Non Professional grade supplements are often contaminated with fillers and toxins

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R E S E A R C H A R T I C L E Open Access

DNA barcoding detects contamination andsubstitution in North American herbal productsSteven G Newmaster1*, Meghan Grguric2, Dhivya Shanmughanandhan3, Sathishkumar Ramalingam3

and Subramanyam Ragupathy1*

Abstract

Background: Herbal products available to consumers in the marketplace may be contaminated or substituted with

alternative plant species and fillers that are not listed on the labels. According to the World Health Organization,

the adulteration of herbal products is a threat to consumer safety. Our research aimed to investigate herbal product

integrity and authenticity with the goal of protecting consumers from health risks associated with productsubstitution and contamination.

Methods: We used DNA barcoding to conduct a blind test of the authenticity for (i) 44 herbal products

representing 12 companies and 30 different species of herbs, and (ii) 50 leaf samples collected from 42 herbal

species. Our laboratory also assembled the first standard reference material (SRM) herbal barcode library from 100

herbal species of known provenance that were used to identify the unknown herbal products and leaf samples.

Results: We recovered DNA barcodes from most herbal products (91%) and all leaf samples (100%), with 95%

species resolution using a tiered approach (rbcL + ITS2). Most (59%) of the products tested contained DNA barcodes

from plant species not listed on the labels. Although we were able to authenticate almost half (48%) of the

products, one-third of these also contained contaminants and or fillers not listed on the label. Product substitution

occurred in 30/44 of the products tested and only 2/12 companies had products without any substitution,

contamination or fillers. Some of the contaminants we found pose serious health risks to consumers.

Conclusions: Most of the herbal products tested were of poor quality, including considerable product substitution,contamination and use of fillers. These activities dilute the effectiveness of otherwise useful remedies, lowering the

perceived value of all related products because of a lack of consumer confidence in them. We suggest that the

herbal industry should embrace DNA barcoding for authenticating herbal products through testing of raw materials

used in manufacturing products. The use of an SRM DNA herbal barcode library for testing bulk materials could

provide a method for best practices in the manufacturing of herbal products. This would provide consumers with

safe, high quality herbal products.

Background

Globalization of trade is expanding natural productcommodity markets that sustain life and promote goodhealth, yet the challenging financial climate is squeezing

profit margins and exacerbating the propensity for con-tamination, fraudulent market substitution and the use ofunlabeled fillers. This comes at a time when consumersare becoming increasingly concerned about the authenti-city of the products they purchase. North America is a

major provider of goods such as herbal products, a com-modity that has come under scrutiny recently in themedia due to product substitution [1-3].

The International Trade in herbal products is a major

force in the global economy and the demand is increas-ing in both developing and developed nations. There arecurrently more than 1,000 companies producing medi-cinal plant products with annual revenues in excess ofUS$60 billion. Notably, medicinal herbs now constitutethe most rapidly growing segment of the North Americanalternative medicine market [4,5], with over 29,000herbal substances [6,7] generating billions of dollars intrade. These statistics are indicative of the rapid growth

* Correspondence: [email protected]; [email protected] for Biodiversity Genomics, Biodiversity Institute of Ontario (BIO),

University of Guelph, Guelph, Ontario N1G 2W1, Canada

Full list of author information is available at the end of the article

2013 Newmaster et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of theCreative Commons Attribution License (http://creativecommons.org/licenses/by/2.0) , which permits unrestricted use,distribution, and reproduction in any medium, provided the original work is properly cited.

Newmaster et al. BMC Medicine 2013, 11:222

http://www.biomedcentral.com/1741-7015/11/222
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(>15%/year) in the marketplace for natural plant productsand the broadening consumer base interested in herbalproducts from India, Canada, USA and Europe. Yet, ashighlighted in recent media reports, product adulterationand ingredient substitution is not uncommon as speciesof a lower market value are substituted for those of ahigher value. This practice constitutes not only productfraud, but according to the World Health Organization(WHO), the adulteration of herbal products is a threat toconsumer safety (see Discussion). Eroding consumer con-fidence is driving the demand for research and market

testing on herbal product authentication.There are currently no best practices in place for iden-

tifying the species of the various ingredients used inherbal products. This is because the diagnostic morpho-logical features of the plants on which the currentLinnaean taxonomic system is based cannot typically

be assessed from powdered or otherwise processed bioma-terials. As a result, the marketplace is prone to contamin-ation and possible product substitution, which dilute theeffectiveness of otherwise useful remedies, lowering theperceived value of all related products because of a lack ofconsumer confidence in them. Herbal product substitu-

tion has been documented for many individual medicinalplant species [8-10], teas [1] and nutraceuticals [11,12].Although there is limited research available, the frequencyof product mislabeling in herbal products has been esti-mated at 14% to 33% from previous studies [1-3]. Thereare legitimate health concerns for consumers which re-

sults in a lack of confidence in safe, high quality herbalproducts.A broadly accessible commercial tool to detect prod-

uct substitution and develop best practices for au-thentication of many commercial products is beginning toemerge. DNA barcoding has been used to test a numberof natural products [1,3,13]. Barcoding uses state-of-the-

art biotechnology to help identify plant material based onshort, standardized gene sequences, in a rapid, accurateand cost-effective manner. For example, Srirama et al.[10] has demonstrated the efficiency of DNA barcodingin identifying adulterants in the raw drug trade ofPhyllanthus (Euphorbiaceae). Phyllanthus is one of the

most important groups of species traded as a raw herbaldrug treatment for liver disorders [14]. This study showedthat plant DNA barcoding can effectively discriminatePhyllanthus species, and hence can be used to resolvespecies admixtures in the raw drug trade samples; thereare several phenotypically similar species that could easilybe misidentified and mixed within herbal products. Overthe last 3 years, studies of Chinese plant medicine [15-20]have documented the potential scope and magnitude ofmarket substitution using this biotechnology. How-ever, there are only a few studies [1,3] available, andthey provide a limited assessment of the authenticity

of commercial herbal products within the market-place. Currently there is a gap in our understandingto the extent of herbal product substitution, contam-ination and use of fillers. Also, there are no studiesthat have used a standard reference material (SRM)herbal barcode library and barcode regions that facili-tate species resolution.

This study utilized blind sampling of commerciallyavailable herbal products, which were tested for authen-tication of plant ingredients using an SRM herbal DNAbarcode library. Our research questions focused on thefollowing three areas. (1) Authentication: is the herbalspecies on the label found in the product? (2) Substitu-tion: is the main herbal ingredient substituted by otherspecies? (3) Fillers: are there any unlabeled fillers used?

Methods

Study designWe tested the authenticity of 44 (41 capsules; 2 powders;1 tablet) herbal products representing 12 companies.The samples were submitted in a blind test for authenti-cation (product no. label only) using DNA barcodingat the Centre for Biodiversity Genomics (CBG) within

the Biodiversity Institute of Ontario (BIO), Universityof Guelph.

The project also included a second blind test in whichwe barcoded 50 leaf samples (blind labels) collectedfrom living samples of known provenance from horticul-tural greenhouses. These leaf samples represented 42

species including all those listed on the herbal productlabels and some closely related species. This provided anindependent test of the ability of barcoding to differenti-ate all the species on the herbal product labels used inthis study. All of the samples are in the Barcode ofLife Database (BOLD) (Additional file 1: Table S1) and

GenBank (Additional file 2: Table S2). All vouchers arescanned and recorded in the BOLD and deposited in theBIO Herbarium at the University of Guelph.

Herbal product sampling

In North America, herbal products are available in a var-iety of mainstream outlets such as supermarkets, health

food stores, pharmacies and can be ordered online fromherbal supply companies. In our study, samples werecollected in the greater Toronto area (GTA) in Canada,with several samples mailed from distributors in theUSA. All products are available to consumers in both

Canada and the USA. The herbal product samples repre-sented 30 herbal species that were each represented by 2or 3 different companies.

DNA barcoding (DNA extraction and sequencing)

We barcoded triplicate samples of plant product (pow-der) from the 44 herbal products and 50 herbal leaf

Newmaster et al. BMC Medicine 2013, 11:222 Page 2 of 13



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samples using a tiered approach (rbcL + ITS2) [21]. Mul-tiple DNA extracts (five to ten) were taken from each ofthese triplicate samples of which these DNA extractswere barcoded to make sure we did not miss any speciesDNA. Since these were only single ingredient herbalproducts we did not expect to detect more than oneplant species barcode unless it was a contaminant. How-ever, our intensive DNA extract sampling allowed us tosample for other plant polymerase chain reaction (PCR)products, which were then sequenced separately. This in-cluded building barcode sample by species curves to see

how many times we needed to sample to get an accurateestimate of species in a sample. As a result some of theproducts yielded two difference sequences, each fromDNA extracts taken from the same sample product.

Total genomic DNA was isolated from 100 mg of thedry material using the Nucleospin Plant II Mini DNA

Extraction kit. The PCR amplification was performed ina 20 L reaction mixture that contained 2.5 L of genomicDNA, 2.5 L of 10 Pfu buffer with MgSO4 (Fermentas),2.5 L of 2 mM dNTPs (Fermentas), 0.5 L each of forwardand reverse primers (10 pM) and 0.2 L of 2.5 UPfu DNAPolymerase (Fermentas) and 2 L 0.5% dimethylsulfoxide(DMSO). The primers and the reaction conditions are pre-

viously published for rbcL [22] and ITS2 [16]. TouchdownPCR was carried out. The PCR products obtained fromthese reactions were subjected to Big Dye (version 3.1)sequencing reactions that had a total volume of 10 Land included 10 pMol of each primer. The sequencing

amplification protocol consisted of 1 cycle of 1 minute at96C, followed by 30 cycles of 10 s at 96C, 5 s at 55C,and 4 minutes at 60C. The amplicons were bidirectionallysequenced using ABI 377 sequencer (Applied Biosystems).The chromatographic traces were aligned and codon readin the Codoncode Aligner ver. 3.0 (CodonCode, Dedham,

MA, USA) and contigs were generated. Sequences weredeposited in BOLD and GenBank.

SRM herbal barcode library

An SRM herbal barcode library was prepared and usedto identify the unknown herbal products and leaf sam-ples. This library was assembled from 100 herbal species

of known provenance that were identified by taxonomicexperts. These samples were prepared into herbarium

vouchers and are archived at the BIO Herbarium and inthe BOLD. Although our library contained 100 herbalspecies in this study it is part of a larger project in which

we are currently expanding the SRM herbal barcode li-brary to include the main species used within herbalproduct industry in North America.

Product identification and analysis

Identification of unknown barcodes from products andleaves was conducted by comparing Basic Local Alignment

Search Tool (BLAST) data against our SRM herbalbarcode library with a minimum BLAST cut off of 97%identity for a top match [3]. These results were verified byneighbor-joining tree analysis in which we compared thebranches of unknown specimens to sequences of referencespecies. A species is considered as taxonomically resolvedif its members showed at least one diagnostic differencefrom any other taxon in the study and formed a monophy-letic clade.

The definitions of terms used when assessing the iden-tity of DNA barcodes recovered from herbal productsare as outlined below.

Authentic

A product is authentic if it contains a DNA barcode fora species that is the main ingredient on the label of atested herbal product.

Contamination

A product is contaminated if a DNA barcode is foundfor a species other than what is labeled on the testedproduct, in addition to the authentic barcode; this couldinclude known herbal product filler species (see below).

Substitution

Product substitution occurs when a DNA barcode isfound for a species other than what is labeled on thetested product, AND there is NOT a barcode recoveredfor the main ingredient listed. Note that this strict defin-

ition does not consider whether it is an accidental mis-identification of a bulk product or a fraudulent marketsubstitution for a cheaper product.

Filler

A product contains fillers if a DNA barcode is found for

known herbal product filler species including rice (Oryzasativa), soybean (Glycine max) and wheat (Triticumspp.). Fillers may be found in place of (substitution) orin addition to the barcode of the main product ingredi-ent (contamination).

Results

DNA barcode success

Recovery of DNA barcodes from various herbal productswas successful. High quality DNA barcodes were obtain-able for both rbcL and ITS2 regions for 100% of the 100

vouchered herbal species (Ontario Agricultural College

Herbarium), which served as a high quality SRM herbalbarcode library. This library provided a database thatcould be used to identify unknown haplotypes (barcodes)recovered from herbal products. The SRM classificationtree (Figure 1) shows that all herbal species have 100%species resolution, with each species presenting at leastone diagnostic difference from any other taxon and




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formed a monophyletic clade (Figure 1). Herbal productDNA barcode success rate was 91% (40/44 products) forthe combined regions, resulting in the recovery of 34 rbcLand 40 ITS2 sequences. Species resolution for barcodes re-covered from herbal products was 66% (rbcL) and 93%

(ITS2). Low resolution in rbcL was expected, because it isknown to have insufficient nucleotide sequence variabilityto distinguish among closely related species. The three un-resolved species with ITS2 was due to three samples thathad product substitution with species that were not in ourSRM library; these unknown species barcodes did matchthe nearest family and genus. We also barcoded blindly

labeled herbal leaf samples collected from living samplesfrom horticultural greenhouses. This provided an inde-pendent test of the ability of barcoding to differentiate allthe species on the herbal product labels. Species reso-lution for barcodes recovered from the herbal leaf samples

was 57% (rbcL) and 100% (ITS2), with perfect matches tothe herbal SRM barcode library using BLAST.

Authentication: is the herbal species on the label found in

the product?

What is listed on the label of herbal products is not al-ways what is found within the product. DNA barcoding

Figure 1 Standard reference material (SRM) herbal barcode library consensus tree (MP-ratchet analysis) for combined barcode data

from rbcL and ITS2 from 100 herbal species of known provenance that were identified by taxonomic experts.




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was able to authenticate 48% of the products tested(Figure 2). However, most (59%) of the herbal prod-ucts tested contained species of plants not listed onthe labels (Figure 2). Some (33%) of the authenticatedherbal products also contained contaminants and or fillersnot listed on the label (Figure 2). Some of the DNA se-quences recovered were only identified to family as ourSRM herbal DNA barcode library is not yet complete (seenote above). However, an incomplete SRM barcode librarystill allows a correct identity in a hierarchical way to familyand genera depending on the level of best match within

the classification tree. Some of the contaminants wereof unrelated species from entirely different plant fam-ilies (Table 1). Although many of the herbal companies(75%) whose product we tested did have some authenticproducts, only 2 of the 12 companies provided authenticproducts with no substitution, contaminants or fillers.

Conversely, we were unable to authenticate any of theproducts for 25% of the companies (Figure 3). Althoughthese numbers shed light on the number of companieswith authenticate products we only sampled three or fourproducts per company and therefore differences amongcompanies are likely due to chance.

Substitution: is the main herbal ingredient substituted for

some other species?

Our barcoding analysis of 44 herbal products representing30 medicinal plants species identified product substitutionin 32% of the samples (Figure 2; Table 1). The criteria we

used to determine product substitution was the identity ofother plant taxa in place of the products main labeled in-gredient, which was NOT detected within the sample. Wedetected product substitution within most (83%) of the 12companies that we tested (Figure 3). It should be notedthat for 90% of the herbal species in this study, there were

product(s) from at least one company that yielded acorrect identity (indicating an authentic product). Thisis confirmation that processing is not an impedimentto recovering barcodes. It should also be noted that wesampled each product intensively without replacement;multiple samples per product were extracted and barcodedto make sure we did not miss any species DNA. Sincethese were only single ingredient herbal products wedid not expect to detect more than one plant speciesbarcode unless it was a contaminant. Our intensive sam-pling yielded a maximum of two species barcodes per

herbal product of which one was either the correct herbalproduct species or a substitute, and the other was either afiller or contaminant.

Fillers: are there unlabeled fillers used in herbal products?

Some of the herbal products contained fillers that were

not listed on the labels. Our definition is limited to the de-tection of DNA barcodes from O. sativa and G. max.These filler species were detected within 21% of the prod-ucts. We also detected grasses in the samples, which alsoincluded wheat (Triticum spp.). It is not known whetheror not these plants were intentionally used as fillers or if

they are contaminants; we are currently investigating theuse of fillers in processing by each individual company. In9% of the samples only rice or wheat barcodes weredetected; there were no barcodes detected for the herbalspecies on the label. The use of fillers was limited to one-third of companies whose products we tested (Figure 3).

DiscussionContamination and substitution in North American herbal

products

Few studies have attempted to estimate how prevalentherbal product contamination and substitution is in the

Figure 2 DNA barcode results from blind testing of the 44 herbal products representing 30 medicinal species of plants.




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Table 1 DNA barcode results listed for individual samples from blind testing of the 44 herbal products and 50 herbal

leaf samples representing 42 medicinal species of plants

Market label or Herb leafspecies

Samplefrom

Barcode ID ITS2 ID rbcL ID

Achillea millefolium (Yarrow) HP64C Achillea millefolium + Medicago sativaa Achillea millefolium + Medicago sativaa Medicago sativaa

Achillea millefolium HP56L Achillea millefolium Achillea millefolium Achillea sp.

Actaea racemosa (Cohosh) HP42C Actaea asiaticaa Actaea asiaticaa Actaea asiaticaa

Actaea racemosa HP20C NS NS NS

Actaea racemosa HP45L Actaea racemosa Actaea racemosa Actaea racemosa



Amaranthus sp. (Amaranth) HP89P Amaranthus sp. Amaranthus sp. Amaranthus sp.

Amaranthus albus HP79L Amaranthus albus Amaranthus albus Amaranthus sp.

Amaranthus retroflexus HP49L Amaranthus retroflexus Amaranthus retroflexus Amaranthus sp.

Apium graveolens (Celery) HP34C Amaryllidaceae + Oryza sativaa Amaryllidaceaea Oryza sativab

Apium graveolens HP32C NS NS NSApium graveolens HP43C NS NS NS

Apium graveolens HP13L Apium graveolens Apium graveolens Apiaceae



Arctium lappa (Burdock) HP6C Arctium lappa Arctium lappa Arctium lappa

Arctium lappa HP22C Oryza sativaa Oryza sativab Oryza sativab

Arctium lappa HP21C Ranunculaceaea Ranunculaceaea NS

Arctium lappa HP4L Arctium lappa Arctium lappa Arctium lappa

Arctium lappa HP92L Arctium lappa Arctium lappa Arctium lappa

Cinnamomum cassia

(Cinnamon)

HP86T Ranunculaceaea Ranunculaceaea NS

Cinnamomum cassia(Cinnamon)

HP60L Cinnamomum cassia Cinnamomum cassia Cinnamomum sp.

Echinacaea purpurea(Echinacaea)

HP35C Echinacaea purpurea + Partheniumhysterophorusa

Echinacaea purpurea + Partheniumhysterophorusa

Asteraceaea

Echinacaea purpurea HP27C Echinacaea purpurea + Partheniumhysterophorusa

Echinacaea purpurea + Partheniumhysterophorusa

Asteraceaea

Echinacaea purpurea HP88L Echinacaea purpurea Echinacaea purpurea Echinacaeapurpurea




Ginkgo biloba (Ginkgo) HP63C Ginkgo biloba Ginkgo biloba Ginkgo biloba

Ginkgo biloba HP28C Juglans nigraa Juglans nigraa NS*

Ginkgo biloba HP81C Oryza sativaa Oryza sativab Oryza sativab

Ginkgo biloba HP33L Ginkgo biloba Ginkgo biloba Ginkgo biloba




Hypericum perforatum(St. Johns wort)

HP8C Fabaceaea Fabaceaea NS




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Table 1 DNA barcode results listed for individual samples from blind testing of the 44 herbal products and 50 herbal

leaf samples representing 42 medicinal species of plants (Continued)

Hypericum perforatum HP46C Hypericum perforatum Hypericum perforatum Hypericumperforatum

Hypericum perforatum HP82C Oryza sativaa Oryza sativab Oryza sativab

Hypericum perforatum HP39L Hypericum perforatum Hypericum perforatum Hypericumperforatum

Hypericum perforatum HP94L Hypericum perforatum Hypericum perforatum Hypericumperforatum

Juglans nigra (Walnut) HP40C Juglans nigra Juglans nigra NS

Juglans nigra HP58C NS NS NS

Juglans nigra HP84C Oryza sativaa NS Oryza sativab

Juglans nigra HP30L Juglans nigra Juglans nigra Juglans nigra



Matricaria recutita (Chamomile) HP37C Matricaria recutita Matricaria recutita Matricaria recutita

Matricaria recutita (Chamomile) HP91C Matricaria recutita + Medicago sativa Matricaria recutita + Medicago sativa Medicago sativa

Matricaria recutita (Chamomile) HP36C Matricaria recutita + Glycine max Matricaria recutita Glycine max

Matricaria recutita HP76L Matricaria recutita Matricaria recutita Matricaria recutita

Mentha arvensis (Mint) HP3C Mentha arvensis Mentha arvensis Lamiaceae

Mentha arvensis HP52L Mentha arvensis Mentha arvensis Lamiaceae

Mentha spicata HP15L Mentha spicata Mentha spicata Lamiaceae

Nepeta cataria HP9L Nepeta cataria Nepeta cataria Lamiaceae

Oenothera biennis (EveningPrimrose)

HP48C Oenothera biennis Oenothera biennis Oenothera biennis

Origanum vulgare (Oregano) HP10C Origanum vulgare + Lamiaceaea Origanum vulgare + Lamiaceaea Lamiaceaea

Oenothera biennis HP47L Oenothera biennis Oenothera biennis Oenothera biennis

Origanum vulgare HP26L Origanum vulgare Origanum vulgare Origanum vulgare

Origanum vulgare HP50L Origanum vulgare Origanum vulgare Origanum vulgare

Oxalis Corniculata(WoodSorrel)

HP1P Oxalis Corniculata + Medicago sativaa Oxalis Corniculata + Medicago sativaa Oxalis sp.

Oxalis Corniculata HP72L Oxalis Corniculata Oxalis Corniculata Oxalis sp.

Oxalis stricta HP57L Oxalis stricta Oxalis stricta Oxalis sp.

Oxalis acetosella HP18L Oxalis acetosella Oxalis acetosella Oxalis sp.

Plantago ovate (Psyllium) HP69C Plantago ovate Plantago ovate NS

Plantago ovate HP71L Plantago ovate Plantago ovate Plantago sp.

Plantago major HP66L Plantago major Plantago major Plantago sp.

Plantago lanceolata HP14L Plantago lanceolata Plantago lanceolata Plantago sp.

Portulaca oleracea (Purslane) HP83C Portulaca oleracea Portulaca oleracea Portulaca oleracea

Portulaca oleracea HP65C Portulaca oleracea + Glycine maxb Portulaca oleracea + Glycine maxb Glycine maxb

Portulaca oleracea HP38L Portulaca oleracea Portulaca oleracea Portulaca oleracea

Portulaca oleracea HP93L Portulaca oleracea Portulaca oleracea Portulaca oleracea

Rubus idaeus (Raspberry) HP24C Rubus idaeus Rubus idaeus Rubus sp.

Rubus idaeus HP12C Rubus idaeus + Medicago sativaa Rubus idaeus + Medicago sativaa Medicago sativaa

Rubus idaeus HP61L Rubus idaeus Rubus idaeus Rubus sp.

Rubus parviflorus HP29L Rubus parviflorus Rubus parviflorus Rubus sp.

Rudbeckia hirta(black-eyed Susan)

HP62C Rudbeckia hirta Rudbeckia hirta Rudbeckia hirta




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marketplace. To date there are only a few [1-3] studiesof herbal product substitution, representing


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supplement products labeled as black cohosh (Actaearacemosa) found that 75% of the samples were authen-tic black cohosh, whereas 25% were substituted with 4closely related Asian Actaea species (A. asiatica , A.cimicifuga, A. dahurica, and A. simplex). We alsofound substitution of black cohosh for Asian Actaea,as did another study by Wallace et al. [3]. Wallaceet al. [3] also documented that 50% of ginseng prod-ucts labeled Korean ginseng (Panax ginseng) wereactually American ginseng (Panax quinquefolius). Al-though the DNA barcode success rate was low (48%,

see below) in Wallaces study, they were still able todetect product substitution in 14% of the herbal sam-ples tested, representing 7 species of herbs in 14products. This study also detected fillers in 21% ofthe herbal products [3]. We found alfalfa (Medicagosativa) in 16% of the products tested, which was also

previously detected in teas [1]. It is unlikely that thiswas a contaminant as it was found in so many of theproducts in addition to the main ingredient. It maybe possible that this species is used by some compan-ies as a cheap filler as it is commonly grown andbailed in agricultural areas, and available in large

quantities throughout the year.Contamination and substitution in herbal products

present considerable health risks for consumers. In ourstudy, we found contamination in several products withplants that have known toxicity, side effects and/ornegatively interact with other herbs, supplements, or

medications. For example, we found that one product(HP8) labeled as St. Johns wort (Hypericum perforatum)was substituted with Senna alexandrina (fabaceae); itcontained only senna barcodes and no St. Johns wortbarcodes. This is a serious health risk as senna is aFood and Drug Administration (FDA)-approved non-prescription herbal laxative, which is not for prolonged

use as it can cause adverse effects such as chronic diar-rhea, cathartic colon, liver damage, abdominal pain, epi-dermal breakdown and blistering [23,24]. Senna containsseveral unique glycosides called sennosides that interactwith immune cells in the colon [25]. We also found con-tamination of several herbal products (Table 1) with

Parthenium hysterophorus (feverfew). Feverfew is native toEurasia and is an invasive weed in Europe, the Mediterra-nean, North America and Chile. Although feverfew hasbeen used to treat fever, migraine headaches and arthritis,it does have negative side effects such as swelling andnumbness of the mouth, oral ulcers, nausea, vomiting, ab-dominal pain, diarrhea, and flatulence; some users experi-enced withdrawal syndromes when discontinuing use,such as rebound headaches and muscle and joint pain[26,27]. Feverfew reacts with a variety of medicationsmetabolized by the liver and may also increase therisk of bleeding, especially if taken with blood-thinning

medications such as warfarin or aspirin [28,29]. Pregnantwomen should not consume any amount of feverfew [29].Feverfew can also cause allergic reactions, including con-tact dermatitis due to a toxin found in this plant speciescalled parthenin [30,31]. Although it is not known howcontamination with feverfew occurred in this product, it ispossible that it was a common weed in the crop used tomake this herbal product.

We also found contamination of a Ginkgo productwith Juglans nigra (black walnut). Wallace et al. [3] alsofound contamination of an Echinacea product with wal-

nut and advised that such contamination would be par-ticularly dangerous for a consumer with nut allergies.However, we feel it is unlikely that nuts are the sourceof contamination, but rather leaves that often litter a

very large area surrounding a tree in mid to late sum-mer. It is conceivable that there may have been a field of

commercial herbs bordered by walnuts (a commonoccurrence on the landscape), and that the target cropwas harvested along with walnut leaves. Walnut leaves,woods, bark and fruits all contain juglone [32], which istoxic; juglone can lead to oxidative stress or electrophi-licity [33-35]. The Registry of Toxic Effects of Chemical

Substances (RTECS) [36] defines juglone as an equivo-cal tumorigenic agent (lungs, thorax and respiration,skin and appendages), which has been shown to pro-mote skin tumors [37-39]. Ours and other studies [1,3]of herbal product contamination in the marketplacehave not been able to identify all the contaminants to

species-level resolution due to the lack of a completeSRM barcode library for herbal plants; a complete SRMlibrary would include herbal species, related species andknown toxic plants. It is possible that there were othercontaminants that were missed because they could onlybe identified to family rather than to species.

Unlabeled plant fillers may also be found in herbal prod-

ucts, and these fillers are in some cases a potential healthrisk for consumers. DNA barcoding in our study identifiedseveral potential fillers, including rice (O. sativa), soybean(G. max) and various grasses such as wheat (Triticumspp.). This is a health concern for people allergic to theseplants, as well as for people seeking gluten free products.

Wallace et al. [3] found rice and soybean fillers in naturalplant and animal products and suggested that these fillersmay produce a mixed signal during the sequencingprocess, contributing to a rather high percentage of failedsequencing reactions in capsulated products. We did notshare this high percentage of sequence failures and foundbarcodes from both the herbal species on the label andfillers within the same product, suggesting that there maybe fillers substituted for authentic herbal species. It isprobable that barcoding detected rice and soybean, whichis a common microcrystalline cellulose and gelatin used asadditives in preparing the capsules that contain the herbal




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product [40]. It is also common practice in the naturalproducts industry to use fillers such as those listedabove, which are mixed with the active ingredients.Nonetheless, the consumer has a right to see all of theplant species used in producing a natural product on thelist of ingredients.

Challenges and biotechnical advances in DNA barcoding

of herbal products

Authentication of herbal products is challenging, butnew DNA barcoding methods are providing tools forroutine market analysis [41]. Several major challengesinclude the lack of an SRM herbal barcode library, anduse of only plastid barcode regions, which has resultedin low species resolution. The original definition of a re-liable DNA identification of species requires (i) recoveryof a barcode sequence from the sample, (ii) representa-

tion of relevant species in the reference database, and(iii) sufficient nucleotide sequence variability to distin-guish among closely related species [42]. We are design-ing new protocols for recovering DNA barcodes fromherbal samples, which satisfies the first requirement, butthe second requirement of a reference database has yet

to be satisfied. This problem has been discussed in pre-vious market studies that have tested the authenticity ofherbal products without a herbal SRM barcode library[1-3]. These studies have defined the uncertainties ofassigning unknown haplotypes from herbal products usingincomplete reference barcode databases in GenBank and

BOLD. The use of GenBank is inappropriate given thatmany of the DNA sequences do not have vouchers toprofessionally identified specimens archived in a herb-arium. Therefore the reference sequence in GenBankmay be from an incorrectly identified plant and thereis no way to verify the specific origin of that DNA.Similarly, the Medicinal Materials DNA Barcode

Database [43] provides barcodes for many species ofmedicinal plants for which there is no record of vouchersto confirm identity; this is an essential component of anyDNA Barcode [42]. Our study is the first to build a partialherbal SRM barcode library using vouchered samples ofknown provenance that was used to test the identity of

unknown barcodes recovered from herbal products. Al-though we only have 100 species in the SRM library, weexpect to expand this to over 200 species by the end of2013, and 1,000 species by 2015. However, this will onlyadd up to 55% of the 1,800 medicinal plant speciesthat are commercially available [44]. If we want tohave reliable identifications using DNA barcodes wemust build an SRM herbal barcode library that has allsister species for the 1,800 known medicinal speciesused in commercial products. This is one of the goalsof our Herb-BOL (barcode of life) research programin the next 5 years.

The use of only plastid barcode regions is problematicfor testing herbal products. Previous studies have notsatisfied Heberts third criteria for a reliable barcode: suf-ficient nucleotide sequence variability to distinguishamong closely related species [42]. Other studies haveadopted the core plant barcodes of rbcLa and matK

genes [45]. We do not recommend using matK becauseof associated problems we encountered with PCR ampli-fication while working on nutmeg [46]. Other publishedstudies have also encountered similar problems [47-51].We recommend a tiered [21] approach to analysis asone way to overcome the issue of alignment with non-

coding regions, while providing the most variability fromtwo-barcode regions for identifying closely related taxa.The tiered approach is based on the use of a common,easily amplified, and aligned region such as rbcL thatcan act as a scaffold on which to place data from a

highly variable region such as ITS2. The use of rbcL as aprimary tier barcode is appropriate because of its univer-sality, ease of amplification, ease of alignment, and be-cause there is a significant body of data available forevaluation [51,52]. We prefer to use ITS2 as the secondtier as proposed by Chen et al. [16] for use in identifyingmedicinal plants because (i) it provides high speciesresolution, (ii) it is from the nuclear genome, whichevolves at a different rate than the plastid genome, and(iii) it is a much shorter sequence allowing higher recov-ery from processed plant materials found within herbalproducts. The use of rbcL + ITS2 in our study resulted

in 95% species resolution of barcodes recovered fromherbal products, which is much higher than previousstudies that used rbcL + matK (42% in Wallace et al. [3];66% in Stoeckle et al. [1]). DNA success rate was high inour study (91%) and in that of Stoeckle et al. [1] wherethey recovered barcodes from 90% of commercial herbaltea products. DNA barcode success rate was relativelylow (48%) in the study by Wallace et al. [3]. This may beattributed to different manufacturing protocols, the typeof plant material (for example, leaf, stem, roots and soon) used in the herbal preparation, or insufficient labora-tory protocols used to extract, amplify and sequencehaplotypes from herbal products. Herbal products contain

plant secondary metabolites that may prevent barcode suc-cess. Herbal extracts contain complicated mixtures of or-ganic chemicals (for example, fatty acids, sterols, alkaloids,flavonoids, glycosides, saponins, tannins and terpenes) thatoften result in PCR inhibition [53]. In addition, degrad-

ation at primer binding sites may also contribute to differ-ential amplification success of selected genes in sampleswith potentially degraded DNA. In the Baker et al. [2]study, four dietary supplements could not be identifiedusing the laboratorys PCR amplification protocol, pre-sumably because the DNA was degraded, possibly whenheat was applied during the manufacturing process.




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Amplification failure in some samples, especially in rbcLgene, can be explained by the fact that primer sets used inthis analysis may not be suitable for amplification of allspecies; we are developing new primer sets for mini-rbcLbarcodes that will be easier to recover from degraded sam-ples such as herbal products. The ideal protocol forbarcoding herbal products will be realized as we build amore complete SRM herbal barcode library and continueto test more commercial products in the marketplace.Our study and others have documented that DNA canbe routinely extracted from common forms of herbal

dietary supplement extracts and powders, supportinga continued effort to explore DNA-based methods forquality assurance and quality control of herbal dietarysupplements [1-3,54].

Many herbal products contain mixtures, which areparticularly difficult to barcode. This difficulty is due to

varied PCR success of selected genes in samples with po-tentially degraded DNA due to varied gene copy numberand PCR bias; the chemical and physical properties ofeach piece of DNA can selectively amplify certain se-quences more than others [55]. Several approaches couldbe developed for testing herbal mixtures such as real-

time multiplex PCR and the use of next-generationsequencing [56]. In our study we chose to test only sin-gle ingredient herbal products. However, some of theseproducts could contain more species than what waslabeled. In order to search for other possible DNAbarcodes we extracted multiple barcodes per sample in an

attempt to obtain the most accurate estimate of identitywithin a sample. This included building barcode sampleby species curves to see how many times we needed tosample to get an accurate estimate of species in a sample.Although we arrived at a figure of five to ten samples, weare currently using other molecular methods to verifythe number of barcodes recovered from all of our

herbal products that will overcome PCR bias and pro-vide appropriate methods for testing herbal mixtures.Recent innovative approaches have combined mor-phological, molecular, and chemical techniques inorder to identify the plant and chemical compositionof some previous-generation smart drugs [57]. Such a

multidisciplinary approach is proposed as a methodfor the identification of herbal blends of uncertaincomposition, which are widely available and representa serious hazard to public health [57].

Conclusions

Currently there are no standards for authentication ofherbal products. Although there is considerable evidenceof the health benefits of herbal medicine [53,58-66], theindustry suffers from unethical activities by some ofthe manufacturers, which includes false advertising,product substitution, contamination and use of fillers.

This practice constitutes not only food fraud, butaccording to the WHO, serious health risks for consumers[67]. A study of health claims made by herbal productmanufacturers on the internet found that 55% of manufac-turers illegally claimed to treat, prevent, diagnose or curespecific diseases [68]. Regulators such as the FDA andCanadian Food Inspection Agency (CFIA) may not havethe resources to adequately monitor the dietary supple-ment manufacturers and their advertising claims, andthere are concerns that the current regulatory system isnot effective in protecting consumers from the risks asso-

ciated with certain herbal products [69,70]. Chemicalresearch studies have documented poor quality controland high content variability of active ingredients amongproducts from a variety of manufacturers of herbal supple-ments [71,72]. This is partly because herbs contain com-plicated mixtures of organic chemicals, the levels of which

may vary substantially depending upon many factorsrelated to the growth, production and processing of eachspecific herbal product [73]. Although many manufacturersprovide products with consistent levels of active ingredientsthrough a process known as chemical standardization, thistechnique has uncertain effects on the safety and efficacy of

the final product [71,74,75]. Many of the dangers of com-mercial plant medicine have been brought to light by DNAtechnology based studies that have identified contaminationof herbal products with poisonous plants [70]. Eroding con-sumer confidence is driving the demand for a product au-thentication service that utilizes molecular biotechnology.

One approach to vetting herbal product substitutionand contamination is product authentication usingDNA barcoding. Research studies such as ours andothers [1-3] reinforce the importance of using DNAbarcoding in the authentication of socioeconomically im-portant herbal species [18]. We suggest that the herbal in-dustry should voluntarily embrace DNA barcoding for

authenticating herbal products through testing of raw ma-terials used in manufacturing products, which would sup-port sovereign business interests and protect consumers.This would be a minor cost to industry with a limitedamount of bulk product testing, which would certify ahigh quality, authentic product. If the herb is known to

have health benefits and it is in the product, then thiswould provide a measure of quality assurance in additionto consistent levels of active ingredients. Currently we arebuilding an SRM DNA barcode library for commercialherbal species and standard testing procedures that couldbe integrated into cost effective best practices in themanufacturing of herbal products. This would provide in-dustry with a competitive advantage as they could adver-tise that they produce an authentic, high quality product,which has been tested using DNA-based species identifi-cation biotechnology, therefore gaining consumer confi-dence and preference. This approach would support the




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need to address considerable health risks to consumerswho expect to have access to high quality herbal productsthat promote good health.

Additional files

Additional file 1: Table S1. Standard reference material (SRM) herbal

Barcode of Life Database (BOLD) accession numbers.

Additional file 2: Table S2. List of GenBank accession numbers.

Competing interests

The authors declare that they have no competing interests.

Authors contributions

SGN, SR and DS conceived of and designed the study, and carried out wet

lab analysis and writing of the manuscript. MG carried out the fieldwork

and contributed to the study design and writing of manuscript. SGN, SR

and RS contributed to final editing and submission of the final manuscript.

All authors read and approved the final manuscript.

AcknowledgementsThis research was supported by the grants to SGN from th e International

Science and Technology Partnership Canada and Humanities Research

Council of Canada, Genome Canada through the Ontario Genomics Institute,

and the Canadian Foundation for Innovation. We would like to thank Mr

Richard Desylva, Managing Director, The Herb Works and Mr Joel Thuna,

General Manager, Global Botanical, Ontario, Canada for offering valuable

advice and reviewing a previous version of the manuscript. We thank the

Administrators and Vice Chancellor of the Bharathiar University, India for

their administrative help and support. Thanks to Kawa Cheng and Kevan

Berg for providing assistance with editing and referencing.

Author details1Centre for Biodiversity Genomics, Biodiversity Institute of Ontario (BIO),

University of Guelph, Guelph, Ontario N1G 2W1, Canada. 2Bachelor of Arts

and Science Program, University of Guelph, Guelph, Ontario N1G 2W1,

Canada. 3Plant Genetic Engineering Laboratory, Department ofBiotechnology, Bharathiar University, Coimbatore 641 046, Tamil Nadu, India.

Received: 2 July 2013 Accepted: 12 September 2013

Published: 11 October 2013

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doi:10.1186/1741-7015-11-222Cite this article as: Newmaster et al.: DNA barcoding detectscontamination and substitution in North American herbal products.

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