ABC Review and Critique of the Research Article "DNA Barcoding Detects Contamination and Substitution In North American Herbal Products" by Newmaster et al.
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.
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.
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