Data_Sheet_7_DNA Metabarcoding Authentication of Ayurvedic Herbal Products on the European Market Raises Concerns of Quality and Fidelity.PDF

Ayurveda is one of the oldest systems of medicine in the world, but the growing commercial interest in Ayurveda based products has increased the incentive for adulteration and substitution within this herbal market. Fraudulent practices such as the use of undeclared fillers and use of other species of inferior quality is driven both by the increased as well as insufficient supply capacity of especially wild plant species. Developing novel strategies to exhaustively assess and monitor both the quality of raw materials and final marketed herbal products is a challenge in herbal pharmacovigilance. Seventy-nine Ayurvedic herbal products sold as tablets, capsules, powders, and extracts were randomly purchased via e-commerce and pharmacies across Europe, and DNA metabarcoding was used to assess the ability of this method to authenticate these products. Our analysis reveals that only two out of 12 single ingredient products contained only one species as labeled, eight out of 27 multiple ingredient products contained none of the species listed on the label, and the remaining 19 products contained 1 to 5 of the species listed on the label along with many other species not specified on the label. The fidelity for single ingredient products was 67%, the overall ingredient fidelity for multi ingredient products was 21%, and for all products 24%. The low level of fidelity raises concerns about the reliability of the products, and detection of threatened species raises further concerns about illegal plant trade. The study highlights the necessity for quality control of the marketed herbal products and shows that DNA metabarcoding is an effective analytical approach to authenticate complex multi ingredient herbal products. However, effort needs to be done to standardize the protocols for DNA metabarcoding before this approach can be implemented as routine analytical approaches for plant identification, and approved for use in regulated procedures.</p

Data_Sheet_1_DNA Metabarcoding Authentication of Ayurvedic Herbal Products on the European Market Raises Concerns of Quality and Fidelity.PDF

Ayurveda is one of the oldest systems of medicine in the world, but the growing commercial interest in Ayurveda based products has increased the incentive for adulteration and substitution within this herbal market. Fraudulent practices such as the use of undeclared fillers and use of other species of inferior quality is driven both by the increased as well as insufficient supply capacity of especially wild plant species. Developing novel strategies to exhaustively assess and monitor both the quality of raw materials and final marketed herbal products is a challenge in herbal pharmacovigilance. Seventy-nine Ayurvedic herbal products sold as tablets, capsules, powders, and extracts were randomly purchased via e-commerce and pharmacies across Europe, and DNA metabarcoding was used to assess the ability of this method to authenticate these products. Our analysis reveals that only two out of 12 single ingredient products contained only one species as labeled, eight out of 27 multiple ingredient products contained none of the species listed on the label, and the remaining 19 products contained 1 to 5 of the species listed on the label along with many other species not specified on the label. The fidelity for single ingredient products was 67%, the overall ingredient fidelity for multi ingredient products was 21%, and for all products 24%. The low level of fidelity raises concerns about the reliability of the products, and detection of threatened species raises further concerns about illegal plant trade. The study highlights the necessity for quality control of the marketed herbal products and shows that DNA metabarcoding is an effective analytical approach to authenticate complex multi ingredient herbal products. However, effort needs to be done to standardize the protocols for DNA metabarcoding before this approach can be implemented as routine analytical approaches for plant identification, and approved for use in regulated procedures.</p

Data_Sheet_4_DNA Metabarcoding Authentication of Ayurvedic Herbal Products on the European Market Raises Concerns of Quality and Fidelity.PDF

Ayurveda is one of the oldest systems of medicine in the world, but the growing commercial interest in Ayurveda based products has increased the incentive for adulteration and substitution within this herbal market. Fraudulent practices such as the use of undeclared fillers and use of other species of inferior quality is driven both by the increased as well as insufficient supply capacity of especially wild plant species. Developing novel strategies to exhaustively assess and monitor both the quality of raw materials and final marketed herbal products is a challenge in herbal pharmacovigilance. Seventy-nine Ayurvedic herbal products sold as tablets, capsules, powders, and extracts were randomly purchased via e-commerce and pharmacies across Europe, and DNA metabarcoding was used to assess the ability of this method to authenticate these products. Our analysis reveals that only two out of 12 single ingredient products contained only one species as labeled, eight out of 27 multiple ingredient products contained none of the species listed on the label, and the remaining 19 products contained 1 to 5 of the species listed on the label along with many other species not specified on the label. The fidelity for single ingredient products was 67%, the overall ingredient fidelity for multi ingredient products was 21%, and for all products 24%. The low level of fidelity raises concerns about the reliability of the products, and detection of threatened species raises further concerns about illegal plant trade. The study highlights the necessity for quality control of the marketed herbal products and shows that DNA metabarcoding is an effective analytical approach to authenticate complex multi ingredient herbal products. However, effort needs to be done to standardize the protocols for DNA metabarcoding before this approach can be implemented as routine analytical approaches for plant identification, and approved for use in regulated procedures.</p

Data_Sheet_6_DNA Metabarcoding Authentication of Ayurvedic Herbal Products on the European Market Raises Concerns of Quality and Fidelity.PDF

Ayurveda is one of the oldest systems of medicine in the world, but the growing commercial interest in Ayurveda based products has increased the incentive for adulteration and substitution within this herbal market. Fraudulent practices such as the use of undeclared fillers and use of other species of inferior quality is driven both by the increased as well as insufficient supply capacity of especially wild plant species. Developing novel strategies to exhaustively assess and monitor both the quality of raw materials and final marketed herbal products is a challenge in herbal pharmacovigilance. Seventy-nine Ayurvedic herbal products sold as tablets, capsules, powders, and extracts were randomly purchased via e-commerce and pharmacies across Europe, and DNA metabarcoding was used to assess the ability of this method to authenticate these products. Our analysis reveals that only two out of 12 single ingredient products contained only one species as labeled, eight out of 27 multiple ingredient products contained none of the species listed on the label, and the remaining 19 products contained 1 to 5 of the species listed on the label along with many other species not specified on the label. The fidelity for single ingredient products was 67%, the overall ingredient fidelity for multi ingredient products was 21%, and for all products 24%. The low level of fidelity raises concerns about the reliability of the products, and detection of threatened species raises further concerns about illegal plant trade. The study highlights the necessity for quality control of the marketed herbal products and shows that DNA metabarcoding is an effective analytical approach to authenticate complex multi ingredient herbal products. However, effort needs to be done to standardize the protocols for DNA metabarcoding before this approach can be implemented as routine analytical approaches for plant identification, and approved for use in regulated procedures.</p

Data_Sheet_5_DNA Metabarcoding Authentication of Ayurvedic Herbal Products on the European Market Raises Concerns of Quality and Fidelity.PDF

Ayurveda is one of the oldest systems of medicine in the world, but the growing commercial interest in Ayurveda based products has increased the incentive for adulteration and substitution within this herbal market. Fraudulent practices such as the use of undeclared fillers and use of other species of inferior quality is driven both by the increased as well as insufficient supply capacity of especially wild plant species. Developing novel strategies to exhaustively assess and monitor both the quality of raw materials and final marketed herbal products is a challenge in herbal pharmacovigilance. Seventy-nine Ayurvedic herbal products sold as tablets, capsules, powders, and extracts were randomly purchased via e-commerce and pharmacies across Europe, and DNA metabarcoding was used to assess the ability of this method to authenticate these products. Our analysis reveals that only two out of 12 single ingredient products contained only one species as labeled, eight out of 27 multiple ingredient products contained none of the species listed on the label, and the remaining 19 products contained 1 to 5 of the species listed on the label along with many other species not specified on the label. The fidelity for single ingredient products was 67%, the overall ingredient fidelity for multi ingredient products was 21%, and for all products 24%. The low level of fidelity raises concerns about the reliability of the products, and detection of threatened species raises further concerns about illegal plant trade. The study highlights the necessity for quality control of the marketed herbal products and shows that DNA metabarcoding is an effective analytical approach to authenticate complex multi ingredient herbal products. However, effort needs to be done to standardize the protocols for DNA metabarcoding before this approach can be implemented as routine analytical approaches for plant identification, and approved for use in regulated procedures.</p

Beta diversity according to the ancestral HLA haplotype AH8.1 status.

<p>A principal coordinate plot of the beta diversity measure unweighted unifrac shows all samples colored according to the HLA genotype groups. There were no significant differences between the groups. <i>Yellow</i>: AH8.1 homozygous, i.e. homozygosity for both HLA-B*08 and HLA-DRB1*03. <i>Violet</i>: AH8.1 heterozygotes, i.e. at least 1 copy of HLA-B*08 and HLA-DRB1*03. <i>Turquoise</i>: Non AH8.1 heterozygotes, i.e. HLA-B*08 and HLA-DRB1*03 both not present. <i>Grey</i>: HLA-DRB1 homozygous but non AH8.1.</p

Characteristics of the study population according to HLA genotype^a.

<p>^aFour different groups were included to represent homozygosity and heterozygosity for the AH8.1 haplotype (as defined by the presence of HLA-B*0801 and DRB1*0301), absence of this haplotype as well as homozygosity for other DRB1 haplotypes. There were no significant differences (all p>0.23) for any of the parameters when applying Kruskal-Wallis test for continuous and 2x4 chi square test for categorical parameters.</p><p>^bTransport time as defined by time from sampling to the freezer.</p><p>Characteristics of the study population according to HLA genotype<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133804#t002fn001" target="_blank">^a</a>.</p

The Influence of the Autoimmunity-Associated Ancestral HLA Haplotype AH8.1 on the Human Gut Microbiota: A Cross-Sectional Study

<div><p>Multiple immune-related genes are encoded in the HLA complex on chromosome 6p21. The 8.1 ancestral haplotype (AH8.1) include the classical HLA alleles HLA-B*08:01 and HLA-DRB1*03:01, and has been associated with a large number of autoimmune diseases, but the underlying mechanisms for this association are largely unknown. Given the recently established links between the gut microbiota and inflammatory diseases, we hypothesized that the AH8.1 influences the host gut microbial community composition. To study this further, healthy individuals were selected from the Norwegian Bone Marrow Donor Registry and categorized as either I. AH8.1 homozygote (n=34), II. AH8.1 heterozygote (n=38), III. Non AH8.1 heterozygote or IV. HLA-DRB1 homozygote but non AH8.1 (n=15). Bacterial DNA from stool samples were subjected to sequencing of the V3–V5 region of the 16S rRNA gene on the 454 Life Sciences platform and data analyzed using Mothur and QIIME. The results showed that the abundances of different taxa were highly variable within all pre-defined AH8.1 genotype groups. Using univariate non-parametric statistics, there were no differences regarding alpha or beta diversity between AH8.1 carriers (categories I and II) and non-carriers (categories III and IV), however four different taxa (<i>Prevotellaceae</i>, <i>Clostridium XVIII</i>, <i>Coprococcus</i>, <i>Enterorhabdus</i>) had nominally significant lower abundances in AH8.1 carriers than non-carriers. After including possible confounders in a multivariate linear regression, only the two latter genera remained significantly associated. In conclusion, the overall contribution of the AH8.1 haplotype to the variation in gut microbiota profile of stool in the present study was small.</p></div

Enterotype groups according to ancestral HLA haplotype AH8.1 status.

<p>The figure shows the individual samples as colored symbols according to their HLA genotype groups (see below) distributed according to similarity of their distribution of bacterial genera in a two-dimensional plot according to the methods by Arumugam et al. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133804#pone.0133804.ref034" target="_blank">34</a>]. The samples were classified into three fractions, enterotypes, dominated by either <i>Prevotella</i> (blue), <i>Bacteroides</i> (green) or a mix of bacteria (red), where the colored square indicate the centre of the distribution of the enterotype, the straight lines connect the included samples and the colored ellipses cover the individuals near the center of gravity for each cluster (1.5σ). Bacterial taxa overrepresented in the corresponding enterotypes are listed. As evident from the symbol color of the individual dots, the different HLA genotype groups (see below) were not preferentially distributed to one particular enterotype fraction. <i>Yellow diamonds</i>: AH8.1 homozygotes, i.e. homozygosity for both HLA-B*08 and HLA-DRB1*03. <i>Violet squares</i>: AH8.1 heterozygous, i.e. at least 1 copy of HLA-B*08 and HLA-DRB1*03. <i>Turquoise circles</i>: Non AH8.1 heterozygotes, i.e. HLA-B*08 and HLA-DRB1*03 both not present. <i>Grey triangles</i>: HLA-DRB1 homozygotes but non AH8.1.</p

Genus level taxonomic distribution in 117 Norwegian stool samples according to ancestral HLA haplotype AH8.1 status.

<p>(A) The genus abundances sorted according to the relative abundance of <i>Bacteroides</i>, of n = 72 AH8.1 carriers (left) are shown compared with the n = 45 non-AH8.1 samples (right). In (B) the genus abundances are shown according to all four pre-defined genotype groups; Left: AH8.1 homozygotes, i.e. homozygosity for both HLA-B*08 and HLA-DRB1*03; Middle left: AH8.1 heterozygotes, i.e. at least 1 copy of HLA-B*08 and HLA-DRB1*03; Middle right: Non AH8.1 heterozygotes, i.e. HLA-B*08 and HLA-DRB1*03 both not present; Right: <i>HLA-DRB1</i> homozygous but non AH8.1.</p