Newmaster et al.

BMC Medicine 2013, 11:222

http://www.biomedcentral.com/1741-7015/11/222

RESEARCH ARTICLE Open Access

DNA barcoding detects contamination and

substitution in North American herbal products
Steven 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 product
substitution 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 major provider of goods such as herbal products, a com-

Globalization of trade is expanding natural product
commodity markets that sustain life and promote good
health, yet the challenging financial climate is squeezing
profit margins and exacerbating the propensity for con-
tamination, fraudulent market substitution and the use of
unlabeled fillers. This comes at a time when consumers
are becoming increasingly concerned about the authenti-
city of the products they purchase. North America is a
* Correspondence: snewmast@uoguelph.ca; ragu@uoguelph.ca
1
Centre 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
modity that has come under scrutiny recently in the
media 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 are
currently more than 1,000 companies producing medi-
cinal plant products with annual revenues in excess of
US$60 billion. Notably, medicinal herbs now constitute
the most rapidly growing segment of the North American
alternative medicine market [4,5], with over 29,000
herbal substances [6,7] generating billions of dollars in
trade. These statistics are indicative of the rapid growth

2013 Newmaster et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the
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(>15%/year) in the marketplace for natural plant products
and the broadening consumer base interested in herbal
products from India, Canada, USA and Europe. Yet, as
highlighted in recent media reports, product adulteration
and ingredient substitution is not uncommon as species
of a lower market value are substituted for those of a
higher value. This practice constitutes not only product
fraud, but according to the World Health Organization
(WHO), the adulteration of herbal products is a threat to
consumer 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 in
herbal products. This is because the diagnostic morpho-
logical features of the plants on which the current
Linnaean 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 the
effectiveness of otherwise useful remedies, lowering the
perceived value of all related products because of a lack of
consumer confidence in them. Herbal product substitu-
tion has been documented for many individual medicinal
plant species [8-10], teas [1] and nutraceuticals [11,12].
Although there is limited research available, the frequency
of product mislabeling in herbal products has been esti-
mated at 14% to 33% from previous studies [1-3]. There
are legitimate health concerns for consumers which re-
sults in a lack of confidence in safe, high quality herbal
products.
A broadly accessible commercial tool to detect prod-
uct substitution and develop best practices for au-
thentication of many commercial products is beginning to
emerge. DNA barcoding has been used to test a number
of natural products [1,3,13]. Barcoding uses state-of-the-
art biotechnology to help identify plant material based on
short, standardized gene sequences, in a rapid, accurate
and cost-effective manner. For example, Srirama et al.
[10] has demonstrated the efficiency of DNA barcoding
in identifying adulterants in the raw drug trade of
Phyllanthus (Euphorbiaceae). Phyllanthus is one of the
most important groups of species traded as a raw herbal
drug treatment for liver disorders [14]. This study showed
that plant DNA barcoding can effectively discriminate
Phyllanthus species, and hence can be used to resolve
species admixtures in the raw drug trade samples; there
are several phenotypically similar species that could easily
be misidentified and mixed within herbal products. Over
the last 3 years, studies of Chinese plant medicine [15-20]
have documented the potential scope and magnitude of
market substitution using this biotechnology. How-
ever, there are only a few studies [1,3] available, and
they provide a limited assessment of the authenticity
Page 2 of 13
of commercial herbal products within the market-
place. Currently there is a gap in our understanding
to the extent of herbal product substitution, contam-
ination and use of fillers. Also, there are no studies
that have used a standard reference material (SRM)
herbal barcode library and barcode regions that facili-
tate species resolution.
This study utilized blind sampling of commercially
available herbal products, which were tested for authen-
tication of plant ingredients using an SRM herbal DNA
barcode library. Our research questions focused on the
following three areas. (1) Authentication: is the herbal
species on the label found in the product? (2) Substitu-
tion: is the main herbal ingredient substituted by other
species? (3) Fillers: are there any unlabeled fillers used?
Methods
Study design
We 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 barcoding
at the Centre for Biodiversity Genomics (CBG) within
the Biodiversity Institute of Ontario (BIO), University
of Guelph.
The project also included a second blind test in which
we barcoded 50 leaf samples (blind labels) collected
from living samples of known provenance from horticul-
tural greenhouses. These leaf samples represented 42
species including all those listed on the herbal product
labels and some closely related species. This provided an
independent test of the ability of barcoding to differenti-
ate all the species on the herbal product labels used in
this study. All of the samples are in the Barcode of
Life Database (BOLD) (Additional file 1: Table S1) and
GenBank (Additional file 2: Table S2). All vouchers are
scanned and recorded in the BOLD and deposited in the
BIO 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 from
herbal supply companies. In our study, samples were
collected in the greater Toronto area (GTA) in Canada,
with several samples mailed from distributors in the
USA. 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 2
or 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
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samples using a tiered approach (rbcL + ITS2) [21]. Mul-
tiple DNA extracts (five to ten) were taken from each of
these triplicate samples of which these DNA extracts
were barcoded to make sure we did not miss any species
DNA. Since these were only single ingredient herbal
products we did not expect to detect more than one
plant species barcode unless it was a contaminant. How-
ever, our intensive DNA extract sampling allowed us to
sample 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 accurate
estimate of species in a sample. As a result some of the
products yielded two difference sequences, each from
DNA extracts taken from the same sample product.
Total genomic DNA was isolated from 100 mg of the
dry material using the Nucleospin Plant II Mini DNA
Extraction kit. The PCR amplification was performed in
a 20 L reaction mixture that contained 2.5 L of genomic
DNA, 2.5 L of 10 Pfu buffer with MgSO4 (Fermentas),
2.5 L of 2 mM dNTPs (Fermentas), 0.5 L each of forward
and reverse primers (10 pM) and 0.2 L of 2.5 U Pfu DNA
Polymerase (Fermentas) and 2 L 0.5% dimethylsulfoxide
(DMSO). The primers and the reaction conditions are pre-
viously published for rbcL [22] and ITS2 [16]. Touchdown
PCR was carried out. The PCR products obtained from
these reactions were subjected to Big Dye (version 3.1)
sequencing reactions that had a total volume of 10 L
and included 10 pMol of each primer. The sequencing
amplification protocol consisted of 1 cycle of 1 minute at
96C, followed by 30 cycles of 10 s at 96C, 5 s at 55C,
and 4 minutes at 60C. The amplicons were bidirectionally
sequenced using ABI 377 sequencer (Applied Biosystems).
The chromatographic traces were aligned and codon read
in the Codoncode Aligner ver. 3.0 (CodonCode, Dedham,
MA, USA) and contigs were generated. Sequences were
deposited in BOLD and GenBank.
SRM herbal barcode library
An SRM herbal barcode library was prepared and used
to identify the unknown herbal products and leaf sam-
ples. This library was assembled from 100 herbal species
of known provenance that were identified by taxonomic
experts. These samples were prepared into herbarium
vouchers and are archived at the BIO Herbarium and in
the BOLD. Although our library contained 100 herbal
species 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 herbal
product industry in North America.
Product identification and analysis
Identification of unknown barcodes from products and
leaves was conducted by comparing Basic Local Alignment
Page 3 of 13
Search Tool (BLAST) data against our SRM herbal
barcode library with a minimum BLAST cut off of 97%
identity for a top match [3]. These results were verified by
neighbor-joining tree analysis in which we compared the
branches of unknown specimens to sequences of reference
species. A species is considered as taxonomically resolved
if its members showed at least one diagnostic difference
from 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 products
are as outlined below.
Authentic
A product is authentic if it contains a DNA barcode for
a species that is the main ingredient on the label of a
tested herbal product.
Contamination
A product is contaminated if a DNA barcode is found
for a species other than what is labeled on the tested
product, in addition to the authentic barcode; this could
include known herbal product filler species (see below).
Substitution
Product substitution occurs when a DNA barcode is
found for a species other than what is labeled on the
tested product, AND there is NOT a barcode recovered
for 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 market
substitution for a cheaper product.
Filler
A product contains fillers if a DNA barcode is found for
known herbal product filler species including rice (Oryza
sativa), soybean (Glycine max) and wheat (Triticum
spp.). Fillers may be found in place of (substitution) or
in addition to the barcode of the main product ingredi-
ent (contamination).
Results
DNA barcode success
Recovery of DNA barcodes from various herbal products
was 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 herbal
barcode library. This library provided a database that
could be used to identify unknown haplotypes (barcodes)
recovered from herbal products. The SRM classification
tree (Figure 1) shows that all herbal species have 100%
species resolution, with each species presenting at least
one diagnostic difference from any other taxon and
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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.

formed a monophyletic clade (Figure 1). Herbal product
DNA barcode success rate was 91% (40/44 products) for
the combined regions, resulting in the recovery of 34 rbcL
and 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 is
known to have insufficient nucleotide sequence variability
to distinguish among closely related species. The three un-
resolved species with ITS2 was due to three samples that
had product substitution with species that were not in our
SRM library; these unknown species barcodes did match
the nearest family and genus. We also barcoded blindly
labeled herbal leaf samples collected from living samples
from horticultural greenhouses. This provided an inde-
pendent test of the ability of barcoding to differentiate all
the 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 to
the 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
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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 on
the labels (Figure 2). Some (33%) of the authenticated
herbal products also contained contaminants and or fillers
not listed on the label (Figure 2). Some of the DNA se-
quences recovered were only identified to family as our
SRM herbal DNA barcode library is not yet complete (see
note above). However, an incomplete SRM barcode library
still allows a correct identity in a hierarchical way to family
and genera depending on the level of best match within
the classification tree. Some of the contaminants were
of unrelated species from entirely different plant fam-
ilies (Table 1). Although many of the herbal companies
(75%) whose product we tested did have some authentic
products, only 2 of the 12 companies provided authentic
products with no substitution, contaminants or fillers.
Conversely, we were unable to authenticate any of the
products for 25% of the companies (Figure 3). Although
these numbers shed light on the number of companies
with authenticate products we only sampled three or four
products per company and therefore differences among
companies are likely due to chance.
Substitution: is the main herbal ingredient substituted for
some other species?
Our barcoding analysis of 44 herbal products representing
30 medicinal plants species identified product substitution
in 32% of the samples (Figure 2; Table 1). The criteria we
used to determine product substitution was the identity of
other plant taxa in place of the products main labeled in-
gredient, which was NOT detected within the sample. We
detected product substitution within most (83%) of the 12
companies that we tested (Figure 3). It should be noted
that for 90% of the herbal species in this study, there were
Figure 2 DNA barcode results from blind testing of the 44 herbal products representing 30 medicinal species of plants.
Page 5 of 13
product(s) from at least one company that yielded a
correct identity (indicating an authentic product). This
is confirmation that processing is not an impediment
to recovering barcodes. It should also be noted that we
sampled each product intensively without replacement;
multiple samples per product were extracted and barcoded
to make sure we did not miss any species DNA. Since
these were only single ingredient herbal products we
did not expect to detect more than one plant species
barcode 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 herbal
product species or a substitute, and the other was either a
filler 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 also
included wheat (Triticum spp.). It is not known whether
or not these plants were intentionally used as fillers or if
they are contaminants; we are currently investigating the
use of fillers in processing by each individual company. In
9% of the samples only rice or wheat barcodes were
detected; there were no barcodes detected for the herbal
species on the label. The use of fillers was limited to one-
third of companies whose products we tested (Figure 3).
Discussion
Contamination and substitution in North American herbal
products
Few studies have attempted to estimate how prevalent
herbal product contamination and substitution is in the
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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 leaf Sample Barcode ID ITS2 ID rbcL ID
species from
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
Actaea racemosa HP74L Actaea racemosa Actaea racemosa Actaea racemosa
Actaea racemosa HP77L 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 NS
Apium graveolens HP43C NS NS NS
Apium graveolens HP13L Apium graveolens Apium graveolens Apiaceae
Apium graveolens HP5L Apium graveolens Apium graveolens Apiaceae
Apium graveolens HP51L Apium graveolens Apium graveolens Apiaceae
Arctium lappa (Burdock) HP6C Arctium lappa Arctium lappa Arctium lappa
Arctium lappa HP22C Oryza sativaa Oryza sativab Oryza sativab
a a
Arctium lappa HP21C Ranunculaceae Ranunculaceae NS
Arctium lappa HP4L Arctium lappa Arctium lappa Arctium lappa
Arctium lappa HP92L Arctium lappa Arctium lappa Arctium lappa
a a
Cinnamomum cassia HP86T Ranunculaceae Ranunculaceae NS
(Cinnamon)
Cinnamomum cassia HP60L Cinnamomum cassia Cinnamomum cassia Cinnamomum sp.
(Cinnamon)
Echinacaea purpurea HP35C Echinacaea purpurea + Parthenium Echinacaea purpurea + Parthenium Asteraceaea
(Echinacaea) hysterophorusa hysterophorusa
Echinacaea purpurea HP27C Echinacaea purpurea + Parthenium Echinacaea purpurea + Parthenium Asteraceaea
hysterophorusa hysterophorusa
Echinacaea purpurea HP88L Echinacaea purpurea Echinacaea purpurea Echinacaea
purpurea
Echinacaea purpurea HP80L Echinacaea purpurea Echinacaea purpurea Echinacaea
purpurea
Echinacaea purpurea HP70L Echinacaea purpurea Echinacaea purpurea Echinacaea
purpurea
Echinacaea purpurea HP85L Echinacaea purpurea Echinacaea purpurea Echinacaea
purpurea
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
Ginkgo biloba HP2L Ginkgo biloba Ginkgo biloba Ginkgo biloba
Ginkgo biloba HP78L Ginkgo biloba Ginkgo biloba Ginkgo biloba
Ginkgo biloba HP16L Ginkgo biloba Ginkgo biloba Ginkgo biloba
Hypericum perforatum HP8C Fabaceaea Fabaceaea NS
(St. Johns wort)
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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 Hypericum
perforatum
Hypericum perforatum HP82C Oryza sativaa Oryza sativab Oryza sativab
Hypericum perforatum HP39L Hypericum perforatum Hypericum perforatum Hypericum
perforatum
Hypericum perforatum HP94L Hypericum perforatum Hypericum perforatum Hypericum
perforatum
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
Juglans nigra HP19L Juglans nigra Juglans nigra Juglans nigra
Juglans nigra HP17L 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 (Evening HP48C Oenothera biennis Oenothera biennis Oenothera biennis
Primrose)
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
a a
Oxalis Corniculata HP1P Oxalis Corniculata + Medicago sativa Oxalis Corniculata + Medicago sativa Oxalis sp.
(WoodSorrel)
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
b b
Portulaca oleracea HP65C Portulaca oleracea + Glycine max Portulaca oleracea + Glycine max 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 HP62C Rudbeckia hirta Rudbeckia hirta Rudbeckia hirta
(black-eyed Susan)
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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)
Rudbeckia hirta HP31L Rudbeckia hirta Rudbeckia hirta Rudbeckia hirta
a a
Sambucus nigra (Elderberry) HP23C Sambucus nigra + Medicago sativa Sambucus nigra + Medicago sativa Sambucus sp.
Sambucus nigra HP59L Sambucus nigra Sambucus nigra Sambucus sp.
Sambucus racemosa HP54L Sambucus racemosa Sambucus racemosa Sambucus sp.
Sonchus arvensis (Thistle) HP87C Sonchus arvensis Sonchus arvensis Sonchus arvensis
Sonchus arvensis HP67C Sonchus arvensis + Medicago sativaa Sonchus arvensis + Medicago sativaa Sonchus arvensis
Sonchus arvensis HP73L Sonchus arvensis Sonchus arvensis Sonchus arvensis
Sonchus asper HP41L Sonchus asper Sonchus asper Sonchus asper
Symphytum officinale HP7C Symphytum officinale + Medicago sativaa Symphytum officinale + Medicago sativaa Medicago sativaa
(Comfrey)
Symphytum officinale HP90L Symphytum officinale Symphytum officinale Boraginaceae
a a
Taraxacum officinale HP25C Parthenium hysterophorus Parthenium hysterophorus Asteraceaea
a a
Taraxacum officinale HP11C Taraxacum officinale + Panicum sp. Taraxacum officinale + Panicum sp. Panicum sp. a
Taraxacum officinale HP75C Triticum spp. + Musa sp. a Triticum spp. + Musa sp. a Musaceaea
Taraxacum officinale HP53L Taraxacum officinale Taraxacum officinale Taraxacum
officinale
Urtica dioica (Nettles) HP55C Urtica dioica Urtica dioica Urtica dioica
Urtica dioica HP44C Urtica dioica + Oryza sativab Urtica dioicab Oryza sativab
Urtica dioica HP68L Urtica dioica Urtica dioica Urtica dioica
a
Designates product contamination or substitution.
b
Refers to use of fillers.
C capsule, L leaf, NS no sequence, P powder, T tablet.

marketplace. To date there are only a few [1-3] studies
of herbal product substitution, representing <1% of the
herbal products and <5% of the herbal companies in
North America. These studies document species-level
DNA barcode identification success for 42% to 66% of
the herbal species tested. Our study demonstrates in-
creased testing and success, including (i) species-level
DNA barcode identification success to 95% of the herbal
species tested, and (ii) herbal market testing by approxi-
mately 20% for both products and companies. Although
we provide new estimates of herbal product contamin-
ation and substitution, we caution that these values need
to be refined by further studies as we have yet to sample
>1% market. However, our estimates do corroborate
those of other studies. For example, Stoeckle et al. [1]
investigated contamination in 131 herbal tea products
representing 48 herbal species and found that although
they were able to authenticate 58% of the products, 33%
of the herbal teas tested were contaminated. Similar esti-
mates from a marketplace survey [2] of 40 dietary

Figure 3 DNA barcode results from blind testing of the 44 herbal products representing 12 companies.
Newmaster et al. BMC Medicine 2013, 11:222
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supplement products labeled as black cohosh (Actaea
racemosa) found that 75% of the samples were authen-
tic black cohosh, whereas 25% were substituted with 4
closely related Asian Actaea species (A. asiatica, A.
cimicifuga, A. dahurica, and A. simplex). We also
found substitution of black cohosh for Asian Actaea,
as did another study by Wallace et al. [3]. Wallace
et al. [3] also documented that 50% of ginseng prod-
ucts labeled Korean ginseng (Panax ginseng) were
actually American ginseng (Panax quinquefolius). Al-
though the DNA barcode success rate was low (48%,
see below) in Wallaces study, they were still able to
detect product substitution in 14% of the herbal sam-
ples tested, representing 7 species of herbs in 14
products. This study also detected fillers in 21% of
the herbal products [3]. We found alfalfa (Medicago
sativa) in 16% of the products tested, which was also
previously detected in teas [1]. It is unlikely that this
was a contaminant as it was found in so many of the
products in addition to the main ingredient. It may
be possible that this species is used by some compan-
ies as a cheap filler as it is commonly grown and
bailed in agricultural areas, and available in large
quantities throughout the year.
Contamination and substitution in herbal products
present considerable health risks for consumers. In our
study, we found contamination in several products with
plants that have known toxicity, side effects and/or
negatively 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); it
contained only senna barcodes and no St. Johns wort
barcodes. This is a serious health risk as senna is a
Food 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 contains
several unique glycosides called sennosides that interact
with immune cells in the colon [25]. We also found con-
tamination of several herbal products (Table 1) with
Parthenium hysterophorus (feverfew). Feverfew is native to
Eurasia and is an invasive weed in Europe, the Mediterra-
nean, North America and Chile. Although feverfew has
been used to treat fever, migraine headaches and arthritis,
it does have negative side effects such as swelling and
numbness 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 medications
metabolized by the liver and may also increase the
risk of bleeding, especially if taken with blood-thinning
Page 9 of 13
medications such as warfarin or aspirin [28,29]. Pregnant
women 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 species
called parthenin [30,31]. Although it is not known how
contamination with feverfew occurred in this product, it is
possible that it was a common weed in the crop used to
make this herbal product.
We also found contamination of a Ginkgo product
with Juglans nigra (black walnut). Wallace et al. [3] also
found 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 source
of 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 common
occurrence on the landscape), and that the target crop
was harvested along with walnut leaves. Walnut leaves,
woods, bark and fruits all contain juglone [32], which is
toxic; 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 marketplace
have not been able to identify all the contaminants to
species-level resolution due to the lack of a complete
SRM barcode library for herbal plants; a complete SRM
library would include herbal species, related species and
known toxic plants. It is possible that there were other
contaminants that were missed because they could only
be 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 health
risk for consumers. DNA barcoding in our study identified
several potential fillers, including rice (O. sativa), soybean
(G. max) and various grasses such as wheat (Triticum
spp.). This is a health concern for people allergic to these
plants, as well as for people seeking gluten free products.
Wallace et al. [3] found rice and soybean fillers in natural
plant and animal products and suggested that these fillers
may produce a mixed signal during the sequencing
process, contributing to a rather high percentage of failed
sequencing reactions in capsulated products. We did not
share this high percentage of sequence failures and found
barcodes from both the herbal species on the label and
fillers within the same product, suggesting that there may
be fillers substituted for authentic herbal species. It is
probable that barcoding detected rice and soybean, which
is a common microcrystalline cellulose and gelatin used as
additives in preparing the capsules that contain the herbal
Newmaster et al. BMC Medicine 2013, 11:222
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product [40]. It is also common practice in the natural
products industry to use fillers such as those listed
above, which are mixed with the active ingredients.
Nonetheless, the consumer has a right to see all of the
plant species used in producing a natural product on the
list of ingredients.
Challenges and biotechnical advances in DNA barcoding
of herbal products
Authentication of herbal products is challenging, but
new DNA barcoding methods are providing tools for
routine market analysis [41]. Several major challenges
include the lack of an SRM herbal barcode library, and
use of only plastid barcode regions, which has resulted
in low species resolution. The original definition of a re-
liable DNA identification of species requires (i) recovery
of 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 from
herbal samples, which satisfies the first requirement, but
the 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 of
herbal products without a herbal SRM barcode library
[1-3]. These studies have defined the uncertainties of
assigning unknown haplotypes from herbal products using
incomplete reference barcode databases in GenBank and
BOLD. The use of GenBank is inappropriate given that
many of the DNA sequences do not have vouchers to
professionally identified specimens archived in a herb-
arium. Therefore the reference sequence in GenBank
may be from an incorrectly identified plant and there
is no way to verify the specific origin of that DNA.
Similarly, the Medicinal Materials DNA Barcode
Database [43] provides barcodes for many species of
medicinal plants for which there is no record of vouchers
to confirm identity; this is an essential component of any
DNA Barcode [42]. Our study is the first to build a partial
herbal SRM barcode library using vouchered samples of
known 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, we
expect to expand this to over 200 species by the end of
2013, and 1,000 species by 2015. However, this will only
add up to 55% of the 1,800 medicinal plant species
that are commercially available [44]. If we want to
have reliable identifications using DNA barcodes we
must build an SRM herbal barcode library that has all
sister species for the 1,800 known medicinal species
used in commercial products. This is one of the goals
of our Herb-BOL (barcode of life) research program
in the next 5 years.
Page 10 of 13
The use of only plastid barcode regions is problematic
for testing herbal products. Previous studies have not
satisfied Heberts third criteria for a reliable barcode: suf-
ficient nucleotide sequence variability to distinguish
among closely related species [42]. Other studies have
adopted the core plant barcodes of rbcLa and matK
genes [45]. We do not recommend using matK because
of associated problems we encountered with PCR ampli-
fication while working on nutmeg [46]. Other published
studies have also encountered similar problems [47-51].
We recommend a tiered [21] approach to analysis as
one way to overcome the issue of alignment with non-
coding regions, while providing the most variability from
two-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 that
can act as a scaffold on which to place data from a
highly variable region such as ITS2. The use of rbcL as a
primary 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 for
evaluation [51,52]. We prefer to use ITS2 as the second
tier as proposed by Chen et al. [16] for use in identifying
medicinal plants because (i) it provides high species
resolution, (ii) it is from the nuclear genome, which
evolves 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 herbal
products. The use of rbcL + ITS2 in our study resulted
in 95% species resolution of barcodes recovered from
herbal products, which is much higher than previous
studies that used rbcL + matK (42% in Wallace et al. [3];
66% in Stoeckle et al. [1]). DNA success rate was high in
our study (91%) and in that of Stoeckle et al. [1] where
they recovered barcodes from 90% of commercial herbal
tea products. DNA barcode success rate was relatively
low (48%) in the study by Wallace et al. [3]. This may be
attributed to different manufacturing protocols, the type
of plant material (for example, leaf, stem, roots and so
on) used in the herbal preparation, or insufficient labora-
tory protocols used to extract, amplify and sequence
haplotypes 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) that
often 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 samples
with potentially degraded DNA. In the Baker et al. [2]
study, four dietary supplements could not be identified
using the laboratorys PCR amplification protocol, pre-
sumably because the DNA was degraded, possibly when
heat was applied during the manufacturing process.
Newmaster et al. BMC Medicine 2013, 11:222
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Amplification failure in some samples, especially in rbcL
gene, can be explained by the fact that primer sets used in
this analysis may not be suitable for amplification of all
species; we are developing new primer sets for mini-rbcL
barcodes that will be easier to recover from degraded sam-
ples such as herbal products. The ideal protocol for
barcoding herbal products will be realized as we build a
more complete SRM herbal barcode library and continue
to test more commercial products in the marketplace.
Our study and others have documented that DNA can
be routinely extracted from common forms of herbal
dietary supplement extracts and powders, supporting
a continued effort to explore DNA-based methods for
quality assurance and quality control of herbal dietary
supplements [1-3,54].
Many herbal products contain mixtures, which are
particularly 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 number
and PCR bias; the chemical and physical properties of
each piece of DNA can selectively amplify certain se-
quences more than others [55]. Several approaches could
be developed for testing herbal mixtures such as real-
time multiplex PCR and the use of next-generation
sequencing [56]. In our study we chose to test only sin-
gle ingredient herbal products. However, some of these
products could contain more species than what was
labeled. In order to search for other possible DNA
barcodes we extracted multiple barcodes per sample in an
attempt to obtain the most accurate estimate of identity
within a sample. This included building barcode sample
by species curves to see how many times we needed to
sample to get an accurate estimate of species in a sample.
Although we arrived at a figure of five to ten samples, we
are currently using other molecular methods to verify
the 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 in
order to identify the plant and chemical composition
of some previous-generation smart drugs [57]. Such a
multidisciplinary approach is proposed as a method
for the identification of herbal blends of uncertain
composition, which are widely available and represent
a serious hazard to public health [57].
Conclusions
Currently there are no standards for authentication of
herbal products. Although there is considerable evidence
of the health benefits of herbal medicine [53,58-66], the
industry suffers from unethical activities by some of
the manufacturers, which includes false advertising,
product substitution, contamination and use of fillers.
Page 11 of 13
This practice constitutes not only food fraud, but
according to the WHO, serious health risks for consumers
[67]. A study of health claims made by herbal product
manufacturers on the internet found that 55% of manufac-
turers illegally claimed to treat, prevent, diagnose or cure
specific diseases [68]. Regulators such as the FDA and
Canadian Food Inspection Agency (CFIA) may not have
the resources to adequately monitor the dietary supple-
ment manufacturers and their advertising claims, and
there are concerns that the current regulatory system is
not effective in protecting consumers from the risks asso-
ciated with certain herbal products [69,70]. Chemical
research studies have documented poor quality control
and high content variability of active ingredients among
products 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 factors
related to the growth, production and processing of each
specific herbal product [73]. Although many manufacturers
provide products with consistent levels of active ingredients
through a process known as chemical standardization, this
technique 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 DNA
technology based studies that have identified contamination
of 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 substitution
and contamination is product authentication using
DNA barcoding. Research studies such as ours and
others [1-3] reinforce the importance of using DNA
barcoding 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 limited
amount of bulk product testing, which would certify a
high quality, authentic product. If the herb is known to
have health benefits and it is in the product, then this
would provide a measure of quality assurance in addition
to consistent levels of active ingredients. Currently we are
building an SRM DNA barcode library for commercial
herbal species and standard testing procedures that could
be integrated into cost effective best practices in the
manufacturing 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
Newmaster et al. BMC Medicine 2013, 11:222
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need to address considerable health risks to consumers
who expect to have access to high quality herbal products
that 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.
Acknowledgements
This research was supported by the grants to SGN from the 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 details
1 23. Lemli J: Mechanism of action of sennosides. Bull Acad Natl Med 1995,
Centre 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 of
Biotechnology, Bharathiar University, Coimbatore 641 046, Tamil Nadu, India.
Received: 2 July 2013 Accepted: 12 September 2013
Published: 11 October 2013
References
1. Stoeckle MY, Gamble CC, Kirpekar R, Young G, Ahmed S, Little DP:
Commercial teas highlight plant DNA barcode identification successes
and obstacles. Sci Rep 2011, 1:42.
2. Baker DA, Stevenson DW, Little DP: DNA barcode identification of black
cohosh herbal dietary supplements. J AOAC Int 2012, 95:10231034.
3. Wallace LJ, Boilard SMAL, Eagle SHC, Spall JL, Shokralla S, Hajibabaei M: DNA
barcodes for everyday life: routine authentication of natural health
products. Food Res Int 2012, 49:446452.
4. McCabe S: Complementary herbal and alternative drugs in clinical
practice. Perspect Psychiatr C 2002, 38:98107.
5. Gutierrez S, Ang-Lee MK, Walker DJ, Zacny JP: Assessing subjective and
psychomotor effects of the herbal medication valerian in healthy
volunteers. Pharmacol Biochem Behav 2004, 78:5764.
6. Astin JA, Marie A, Pelletier KR, Hansen E, Haskell WL: A review of the
incorporation of complementary and alternative medicine by
mainstream physicians. Arch Intern Med 1998, 158:23032310.
7. Kaye A, Clarke R, Sabar R, Vig S, Dhawan K, Hofbauer R, Kaye A: Herbal
medicines: current trends in anesthesiology practice - a hospital survey.
J Clin Anesth 2000, 12:468471.
8. Techen N, Crockett SL, Khan IA, Scheffler BE: Authentication of medicinal
plants using molecular biology techniques to compliment conventional
methods. Curr Med Chem 2004, 11:13911401.
9. Song J, Yao H, Li Y, Li X, Lin Y, Liu C, Han J, Xie C, Chen S: Authentication
of the family Polygonaceae in Chinese pharmacopoeia by DNA
barcoding technique. J Ethnopharmacol 2009, 124:434439.
Page 12 of 13
10. Srirama R, Senthilkumar U, Sreejayan N, Ravikanth G, Gurumurthy BR,
Shivanna MB, Sanjappa M, Ganeshaiah KN, Shaanker RU: Assessing species
admixtures in raw drug trade of Phyllanthus, a hepato-protective plant
using molecular tools. J Ethnopharmacol 2010, 130:208215.
11. Jaakola L, Suokas M, Hggman H: Novel approaches based on DNA
barcoding and high-resolution melting of amplicons for authenticity
analyses of berry species. Food Chem 2010, 123:494500.
12. Bruni I, De Mattia F, Galimberti A, Galasso G, Banfi E, Casiraghi M, Labra M:
Identification of poisonous plants by DNA barcoding approach. Int J
Legal Med 2010, 124:595603.
13. Sui X, Huang Y, Tan Y, Guo Y, Long C: Molecular authentication of the
ethnomedicinal plant Sabia parviflora and its adulterants by DNA
barcoding technique. Planta Med 2011, 77:492496.
14. Ved DK, Goraya GS: Demand and Supply of Medicinal Plants in India.
Bangalore, India: NMPB, New Delhi & FRLHT; 2007.
15. Gao T, Yao H, Song J, Liu C, Zhu Y, Ma X, Pang X, Xu H, Chen S:
Identification of medicinal plants in the family Fabaceae using a
potential DNA barcode ITS2. J Ethnopharmacol 2010, 130:116121.
16. Chen S, Yao H, Han J, Liu C, Song J, Shi L, Zhu Y, Ma X, Gao T, Pang X, Luo K, Li
Y, Li X, Jia X, Lin Y, Leon C: Validation of the ITS2 region as a novel DNA
barcode for identifying medicinal plant species. PLoS ONE 2010, 5:e8613.
17. Guo X, Wang X, Su W, Zhang G, Zhou R: DNA barcodes for discriminating
the medicinal plant Scutellaria baicalensis (Lamiaceae) and its
adulterants. Biol Pharm Bull 2011, 34:11981203.
18. Li M, Cao H, But PP-H, Shaw P-C: Identification of herbal medicinal
materials using DNA barcodes. J Syst Evol 2011, 49:271283.
19. Xue C-Y, Li D-Z: Use of DNA barcode sensu lato to identify traditional
Tibetan medicinal plant Gentianopsis paludosa (Gentianaceae). J Syst Evol
2011, 49:267270.
20. Pang X, Song J, Xu H, Yao H: Using ITS2 barcode to identify ephedrae
herba [in Chinese]. Zhongguo Zhong Yao Za Zhi 2012, 37:11181121.
21. Newmaster SG, Fazekas AJ, Ragupathy S: DNA barcoding in the land
plants: evaluation of rbcL in a multigene tiered approach. Can J Bot 2006,
84:335341.
22. Fazekas A, Kuzmina M, Newmaster S: DNA Barcoding methods for land
plants. In DNA Barcodes. New York, NY: Springer; 2012.
179:16051611.
24. Spiller HA, Winter ML, Weber JA, Krenzelok EP, Anderson DL, Ryan ML: Skin
breakdown and blisters from senna-containing laxatives in young
children. Ann Pharmacother 2003, 37:636639.
25. Hietala P, Marvola M, Parviainen T, Lainonen H: Laxative potency and acute
toxicity of some anthraquinone derivatives, senna extracts and fractions
of senna extracts. Pharmacol Toxicol 1987, 61:153156.
26. Klepser TB, Klepser ME: Unsafe and potentially safe herbal therapies. Am J
Health Syst Pharm 1999, 56:125138. quiz 139141.
27. Ernst E, Pittler MH: The efficacy and safety of feverfew (Tanacetum
parthenium L.): an update of a systematic review. Public Health Nutr 2000,
3:509514.
28. Miller LG: Herbal medicinals: selected clinical considerations focusing on
known or potential drug-herb interactions. Arch Intern Med 1998,
158:22002211.
29. Heck AM, DeWitt BA, Lukes AL: Potential interactions between alternative
therapies and warfarin. Am J Health Syst Pharm 2000, 57:12211227. quiz
12281230.
30. Yao M, Ritchie HE, Brown-Woodman PD: A reproductive screening test of
feverfew: is a full reproductive study warranted? Reprod Toxicol 2006,
22:688693.
31. Killoran CE, Crawford GH, Pedvis-Leftick A: Two cases of compositae
dermatitis exacerbated by moisturizer containing feverfew. Dermatitis
2007, 18:225229.
32. Daglish C: The determination and occurrence of a hydrojuglone
glucoside in the walnut. Biochem J 1950, 47:458462.
33. Grant TW, Doherty MD, Odowole D, Sales KD, Cohen GM: Semiquinone
anion radicals formed by the reaction of quinones with glutathione or
amino acids. FEBS Lett 1986, 201:296300.
34. Varga Z, Bene L, Pieri C, Damjanovich S, Gspr R Jr: The effect of juglone
on the membrane potential and whole-cell K+ currents of human
lymphocytes. Biochem Biophys Res Commun 1996, 218:828832.
35. YouLun P, DongMei L, Xi L, GuangMing L: Advances in the phytochemistry
and pharmacology researches of Juglans Linn. Med Plant 2012, 3:6466.
Newmaster et al. BMC Medicine 2013, 11:222
http://www.biomedcentral.com/1741-7015/11/222
36. NLM: Registry of Toxic Effects of Chemical Substances (RTECS). Bethesda, MD:
National Library of Medicine; 2005.
37. Van Duuren BL, Segal A, Tseng SS, Rusch GM, Loewengart G, Mat U, Roth
D, Smith A, Melchionne S, Seidman I: Structure and tumor-promoting
activity of analogues of anthralin (1,8-dihydroxy-9-anthrone). J Med Chem
1978, 21:2631.
38. Mller W-U, Leistner E: Metabolic relation between naphthalene
derivatives in Juglans. Phytochemistry 1978, 17:17351738.
39. Aithal BK, Sunil Kumar MR, Rao BN, Upadhya R, Prabhu V, Shavi G,
Arumugam K, Sajankila SP, Udupa N, Satyamoorthy K, Satish Rao BS:
Evaluation of pharmacokinetic, biodistribution, pharmacodynamic, and
toxicity profile of free juglone and its sterically stabilized liposomes.
J Pharm Sci 2011, 100:35173528.
40. Foster BC, Sockovie ER, Vandenhoek S, Bellefeuille N, Drouin CE, Krantis A,
Budzinski JW, Livesey J, Arnason JT: In vitro activity of St. Johns wort against
cytochrome P450 isozymes and glycoprotein. Pharm Biol 2004, 42:159169.
41. Sucher NJ, Carles MC: Genome-based approaches to the authentication
of medicinal plants. Planta Med 2008, 74:603623.
42. Hebert PDN, Stoeckle MY, Zemlak TS, Francis CM: Identification of birds
through DNA barcodes. PLoS Biol 2004, 2:e312.
43. Lou SK, Wong KL, Li M, But PP, Tsui SK, Shaw PC: An integrated web
medicinal materials DNA database: MMDBD (Medicinal Materials DNA
Barcode Database). BMC Genomics 2010, 11:402.
44. Muller J, Clauson K: Top herbal products encountered in drug
information requests (part 1). Drug Ben Trends 1998, 10:2123.
45. CBOL Plant Working Group: A DNA barcode for land plants. Proc Natl Acad
Sci U S A 2009, 106:1279412797.
46. Newmaster SG: Testing candidate plant barcode regions in the
Myristicaceae. Mol Ecol Resour 2008, 8:480490.
47. Fazekas AJ, Burgress KS, Kesanakurti PR, Percy DM, Hajibabaei M, Graham
SW, Husband BC, Barrett SCH, Newmaster SG: Assessing the utility of
coding and non-coding genomic regions for plant DNA barcoding.
PLoS ONE 2008, 3:112.
48. Yao H, Song J, Liu C, Luo K, Han J, Li Y, Pang X, Xu H, Zhu Y, Xiao P, Chen S:
Use of ITS2 region as the universal DNA barcode for plants and animals.
PLoS ONE 2010, 5:e13102.
49. Hollingsworth PM, Graham SW, Little DP: Choosing and using a plant DNA
barcode. PLoS ONE 2011, 6:e19254.
50. China Plant BOL Group, Li D-Z, Gao L-M, Li H-T, Wang H, Ge X-J, Liu J-Q,
Chen Z-D, Zhou S-L, Chen S-L, Yang J-B, Fu C-X, Zeng C-X, Yan H-F, Zhu Y-J,
Sun Y-S, Chen S-Y, Zhao L, Wang K, Yang T, Duan G-W: Comparative
analysis of a large dataset indicates that internal transcribed spacer (ITS)
should be incorporated into the core barcode for seed plants. Proc Natl
Acad Sci U S A 2011, 108:1964119646.
51. Kool A, de Boer HJ, Krger , Rydberg A, Abbad A, Bjrk L, Martin G:
Molecular identification of commercialized medicinal plants in Southern
Morocco. PLoS ONE 2012, 7:e39459.
52. Chase MW, Salamin N, Wilkinson M, Dunwell JM, Kesanakurthi RP, Haidar N,
Savolainen V: Land plants and DNA barcodes: short-term and long-term
goals. Philos Trans R Soc Lond B Biol Sci 2005, 360:18891895.
53. Rotblatt M, Ziment I: Evidence-based Herbal Medicine. 1st edition.
Philadelphia, PA: Hanley & Belfus; 2002.
54. Cimino MT: Successful isolation and PCR amplification of DNA from
National Institute of Standards and Technology herbal dietary
supplement standard reference material powders and extracts. Planta
Med 2010, 76:495497.
55. Fazekas AJ, Kesanakurti PR, Burgess KS, Percy DM, Graham SW, Barrett SCH,
Newmaster SG, Hajibabaei M, Husband BC: Are plant species inherently
harder to discriminate than animal species using DNA barcoding
markers? Mol Ecol Resourc 2009, 9:130139.
56. Shokralla S, Spall JL, Gibson JF, Hajibabaei M: Next-generation sequencing
technologies for environmental DNA research. Mol Ecol 2012, 21:17941805.
57. Cornara L, Borghesi B, Canali C, Andrenacci M, Basso M, Federici S, Labra M:
Smart drugs: green shuttle or real drug? Int J Legal Med 2013, 6:115.
58. Winslow LC, Kroll DJ: Herbs as medicines. Arch Intern Med 1998,
158:21922199.
59. Calixto JB: Efficacy, safety, quality control, marketing and regulatory
guidelines for herbal medicines (phytotherapeutic agents). Braz J Med
Biol Res 2000, 33:179189.
60. Vickers A, Zollman C, Lee R: Herbal medicine. West J Med 2001,
175:125128.
Page 13 of 13
61. Zollman C, Vickers AJ, Richardson J: ABC of Complementary Medicine.
Chichester, UK: John Wiley & Sons; 2009.
62. Swerdlow J: Medicine Changes: late 19th to early 20th century. In Natures
Medicine: Plants that Heal. Washington, DC: National Geographic Society;
2000:110191.
63. De Smet PAGM: Herbal remedies. N Engl J Med 2002, 347:20462056.
64. Hoffmann D: Medical Herbalism. The Science and Practice of Herbal Medicine.
Rochester, VT: Inner Traditions/Bear & Co; 2003.
65. Bent S: Herbal medicine in the United States: review of efficacy, safety,
and regulation: grand rounds at University of California, San Francisco
Medical Center. J Gen Intern Med 2008, 23:854859.
66. Nikam PH, Kareparamban J, Jadhav A, Kadam V: Future trends in
standardization of herbal drugs. JAPS 2012, 2:3844.
67. WHO: WHO Traditional Medicine Strategy 20022005. http://whqlibdoc.
who.int/hq/2002/who_edm_trm_2002.1.pdf.
68. Morris CA, Avorn J: Internet marketing of herbal products. JAMA 2003,
290:15051509.
69. DeAngelis CD, Fontanarosa PB: Drugs alias dietary supplements. JAMA
2003, 290:15191520.
70. Coghlan ML, Haile J, Houston J, Murray DC, White NE, Moolhuijzen P,
Bellgard MI, Bunce M: Deep sequencing of plant and animal DNA
contained within traditional Chinese medicines reveals legality issues
and health safety concerns. PLoS Genet 2012, 8:e1002657.
71. Bent S, Ko R: Commonly used herbal medicines in the United States: a
review. Am J Med 2004, 116:478485.
72. Zhang J, Wider B, Shang H, Li X, Ernst E: Quality of herbal medicines:
challenges and solutions. Complement Ther Med 2012, 20:100106.
73. Wolsko PM, Solondz DK, Phillips RS, Schachter SC, Eisenberg DM: Lack of
herbal supplement characterization in published randomized controlled
trials. Am J Med 2005, 118:10871093.
74. Marcus DM, Grollman AP: Botanical medicinesthe need for new
regulations. N Engl J Med 2002, 347:20732076.
75. Raynor DK, Dickinson R, Knapp P, Long AF, Nicolson DJ: Buyer beware?
Does the information provided with herbal products available over the
counter enable safe use? BMC Med 2011, 9:94.
doi:10.1186/1741-7015-11-222
Cite this article as: Newmaster et al.: DNA barcoding detects
contamination and substitution in North American herbal products.
BMC Medicine 2013 11:222.
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