health Canada (hEalth can., 2015) as medical products (loW et al., 2017). The lack of a clear and shared global regulation and the large market demand of high-quality plant- based items led to safety problems with the increase of substitution, fraud and adulteration cases. Anyway, more attention is required to guarantee a high quality level of MAPs which necessitates a stable raw material and its assurance. As a matter of fact, frequently, valu- able plants are substituted with cheaper raw materials, such as the case of saffron substituted with safflower (BosMali et al., 2017). However, adulteration is not necessarily intentional, and herbal pro- ducts may be altered due to inadvertent substitution, misidentification or confusion resulting from the use of different vernacular names in the countries of production. According to the World Health Organization (WHO), the adultera- tion of herbal products is a potential threat to consumers’ safety. This condition opens two key issues which refer to the definition of suitable toxicological evaluations to estimate the risks for human health and the setup of an efficient identification system to trace the herbal products from the field to the traded MAPs items. Usually, macroscopic and microscopic examinations are the classic strategies adopted to verify the identity of fresh plants or the origin of plant portions. These tools can be used to trace the herbal products when the plants are processed immediately after being harvested. How- ever, when the herbs undergo drying, fragmentation and pulveri- zation processes the morphological traits cannot be longer used to reliably assess the botanic source. Moreover, many herbal ingredi- ents are obtained by infusion, maceration, distillation or pressing. In these cases, only dedicated chemical analyses of the complex mix- tures could permit to achieve a reliable plant identification. In the last years the DNA barcoding approach was proposed as a valid molecular identification method to provide species-level reso- lution and it is now more and more used in the authentication of taxonomic provenance of herbal and food products (nEWsMastEr et al., 2013; GaliMBErti et al., 2013; MohaMMEd et al., 2017). How- ever, the most important limit of this molecular tool is that it can preferentially be adopted on unprocessed material (e.g., dry, frag- mented and shredded plant portions) and several difficulties are encountered when dealing with extracts or with any other process that results in the degradation of the DNA. Recently, some manu- scripts described the efficacy of minibarcode regions (i.e., the analy- sis of smaller genome portions − 100-150 bp − usually associated to the largest DNA barcodes) for the identification of processed plant extracts (raclariu et al., 2017; littlE, 2014). To date, any study addressed the efficacy of a DNA barcoding-based approach to trace herbal products along the entire production chain. In this work, we selected Medicinal and Aromatic Plants in the form of phytoextracts obtained by several industrial companies and sub- jected to different kind of industrial processes and phytoextraction strategies. The objective of this survey was to evaluate whether or not the DNA barcoding approach (using standard barcodes or minibarcode regions) could be applied to trace the plant species from 34 J. Frigerio, T. Gorini, A. Galimberti, Il. Bruni, N. Tommasi, V. Mezzasalma, M. Labra the field to the finished commercial product in the case of food sup- plements. Therefore, we evaluated which are the industrial processes mostly affecting the efficacy of DNA analysis such as sample pre- treatment methods, solvents used for extraction and which are the most suitable DNA markers to achieve a reliable MAPs traceability. Material and methods Study design To test the efficacy of DNA traceability at different steps of the industrial production chain of MAPs, we selected a panel of 28 com- mercial phytoextracts (Tab. 1) sold by three main European com- panies. The selected items were obtained starting from 17 plant species (in the initial form of dried raw material) and were processed by the same companies adopting three main extraction procedures, namely maceration, sonication and percolation. Maceration consists in the solubilization of the plant material in different solvents like water and alcohol (e.g., ethanol), while percolation involves the slow descent of a solvent through the plant raw material until it absorbs the molecules of interest. Both methods rely on liquid filtration and concentration. Differently, the sonication provokes cellular cavita- tion and the release of the phytocomplexes in the solvent used. Three different extraction solvents were considered in this study and spe- cifically, ethanol (i.e., alcoholic), deionized water (i.e., aqueous) and glycerol. During maceration the temperature was maintained under the threshold of 55 °C while in percolation higher temperatures (i.e., > 80 °C) were maintained, and sonication was mainly performed at 30-40 °C. After the percolation process, some phytoextracts are dried at very high temperatures (about 200 °C). Processing details for each tested phytoextract are shown in Tab. 1. To evaluate the efficacy of DNA barcoding to trace the intermediates of industrial production after different steps of phytoextraction, we selected a panel of six commercial products obtained only by alco- holic and aqueous extraction procedures (Tab. 2). For these samples, we collected and molecularly analysed through DNA barcoding, any intermediate of production (Fig. 1). DNA Extraction and DNA barcoding analysis All commercial products from Tab. 1 were tested for authenticity by sequencing three candidate markers, namely the standard DNA barcoding plastidial intergenic spacer psbA-trnH (stEvEn and suB- raManyaM, 2009), the nuclear ITS region (primers ITS p5-u4, chEnG et al., 2016) and the minibarcode region rbcL 1-B (littlE, 2014). Primer details and size of amplified fragments are provided in Tab. A.1. A total of 50 mg of dried plant raw material, 150 μL of phytoex- tract (and intermediate products of phytoextraction, see Tab. 2) were treated for DNA extraction by using the EuroGOLD Plant DNA Mini Kit (Euroclone, Pero, Italy). Each commercial phytoextract product was subjected to DNA extraction in three replicates. Purified DNA concentration of each sample was estimated fluorometrically by us- ing NanoDrop™ One/OneC Microvolume UV-Vis Spectrophoto- meter (Thermo Scientific™). A PCR amplification for each candidate marker was performed us- ing puReTaq Ready-To-Go PCR beads (GE Healthcare Life Sciences, Italy) in a 25 μL reaction volume according to the manufacturer’s instructions containing 1 μL 10mM of each primer and up to 3 μL of DNA template. PCR cycles consisted of an initial denaturation step for 7 min at 94 °C, followed by 35 cycles of denaturation (45 s at 94 °C), annealing (30 s at different temperatures; see Tab. A.1) and extension (1 min at 72 °C), and, hence, a final extension at 72 °C for 7 min. In the case of the intermediates of production listed in Tab. 2, we amplified and sequenced only the minibarcode locus rbcL 1B. Amplicons occurrence was assessed by electrophoresis on agarose gel using 1.5% agarose TAE gel stained with ethidium bromide and amplicon length was measured by comparison against 100 bp ladder. When a sample did not produce any band or showed multiple or non- specific amplicons, the reaction was repeated increasing the amount of template DNA up to 10 μL. Purified amplicons were bidirectionally sequenced using an ABI 3730XL automated sequencing machine at Eurofins Genomics (Ebers- berg, Germany). The 3' and 5' terminal portions of each sequence were clipped to generate consensus sequences for each sample. After manual editing, primer removal and pairwise alignment, the obtained sequences for dried raw material were submitted to the international GenBank through the EMBL platform (see Tab. A.2 for accession numbers). For all the tested samples (Tab. 1 and Tab. 2), the reliability of DNA barcoding identification was assessed by adopting a standard com- parison approach against a GenBank database with BLASTn. Each barcode sequence was taxonomically assigned to the plant species with the nearest matches (maximum identity > 99% and query co- verage of 100%) according to Bruni et al. (2015). We performed the identification separately for the three markers. Results and discussion Good DNA quality (i.e., A260/A230 and A260/A280 absorbance ratios within the range 1.8 - 2.2) and extraction yield (20-40 ng/μl) were obtained from all the 17 raw material samples. The three can- Fig. 1: The industrial flowchart of MAPs production. The numbers indicate the intermediate steps of the industrial process for which the DNA barcoding efficacy has been verified (Tab. 2). DNA barcoding to trace MAPs from the field to the food supplement 35 Sonication 12.94 1.58 × × × EtOH < 40% EtOH > 40% WATER GLYCEROL Tab. 1: List of the analysed MAPs samples with details concerning their industrial processing to obtain the final phytoextracts. Average yield of DNA extraction (with standard deviation) and assessment of positive sequencing of DNA barcoding markers (×) are also reported. SAMPLES Industrial processing DNA Extraction yield DNA BARCODING MARKERS Phytoextraction Solvent Value Standard psbA - trnH ITS rbcL 1 - B Process ng /μl Deviation Achillea millefolium L. Sonication 12.26 1.55 × Echinacea pallida (Nutt.) Nutt. Sonication 16.29 0.97 × Harpagophytum procumbens (Burch.) DC. ex Meisn. Melissa officinalis L. Percolation 44.63 1.32 × Mentha × piperita L. Sonication 12.3 0.83 × × × Tilia platyphyllos Scop. Sonication 9.78 1.28 × × × Zingiber officinale Roscoe Sonication 13.16 2.13 × × × Arctium lappa L. Maceration 1.23 0.2 Echinacea angustifolia DC. Maceration 2.83 0.5 Melissa officinalis L. Percolation 1.4 0.44 Passiflora incarnata L. Maceration 1.74 0.17 Taraxacum officinale Weber ex F.H. Wigg. Maceration 2.47 0.54 Thymus vulgaris L. Maceration 1.78 0.38 Arctostaphylos uva-ursi (L.) Spreng. Percolation 3.1 0.33 Cetraria islandica (L.) Ach. Percolation 2.27 0.94 Echinacea purpurea (L.) Moench Percolation 3.47 0.71 × Epilobium angustifolium L. Percolation 1.88 0.63 Malva sylvestris L. Percolation 2.34 0.69 Arctostaphylos uva-ursi (L.) Spreng. Sonication 13.36 1.05 × Echinacea purpurea (L.) Moench Sonication 46.41 0.81 × × × Epilobium angustifolium L. Sonication 14.78 0.97 × Melissa officinalis L. Sonication 12,73 0,76 × × × Arctium lappa L. Maceration 2.69 0.64 Echinacea angustifolia DC. Maceration 4.73 0.85 × Melissa officinalis L. Maceration 2.55 0.76 Passiflora incarnata L. Maceration 2.09 0.6 Taraxacum officinale Weber ex F.H. Wigg. Maceration 3.12 0.3 Thymus vulgaris L. Maceration 2.71 0.8 Tab. 2: List of six commercial MAPs (phytoextracts) traced along their entire production chain. Each sample (intermediates of industrial production) was treated for DNA extraction and DNA barcoding analysis using the minibarcode region rbcL 1-B. Numbers indicate the industrial processing step as described in Fig. 1. Y= correct plant identification by DNA barcoding at rbcL 1-B locus, N= DNA extraction or amplification failure, - = Sample was not collected and analysed. Plant species Solvent Steps of the industrial production process 1 2 3 4 5 6 Achillea millefolium L. 20% Ethanol Y Y N Y Y Y Zingiber officinale Roscoe 30% Ethanol Y Y N Y Y Y Thymus vulgaris L. 60% Ethanol Y N Y N N - Melissa officinalis L. 70% Ethanol Y N Y N N - Echinacea purpurea (L.) Moench Water Y Y N Y Y - Melissa officinalis L. Water Y Y N Y Y Y 36 J. Frigerio, T. Gorini, A. Galimberti, Il. Bruni, N. Tommasi, V. Mezzasalma, M. Labra didate genetic markers exhibited high PCR success and the obtained PCR products were successfully sequenced with high-quality bi- directional sequences. The BLASTn analysis suggested that all the obtained sequences corresponded with 100% maximum identity to the species declared by each company. We are aware that multiple cases of 100% maximum identity within the same plant genus could occur, especially concerning the DNA minibarcoding rbcL 1B region. For example, this plastid region failed in discriminating Echinacea purpurea (L.) Moench from conge- nerics like Echinacea angustifolia DC. or Echinacea pallida (Nutt.). Such events suggest that in some conditions, the main limit of DNA minibarcoding relies on the reduced discrimination power among congenerics but it allows to detect plant contaminations when the adulterant/s belong to genera different from the target one. Never- theless, it should be considered that when DNA content is expected to be low (or of low quality), the use of shorter DNA barcoding re- gions offers the best compromise between amplification universality, sequence quality and taxonomic discrimination (littlE, 2014). Concerning the 17 dry raw material samples, our results agree with the assumptions of nEWMastEr and co-workers (2013) who sug- gested that a DNA barcoding approach could be successfully applied to verify the identity of commercial herbal products and to reveal cases of contamination or substitution. Therefore, when herbal pro- ducts are directly used as ingredients of complex food, medicine or cosmetics items, they are subjected to “soft” processing actions such as cleaning, drying and cutting and the DNA barcoding (achieved using long barcode fragments > 300 bp) represents a useful tool to trace plant species during the processing (dE Mattia et al., 2011). As expected, the efficacy of DNA extraction and amplification decreased when we analyzed the 28 commercial phytoextracts and their intermediates of industrial processing. Overall, the DNA amount obtained after extraction processes ranged from 1.5 to more than 40 ng/μL (Tab. 1). The extracts obtained through hydroalcoholic treatment, with the lower percentage of ethanol (< 40%), and aqueous processing, at the lowest temperature, contained more DNA than the other samples (Tab. 1 and Tab. 2). In the samples where the DNA barcoding analysis worked well, no contamination or adulteration (i.e., the occurrence of DNA bacodes of other plant species) were observed. Unfortunately, in some groups of extracts, the molecular analysis did not provide reliable DNA ex- traction or high-quality sequences. At technical level, we hypothe- size that in general, the high concentration of ethanol used in the industrial processing steps lead to DNA precipitation. This was con- firmed by the data reported in Tab. 2, where samples of Thymus vul- garis L. and Melissa officinalis L. processed with ethanol at high concentration, showed residual DNA in the extraction waste rather than in the phytoextract. For this reason, both the DNA extraction and DNA barcoding authentication failed when applied to the suc- cessive intermediate products of industrial processing and DNA was no longer available in the final herbal supplements. In this case, we conclude that for this kind of industrial production, a DNA-based approach is not suitable to achieve a reliable traceability of the ini- tial plant raw material. Similarly, high temperatures of water du- ring aqueous extraction, followed by a drying step (about 200 °C) probably lead to DNA fragmentation and degradation (karni et al., 2013) as observed in five of the samples processed with a percolation procedure (Tab. 1). Conversely, the use of more lukewarm water (i.e., < 55° C) allows to achieve a successful DNA extraction, amplifi- cation and sequencing of DNA barcoding markers (Tab. 1). More- over, such conditions also allow the traceability of the intermediate pro-ducts of industrial processing as observed for Echinacea pur- purea (L.) Moench and Melissa officinalis L. extracted using water as solvent. Concerning glycerol extracts, although this solvent does not act di- rectly on DNA molecules, it usually contains ethylhexylglycerin and phenoxyethanol, which are typically used as additives. According to Langsrud and co-workers (2016) these antibacterial agents could be responsible for DNA loss. For this reason, also the analysis of the DNA minibarcode region did not produce amplicons in glycerine extracts (Tab. 1). Concerning the industrial treatments, the sonication seems to keep the DNA of raw materials more intact than the other processes (i.e., maceration and percolation). Our results also show that sonicated samples contained higher amounts of DNA (i.e. from 9.73 to 44 ng/ μl, Tab. 1) compared to the other categories, thus allowing a success- ful amplification and sequencing of the DNA minibarcode marker. Concerning the quality of extracted genetic material, the purity of DNA is more important than the extraction yield to achieve a good amplification and then a reliable identification (sonG et al., 2017). It should also be considered that secondary metabolites, like polyphe- nols and polysaccharides, which are normally extracted along with DNA, may interfere with PCR amplification (sahu, thanGaraj, and kathirEsan, 2012). These molecules could bind DNA cova- lently and make the extraction products impure, with several pro- blems for the successive molecular analysis. For example, tannic acids could bind and inactivate Taq polymerase (oPEl, chunG, and Mccord, 2010). However, in our analysis we hypothesize that the main amplification problem for the phytoextracts is the fragmen- tation of DNA. In all the tested cases, the DNA minibarcode locus rbcL 1-B was most easily amplified and sequenced (Tab. 1) than the other two DNA barcoding markers. This suggests that the DNA ob- tained from phytoextracts are richer in small DNA fragments (80- 200 bp). Such condition is in line with the data reported in recent review articles (MohaMMEd et al., 2017) suggesting that DNA bar- coding is a reliable and suitable technique only for the herbal product that preserve a good quality DNA and with poor fragmentation. In the other cases DNA minibarcoding is the most efficient and reliable tool for traceability purposes (sonG et al., 2017). Nowadays, analytical chemistry methods (TLC, HPLC) represent the most used tools to verify the quality of MAPs, however, these approaches are usually directed to define the concentration of spe- cific bioactive molecules or to estimate chemical contaminants (e.g., heavy metals) rather than to identify the occurrence of plant con- taminants (sGaMMa et al., 2017). Conversely, the DNA barcoding approach is globally recognized as one of the most reliable DNA- based approaches to identify species if a well populated reference dataset of DNA barcode sequences for the target taxa is available (GaliMBErti et al., 2013). Moreover, in the case of contamination (or substitution), DNA analyses also allow to simultaneously iden- tify any species (i.e., DNA metabarcoding) using High Throughput Sequencing (HTS) sequencing systems (GaliMBErti et al., 2015; MEzzasalMa et al., 2017). For these reasons, the Pharmacopoeia guidelines of some countries such as that of UK (British PharMa- coPoEia coMMission, 2017) indicate the DNA barcoding as one of the official traceability systems in the sector of herbal products. Our data support this proposal and the ability of DNA minibarcode makers to provide a reliable tracing of the intermediate products of industrial production. However, it is important to underline that some industrial processes demanding high temperatures and the use of solvents, such as a high concentration of ethanol, can induce DNA degradation and make this molecular tool less effective. In conclusion, this study leads to two main considerations about the future application of DNA barcoding as a quality control tool in the sectors where the Medicinal and Aromatic Plants constitute relevant ingredients (e.g., food, cosmetics and pharmacology). First of all, the current industrial trends promote the adoption of extrac- tion processes from plant raw material, which rely on the reduction of energy consumption (i.e., low temperatures), and on the use of more ‘green’ solvents (e.g., water) to obtain exhausted waste products that can be used in other supply chains (e.g., fertilizers). The adoption and DNA barcoding to trace MAPs from the field to the food supplement 37 spread of this trend should lead to an increased integrity and quality of DNA in MAPs (and related intermediate products) and therefore enhance the success of DNA barcoding as a universal traceability system. Secondly, the continuous advances in High Throughput Sequencing and the resulting possibility of exploring multiple short genetic regions simultaneously (i.e. 150-200 bp), could increase the sensi- tivity of a DNA-based identification.An HTS-DNA metabarcoding approach would allow to check the presence of several plant con- taminants in the same sample, even if occurring at low concentra- tions (nEWMastEr et al., 2013). sGaMMa and co-workers (2017) proposed the introduction of DNA metabarcoding to evaluate the quality and authenticate herbal drug material in the industrial con- text. The authors proposed a dedicated DNA barcoding flowchart for industrial traceability purposes. Our results could be taken into ac- count to improve this flowchart and to also adapt it to the traceability of intermediates of industrial production. Interestingly, valEntini and co-workers (2017) recently proposed an innovative nanoparticle- DNA barcoding hybrid system called NanoTracer that could poten- tially revolutionize the world of traceability as it allows for rapid and naked-eye molecular traceability of any food and requires limited instrumentation and cost-effective reagents. This and other similar applications (aartsE et al., 2017) open the op- portunity to really boost the issue of herbal supplements traceability, not only with the industrial actors as the main stakeholders, but also involving a wider cicle of specialists. Acknowledgments This work was supported by Regione Lombardia in the frame- work of the Program ‘Accordi per la ricerca e l’innovazione’, pro- ject name: Food Social Sensor Network Food NET, grant number: E47F17000020009. 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DOI: 10.1111/j.1755-0998.2009.02642.x valEntini, P., GaliMBErti, a., MEzzasalMa, v., dE Mattia, f., casiraGhi, M., laBra, M., PoMPa, P.P., 2017: DNA barcoding meets nanotechno- logy: Development of a Universal Colorimetric Test for Food Authen- tication. Angew. Chem. Int. Ed. 56(28), 8094-8098. DOI: 10.1002/anie.201702120 Who, 1999: WHO Monographs on selected medicinal plants. Vol. 1, Medicinal Plants, Geneva. http://apps.who.int/medicinedocs/pdf/s2200e/s2200e.pdf Address of the corresponding author: Massimo Labra, Department of Biotechnology and Biosciences, University of Milano-Bicocca, Piazza della Scienza 2, 20126 Milan, Italy E-mail: massimo.labra@unimib.it © The Author(s) 2019. This is an Open Access article distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creative- commons.org/licenses/by/4.0/deed.en). Supplementary material IFrom Appendix Table A.1: List of primer pairs used for DNA barcoding analysis. Locus name Name 5' - 3' Sequence Tm °C Amplified samples size Reference rbcL 1 TTGGCAGCATTYCGAGTAACTCC 50 80-200 bp PALMIERI (2009) rbcL rbcL B AACCYTCTTCAAAAAGGTC 50 psbA GTTATGCATGAACGTAATGCTC 53 300-600 bp STEVEN & SUBRAMANYAM (2009) psbA-trnH trnH CGCGCATGGTGGATTCACAATCC 53 ITS p5 CCTTATCAYTTAGAGGAAGGAG 55 300-750 bp CHENG (2016) ITS ITS u4 RGTTTCTTTTCCTCCGCTTA 55 Table A.2: List of all the analysed plant species. The table include the voucher specimens and GenBank accession numbers of the raw material samples used to authenticate the phytoextracts and their intermediates of industrial production treated in this study. Species Type of sample Specimen voucher Company GenBank Accession Number (rbcL 1-B; psbA-trnH; ITS) Achillea millefolium L. Dried raw material FEM_DBE_001 Company 1 LS999840; LS999856; LS999873 Arctium lappa L. Dried raw material FEM_DBE_007 Company 3 LS999841; LS999857; LS999874 Arctostaphylos uva-ursi (L.) Spreng. Dried raw material FEM_DBE_010 Company 2 LS999842; LS999858; LS999875 Cetraria islandica (L.) Ach. Dried raw material FEM_DBE_013 Company 2 ND; ND; LS999876 Echinacea angustifolia DC. Dried raw material FEM_DBE_015 Company 3 LS999843; LS999859; LS999877 Echinacea pallida (Nutt.) Nutt. Dried raw material FEM_DBE_018 Company 1 LS999844; LS999860; LS999878 Echinacea purpurea (L.) Moench Dried raw material FEM_DBE_020 Company 2 LS999845; LS999861; LS999879 Epilobium angustifolium L. Dried raw material FEM_DBE_026 Company 2 LS999846; LS999862; LS999880 Harpagophytum procumbens (Burch.) Dried raw material FEM_DBE_029 Company 1 LS999847; LS999863; LS999881 Malva sylvestris L. Dried raw material FEM_DBE_031 Company 2 LS999848; LS999864, LS999882 Melissa officinalis L. Dried raw material FEM_DBE_033 Company 2 LS999849; LS999865; LS999883 Mentha x piperita L. Dried raw material FEM_DBE_045 Company 1 LS999850; LS999866; LS99984 Passiflora incarnata L. Dried raw material FEM_DBE_047 Company 3 LS999851; LS999867; LS999885 Taraxacum officinale Weber ex F.H. Wigg. Dried raw material FEM_DBE_050 Company 3 LS999852; LS999868; LS999886 Thymus vulgaris L. Dried raw material FEM_DBE_053 Company 3 LS999853; LS999869; LS999887 Tilia platyphyllos Scop. Dried raw material FEM_DBE_059 Company 1 LS999854; LS999870; LS999888 Zingiber officinale Roscoe Dried raw material FEM_DBE_061 Company 1 LS999855; LS999871; LS999889 Supplementary material Tab. A.1: List of primer pairs used for DNA barcoding analysis. Tab. A.2: List of all the analysed plant species. The table include the voucher specimens and GenBank accession numbers of the raw material samples used to authenticate the phytoextracts and their intermediates of industrial production treated in this study. From Appendix Table A.1: List of primer pairs used for DNA barcoding analysis. Locus name Name 5' - 3' Sequence Tm °C Amplified samples size Reference rbcL 1 TTGGCAGCATTYCGAGTAACTCC 50 80-200 bp PALMIERI (2009) rbcL rbcL B AACCYTCTTCAAAAAGGTC 50 psbA GTTATGCATGAACGTAATGCTC 53 300-600 bp STEVEN & SUBRAMANYAM (2009) psbA-trnH trnH CGCGCATGGTGGATTCACAATCC 53 ITS p5 CCTTATCAYTTAGAGGAAGGAG 55 300-750 bp CHENG (2016) ITS ITS u4 RGTTTCTTTTCCTCCGCTTA 55 Table A.2: List of all the analysed plant species. The table include the voucher specimens and GenBank accession numbers of the raw material samples used to authenticate the phytoextracts and their intermediates of industrial production treated in this study. Species Type of sample Specimen voucher Company GenBank Accession Number (rbcL 1-B; psbA-trnH; ITS) Achillea millefolium L. Dried raw material FEM_DBE_001 Company 1 LS999840; LS999856; LS999873 Arctium lappa L. Dried raw material FEM_DBE_007 Company 3 LS999841; LS999857; LS999874 Arctostaphylos uva-ursi (L.) Spreng. Dried raw material FEM_DBE_010 Company 2 LS999842; LS999858; LS999875 Cetraria islandica (L.) Ach. Dried raw material FEM_DBE_013 Company 2 ND; ND; LS999876 Echinacea angustifolia DC. Dried raw material FEM_DBE_015 Company 3 LS999843; LS999859; LS999877 Echinacea pallida (Nutt.) Nutt. Dried raw material FEM_DBE_018 Company 1 LS999844; LS999860; LS999878 Echinacea purpurea (L.) Moench Dried raw material FEM_DBE_020 Company 2 LS999845; LS999861; LS999879 Epilobium angustifolium L. Dried raw material FEM_DBE_026 Company 2 LS999846; LS999862; LS999880 Harpagophytum procumbens (Burch.) Dried raw material FEM_DBE_029 Company 1 LS999847; LS999863; LS999881 Malva sylvestris L. Dried raw material FEM_DBE_031 Company 2 LS999848; LS999864, LS999882 Melissa officinalis L. Dried raw material FEM_DBE_033 Company 2 LS999849; LS999865; LS999883 Mentha x piperita L. Dried raw material FEM_DBE_045 Company 1 LS999850; LS999866; LS99984 Passiflora incarnata L. Dried raw material FEM_DBE_047 Company 3 LS999851; LS999867; LS999885 Taraxacum officinale Weber ex F.H. Wigg. Dried raw material FEM_DBE_050 Company 3 LS999852; LS999868; LS999886 Thymus vulgaris L. Dried raw material FEM_DBE_053 Company 3 LS999853; LS999869; LS999887 Tilia platyphyllos Scop. Dried raw material FEM_DBE_059 Company 1 LS999854; LS999870; LS999888 Zingiber officinale Roscoe Dried raw material FEM_DBE_061 Company 1 LS999855; LS999871; LS999889