By Stefan
Gafner, PhD,a Mark Blumenthal,a Danica Harbaugh Reynaud,
PhD,b Steven Foster,c Natascha Techen, PhDd
a American Botanical Council,
Austin, TX
b Authentechnologies LLC, Richmond,
CA
c Steven Foster Group, Eureka
Springs, AR
d National Center for Natural
Products Research, University of Mississippi, Oxford, MS
Editor’s note: The American Botanical Council (ABC) previously has published
several peer-reviewed articles in HerbalGram describing the valuable role that DNA-based
analytical techniques can provide to quality control efforts in the botanical
products industry. Consequently, we believed it was important to provide ABC
members and others in the botanical research and products communities with this
critique and commentary of a recently published paper that used DNA-based
methods to analyze commercial herbal products.1,2
The
open-access journal BMC Medicine
published an article online titled “DNA barcoding detects
contamination and substitution in North American herbal products” on
October 11, 2013.3 Steven Newmaster, PhD, associate professor in the
Department of Integrative Biology, and his colleagues from the University of
Guelph in Ontario, Canada, conducted research on deoxyribonucleic acid (DNA)
barcoding of North American herbal products using two DNA regions sanctioned by
the Consortium for the Barcode of Life* as core or supplemental barcodes to
differentiate all plant species.
The study purports that “most of the herbal products tested were of poor
quality, including considerable product substitution, contamination and use of
fillers,” and that “some of the contaminants we found pose serious health
risks to consumers.”3 The article eventually was picked up by many
media outlets with headlines such as “Herbal product contamination
'considerable', DNA tests find,” “That herbal supplement may
not be what you think it is, scientists find,” “Herbal supplements often
contain unlisted ingredients,” and “Herbal supplements may contain
toxic contaminants,” respectively.4-7 Another
article, “Herbal supplements are often not what they seem,”
was published online by the New York Times on November 3.8
The study’s results are based on a method
that uses plant DNA barcodes (barcodes are short genetic markers in an
organism's DNA to identify it as belonging to a particular species) as an
unbiased reproducible method of species identification. Since DNA collects
mutations that are subsequently inherited throughout the process of biological
evolution, these genetic markers can be used to determine the exact plant
species and the relationship among species. In the present study, the authors
used the rbcL and ITS2 regions to obtain DNA barcodes,
which were then compared to the barcodes of what the authors refer to as their
own “standard reference material (SRM)” herbal barcode library to identify the
plant species. (This is one of numerous anomalies in the paper. Standard
Reference Material® is a
trademark registered with the United States Patent and Trademark Office by the
US National Institute of Standards and Technology (NIST). It is a certified
reference material issued by NIST that also meets additional NIST-specific
certification criteria and is issued with a certificate or certificate of
analysis that reports the results of its characterizations and provides
information regarding the appropriate use(s) of the material. A knowledgeable
peer reviewer should have detected this error in the Newmaster et al. paper.)
To be clear, this review and critique is not a criticism of
the applicability of DNA as an appropriate form of botanical identification,
which the authors of this critique consider reliable when properly executed.
The paper by Newmaster et al. is problematic
on a number of levels. The approach taken by the authors was to create a number
of homemade definitions and then evaluate materials against those definitions
using DNA fingerprinting. This would perhaps be acceptable if a review of the
list of cited references had not indicated that the authors are apparently deeply
unaware of herbal quality-assurance procedures and programs and the nature of
commercial botanical products. There are internationally recognized definitions
for identity, authenticity, contamination, and substitution. Invention of new
definitions for these terms by the authors in order to demonstrate the novelty
of their approach and their technical virtuosity is self-referential and,
unfortunately, very possibly self-serving. Their apparent lack of adequate
knowledge in this field has allowed them to create a virtual problem and then, figuratively,
ride to the rescue and solve it.
While there are several clinical trial review
articles and many DNA papers cited in the publication, there are no
publications at all from recent and/or the past century’s phytochemistry or
pharmacognosy literature, and there seems to be no awareness at all that
legally binding compendial standards (including specifications and tests for
identity and quality, as noted in various officially recognized pharmacopeias)
have existed for the better part of two centuries. Included in these monographs
are chemical and physical tests for identification of crude botanicals,
powdered botanicals, and botanical extracts. In addition to the individual
plant monographs found in the official Pharmacopoeia
of the People’s Republic of China, the official Japanese Pharmacopoeia, the official Ayurvedic Pharmacopoeia, the official United States Pharmacopeia (USP), the official European Pharmacopoeia (EP), and the unofficial but highly regarded
USP Dietary Supplements Compendium (USP-DSC) and American
Herbal Pharmacopoeia (AHP), each compendium provides several chapters that
describe the nomenclature of different forms of herbs in commerce (extracts,
native extracts, standardized extracts, etc.).
Many herbal materials in commerce are extracts,
or extracts that are spray-dried onto carriers (such as rice powder) rather
than simply dried powdered plant material. These dried extracts are frequently
sold in the form of capsules and/or tablets. Since the authors did not provide
label information for any of the commercial herbal products they tested, it is
not possible to adequately evaluate their claims that the fillers they found were
‘unlabeled.’ In addition, heat or other preservation technologies applied to
herbal materials can denature DNA while maintaining the natural phytochemical
profile of an herb. Despite the authors' contention to
the contrary, there is no evidence that DNA can be obtained
from botanical extracts, and DNA from relatively highly processed materials
such as finished supplements in tablets and capsules is of poor quality. As a
result, the DNA that is detected in these cases is typically either accidental environmental
contamination, cross-contamination among samples in the lab, and/or the
DNA from the product’s carrier or filler (soy, potato, or rice). Only if
the proper controls are used — including negative controls for the extraction
and polymerase chain reaction (PCR) — can accurate data be generated.
While some of the authors’ findings on
adulterated botanical products may indeed seem very troublesome, like the
substitutions of cinnamon (Cinnamomum
spp., Lauraceae) with plant material from a member of the Amaryllis family
(Amaryllidaceae), burdock (Arctium lappa,
Asteraceae) with a member of the buttercup family (Ranunculaceae), or ginkgo (Ginkgo biloba, Ginkgoaceae) with black walnut
(Juglans nigra, Juglandaceae), the
absence of DNA from the plant species indicated on the product label may be
explained due to extensive processing of said material before it was packaged
for consumer use. Furthermore, the presence of the adulterants can, in some
cases, be explained by cross-contamination from other samples or the lack of
specificity in the regions analyzed. The authors’ discussion
failed entirely to address these potential confounders.
The authors provide no quantitative data for their results. We, the
authors of this critique, understand that accurate quantification would be
challenging, if not impossible, since the
ITS2 region, which was used as a
barcode by Newmaster et al., is present in up to several thousands of identical
copies in plant cells — some plants having more copies, some having less. The
more copies that are present, the higher the chance they are amplified by PCR. Therefore,
it would be misleading to conclude that if more ITS2 PCR product of one species is detected, then more of this
plant material is present. Also, the length of the PCR product from authentic
plant material versus an adulterant is important. In the same PCR reaction mix,
a shorter fragment (e.g., from the adulterant) is preferably amplified over a
larger fragment, which also could be misinterpreted to mean that the sample has
more adulterant material than authentic material.
We thus are at a loss to know whether or not non-target DNA found in a
product is present at levels that would constitute a significant amount of
extraneous material or perhaps a few dandelion (Taraxacum officinale, Asteraceae) leaves commingled with a
hectare’s worth of harvested crop. Similarly, PCR amplification could well be
capable of picking up residual rice DNA left on a piece of processing equipment
from the previous production run. Other principles, such as a systematic approach
to documenting botanical collections and evaluating the sensitivity, accuracy,
and precision of the identification methods the study’s authors used, also are
lacking, although such approaches are readily available in the scientific literature.9-11
The following are various points or actual
statements made by the study’s authors, and the American Botanical Council’s
clarification and/or criticism of them:
1. In
the background information, the authors write:
[T]here
are currently no best practices in place for identifying the species of the
various ingredients used in herbal products. This is because the diagnostic
morphological features of the plants on which the current Linnaean taxonomic
system is based cannot typically be assessed from powdered or otherwise
processed biomaterials. As a result, the marketplace is prone to contamination
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.3
The authors continue that “currently, there
are no standards for authentication of herbal products."
The authors
apparently fail to recognize that reputable reference works such as the USP, EP,
AHP, and other official and
unofficial yet highly credible compendia cited above have published methods for
authentication of botanical raw materials and finished products, including
microscopic, macroscopic, and chemical identification. Compliance with the rules
of cGMPs (current Good Manufacturing Practices) in the Dietary Supplement
Health and Education Act (DSHEA), including the use of adequate identification
testing by manufacturers of dietary supplements, is required for all US manufacturers
of dietary supplements, and for foreign manufacturers exporting finished
dietary supplements into the United States. In Canada, in order to obtain product and
site licenses, specific labeling and packaging requirements in accordance with
the Natural Health Products Regulations
must be met, GMPs must be followed, and proper safety and efficacy evidence must be provided. In addition to the methods in the USP or EP
monographs, many research groups and various organizations have published
validated analytical methods to authenticate botanical materials. With regards
to DNA technology, NIST, along with the National Center for Complementary and
Alternative Medicine (NCCAM), has funded cGMP-compliant industrial laboratories
to validate DNA-based methods of authentication for many commonly used herbs,
which are currently used by major suppliers and manufacturers in the United
States and abroad. 2. With
regard to the presence of fillers, the author’s designate that “a filler is
found if a DNA barcode is found from rice (Oryza
sativa,
Poaceae) and
soybean (Glycine max,
Fabaceae). … [I]t 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 product.” What do they mean? Neither microcrystalline
cellulose nor gelatin is commonly derived from rice or soy. And it is doubtful
that microcrystalline cellulose and gelatin contain high-quality DNA, since
both of them are highly processed materials.
3. It is also difficult to understand their
conclusion that “the industry suffers from unethical activities by some of the
manufacturers, which includes false advertising, product substitution,
contamination and the use of fillers.” Although we are sensitive to the
possibility that some manufacturers may add various unlabeled, low-cost, and
possibly inert fillers to herbal products to lower their costs — thereby
possibly producing a product of questionable activity and benefit — the fact
remains that the legitimate and appropriate use of fillers and other processing
excipients are necessary ingredients for processing of various botanical
powders (dried herb and/or dried herb extract) into capsules and tablets.
Anyone with knowledge of manufacturing technology related to the production of
capsules and tablets probably would agree that this appropriate use of fillers per
se should not be described as an unethical activity.
In
the publication abstract, the authors claim that “most of the herbal products
tested were of poor quality, including considerable product substitution,
contamination and use of fillers.” The authors again seem to indicate that
fillers should not be used, and that they “dilute the effectiveness” of the
product. If it were not for purported “contamination” with fillers, the
majority of the analyzed products would have met the identity specifications according
to their criteria. We believe that this is a significant point, as some of the
media misinterpretations of this paper appear to derive from this issue. If one
accepts a reasonable level of appropriate excipients, flowing agents, and other
“fillers” required for the sake of processing dried herbal materials (whether
dried herb materials or dry extracts), then the erroneous conclusion that many
of these products are “adulterated” is highly mitigated.
4. On the scientific level, it is
incomprehensible that a research group intending to identify the correct
species of plant materials can confuse the scientific name that they ascribe to
“feverfew,” as “Parthenium hysterophorus”
(Asteraceae). However in commerce and generally in natural product research, the
common name feverfew is ascribed to the scientific name Tanacetum parthenium
(Asteraceae), an entirely
different species. When the authors discuss contamination of products
with "Parthenium hysterophorus (feverfew)," and possible adverse effects of “feverfew” they
cite papers published on T. parthenium — not
P. hysterophorus. Did they actually
find P. hysterophorus in Echinacea purpurea (Asteraceae)
products as they claim, or was the purported contaminate T. parthenium, the species for which they cite published literature?
An informed explanation might speculate that the contamination reported
possibly involved P. integrifolium (Asteraceae) — the root of which has been known to be an adulterant or unintentional
substitute for E. purpurea root for more than 100 years, dating to an
article published in 1910?12 The only way to know what the
authors were really studying would be to obtain the actual sample materials and
vouchers used in the study and repeat the testing. The reader is left thoroughly
confused and suspicious of the authenticity of the data presented in the paper.
5. Similarly, according to Table 1 in the
article, from the “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,” one
of the St. John’s wort (Hypericum
perforatum, Hypericaceae) products was allegedly substituted with a member
of the bean family (Fabaceae). Apparently, the species could not be identified
since “some of the DNA sequences recovered were only identified to family as
our SRM [sic] herbal DNA barcode library is not yet complete.” However, in the
discussion, the authors state that “one product labeled as St. John’s wort was
substituted with Senna alexandrina
(Fabaceae): it contained only Senna barcodes
and no St. John’s 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.”
So which is true? Why was S. alexandrina
not listed as adulterant in Table 1 if the authors were able to identify the
material to the species level? Or are the statements in the discussion based on
speculation?
6. Furthermore, in Table 1, the authors used
the scientific name "Plantago ovate." Presumably, they are
referring to P. ovata (Plantaginaceae),
and their spellchecker automatically tried to correct “ovata” to “ovate,” an
error that the authors, and, presumably, peer reviewers, should have detected. Even
though this might seem trivial, and, in some cases, it might be, it implies
that either the authors and/or the peer reviewers are not adequately familiar
with the scientific botanical nomenclature, and presumably, other aspects of
botany and medicinal plants that could have informed a more cohesive and
reliable publication.
It
is not clear from the article which of the scientific names listed in Table 1
were actually listed on the labels of the products they tested, and which might
be assumptions of species present if the label provided only a common name. For
example, Sonchus arvensis (Asteraceae)
is listed associated with the common name “Thistle.” This is a sow thistle
species so it could be present in a field-harvested herbal product as a weedy
contaminant but the table indicates that two samples (HP67C and HP87C) were
commercially available products. However, a search of the online National
Institutes of Health Dietary Supplement Label Database for U.S. products and
the Licensed Natural Health Products Database for Canadian products did not
find a single product with any species of Sonchus
as an ingredient. Searching these databases with the term “thistle”
returned results for Blessed Thistle (Cnicus
benedictus, Asteraceae) and Milk Thistle (Silybum marianum, Asteraceae), and a Google search turned only one
seller of S. arvensis raw material up
located in Indonesia, but no dietary supplement containing this material. Table
1 would be much more useful if it were revised to avoid these ambiguities. 7. In regard to the proper
authentication of the leaf material used to set up the authors’ database, statements
such as “this library was assembled from 100 herbal species of known provenance
that were identified by taxonomic experts,” and “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 horticultural greenhouses,” raise
the question just how well the authentication practices were conducted. Nowhere
in the text is there an indication that these authentications were performed according
to known standard operating procedures.9
8. In the methods section, the
authors described the DNA extraction from which they used 2.5 μL of genomic DNA
as template, which is a very unspecific amount. The authors should have given
the concentration in ng genomic DNA per PCR reaction. In addition,
they did not report that they used any negative or positive controls. Some of
the erroneous results may be due to cross-contamination in the lab. The fact
they obtained only one DNA sequence for one of the G. biloba samples, and J.
nigra for the other, seems rather coincidental since they also
were running fresh black walnut leaves in their lab. Also, it is
highly coincidental that P. hysterophorus would be found in
different samples, which again could mean cross-contamination.
All plant groups, as any molecular botanist
knows, have evolved in very different ways; for some, rbcL and ITS2 may be adequate,
but for many others, the two chosen DNA regions do not have enough variation.
However, this would be apparent only if an appropriate validation study had
been performed, for example, testing the selected genes on many samples of the
target and closely related species. This is obviously something the authors
have not even attempted to do. If they had, they would have noticed that rbcL and ITS2 do not distinguish among species of Echinacea.
9. An example of an obvious
— and some might consider egregious — calculation error is in the abstract: “Product substitution occurred in 30/44 of
the products tested and only 2/12 companies had products without any substitution,
contamination or fillers.” According to page five in the same publication,
substitution occurred in ca. 32% of the analyzed botanical materials, which
were chosen as representative of botanicals in the North American marketplace.
This 32% figure is much less than the 30 out of 44 (68%) reported in the
abstract, so, curiously and unfortunately, the information in the abstract is highly
misleading. According to Table 1, 12 out of 40 products (since DNA in 4 out of
44 products could not be amplified) is substituted, which corresponds to 30%.
10.
On the issue of regulation, the authors refer to the US FDA and the Canadian
Food Inspection Agency (CFIA) on page 11. As Canadians, the authors should be
aware that the CFIA does not regulate
herbal dietary supplements, which in Canada are regulated not as foods but as a
subset of drugs (“Natural Health Products”) by Health Canada’s Natural Health
Products Directorate, with compliance enforcement by the Health Products and
Food Branch Inspectorate. This obvious error
by Canadians regarding relatively simple regulatory information in their
own country is curious and puzzling.
Summary/Conclusion
Besides the various errors and misleading
quotes, perhaps most problematic aspect of the paper is the fact that there is
already a rich literature that demonstrates that quality of herbal products is
a real issue.13-16 In this regard, the authors have not made a
unique contribution to the field of DNA fingerprinting or herbal quality
assurance. Ironically, readers of the article can draw two opposing conclusions
from this paper, neither of which is correct. First, herbal critics will look
at the paper and see confirmation of their preconceived notions about the lack
of competence of the herbal industry. On the other hand, herbal advocates will
use the authors’ indifference to established practice, the extant peer-reviewed
literature, and the current state-of-the-art herbal quality assurance processes
to dismiss this paper and to pretend that quality issues do not exist. Even
if the DNA barcoding technique described is a reasonable and scientifically
valid method for species authentication — which hopefully will be more widely
used in the herbal industry, as ABC has already indicated as a possibility in
articles cited above — in our view, this paper is as much an example of
misrepresentation as the adulterated products that the authors purport to
expose. It is very disappointing that a well-regarded journal like BMC Medicine would allow publication of such
a flawed paper.
Three of the co-authors of
this critique (Dr. Gafner, Blumenthal, and Foster) are involved in the ABC-AHP-NCNPR
Botanical Adulterants Program, a nonprofit international
consortium designed to investigate known and suspected cases of adulteration of
botanical ingredients (including extracts) in the global herbal marketplace. Thus
far, the Program has published five extensive, appropriately peer-reviewed,
open-access papers on adulteration, with more papers forthcoming. Thus, these
authors are cognizant of the extent of much of the ingredient adulteration that
currently is occurring in the North American marketplace and in the global
supply chain, and welcome appropriate efforts to help clarify the extent of
this problem.
However, in our view, and in the opinion of
expert reviewers of this critique, and with all due respect to the authors and BMC Medicine, the journal should retract
this paper, and require that the authors address various errors, ambiguities,
and areas of confusion by appropriately rewriting, correcting, and resubmiting
it to the journal. The editors of the journal should then submit the corrected revision
to an appropriate peer-review process that employs numerous expert reviewers (not
just the two who presumably reviewed the initial paper) who are knowledgeable
not only in the fields of DNA testing, but also in botanical analytics and
related disciplines. Only then, if the paper passes such appropriately expanded
peer review, should the paper be republished. Until then, despite the good
intentions of its authors, this paper creates confusion, promotes false
conclusions, and, unfortunately, may constitute a disservice to scientific
researchers and other responsible members of the botanical products community.
* The
Consortium for the Barcode of Life is an international initiative devoted to
developing DNA barcoding as a global standard for the identification of
biological species. More information is available here.
References
1. Cimino M.
Ensuring the Specific Identity and Quality of
Herbal Products by the Power of DNA HerbalGram.
2010;86:50-57.
2. Ma YC, Chen SL, Thibault ME, Ma J. Enhancing quality
control of botanical medicine in the 21st century from the perspective of industry:
The use of chemical profiling and DNA barcoding to ensure accurate identity. HerbalGram. 2013;97:58-67.
3. Newmaster
SG, Grguric M, Shanmughanandhan D, Ramalingam S, Ragupathy S. DNA barcoding
detects contamination and substitution. BMC
Medicine 2013:11:222. doi:10.1186/1741-7015-11-222.
4. Brunhuber
K. Herbal product contamination 'considerable',
DNA tests find. CBCNews/Health, October 11, 2013. Available at: www.cbc.ca/news/health/herbal-product-contamination-considerable-dna-tests-find-1.1959278. Accessed October 30, 2013.
5. Branswell
H. That herbal supplement may not be what you
think it is, scientists find. The Globe
and Mail, October 11,
2013. Available at: www.theglobeandmail.com/life/health-and-fitness/health/that-herbal-supplement-may-not-be-what-you-think-it-is/article14825662. Accessed October 30, 2013.
6. Rettner R.
Herbal supplements often contain unlisted ingredients. FoxNews.Com., October 11, 2013. Available at: www.foxnews.com/health/2013/10/11/herbal-supplements-often-contain-unlisted-ingredients. Accessed
October 30, 2013.
7. Herbal
supplements may contain toxic contaminants. Business
Standard, October 13, 2013. Available at: www.business-standard.com/article/pti-stories/herbal-supplements-may-contain-toxic-contaminants-113101300284_1.html.
Accessed October 30, 2013.
8. O’Connor A. Herbal Supplements Are Often Not What They Seem. New York Times. November 3, 2013.
9.
Hildreth
J, Hrabeta-Robinson E,
Applequist W, Betz J, Miller J. Standard operating procedure for the collection and preparation of
voucher plant specimens for use in the nutraceutical industry. Anal Bioanal Chem.
2007;389(1):13-17.
10. AOAC International. Appendix K: Guidelines for Dietary Supplements and Botanicals. Part I: AOAC Guidelines for
Single-Laboratory Validation of Chemical Methods for Dietary Supplements and
Botanicals; Part II: AOAC Guidelines for Validation of Botanical Identification
Methods; Part III: Probability of Identification: A Statistical Model for the
Validation of Qualitative Botanical Identification Methods. AOAC International; Gaithersburg,
MD: 2012.
11. Betz JM, Brown
PN, Roman MC. Accuracy, precision, and
reliability of chemical measurements in natural products research. Fitoterapia 2011;82(1):44-52.
12. Moser Jr J. Echinacea and a spurious root
that appeared in the fall of 1909. Am
Jour Pharm. 1910;82:224-226.
13. McCutcheon A. An Exploration of Current Issues in Botanical
Quality: A Discussion Paper for
Natural Health Products Directorate, Health Canada. 2002.
Available at: http://citeseerx.ist.psu.edu/viewdoc/download?doi=10.1.1.118.3505&rep=rep1&type=pdf.
14. Ernst
E. Adulteration
of Chinese herbal medicines with synthetic drugs: a systematic review. J Int Med. 2002;252:107-113.
15. Foster S.
Exploring the peripatetic maze of black cohosh adulteration. HerbalGram 2013;98:32-51.
16. Foster S. The adulteration
of skullcap (Scutellaria lateriflora) with American germander (Teucrium
canadense). HerbalGram 2012:93;34-41.
|