Putting the Cart Before the Horse: Toward the Appropriate Use of DNA Methods for Quality Assurance in the Herbal Supplement Industry

The 2016 International Conference on the Science of Botanicals saw several researchers respond to the New York State Attorney General's attempts to apply DNA barcoding methods to confirm whether herbal supplement products contained the plant material listed on product labels. These presentations, combined with the presence of so many experts at the conference, created an excellent opportunity for real discussion of the underlying science of DNA barcoding in forensic quality assurance. Some clarity on appropriate use is emerging.

The use of genetic (DNA) approaches to determining relatedness of plant species has been around for a while, but is a new approach to assure ingredient identity and product quality in the herbal supplement field. DNA barcoding works by identifying short, characteristic sequences of DNA, and comparing these sequences between taxa. To be useful for taxonomic differentiation, a short sequence must be unique within each taxonomic subunit (e.g., species) and different between dissimilar taxonomic units. Identifying unique DNA sequences and determining that they are truly unique requires knowledge of the entire genome of each of the taxonomic units of interest so that sequences may be identified and comparisons made. These data do not currently exist.

The generic term “DNA barcoding” has been applied to a number of genetic methods. This is both inaccurate and confusing. The method has worked well to identify animal species using a universal animal barcode. This is a specific sequence from the cytochrome c oxidase 1 gene, the composition of which has been shown to differ between animal species.

At the conference, Sara Handy, PhD, of the Food and Drug Administration's (FDA) Center of Food Safety and Nutrition (CFSAN), noted that in an attempt to incorporate genetic tools into FDA's regulatory toolbox, CFSAN is developing a reference DNA database for plants and constructing a library of DNA primers and assays. She noted that each different plant will require a different combination of gene regions. She also reported on the agency's strategies to avoid choosing non-diagnostic DNA markers. ¹

Wendy Applequist, PhD, from the Missouri Botanical Garden, discussed the nature of plant nomenclature and identification. ² She made the point that morphology is the core method for naming plants in the first place, adding that most potential identity markers, including DNA sequences, do not always provide accurate proxies for botanical identification. Any new technique used to identify plant species must generate markers that agree with morphological characterization of identity. The new techniques must also be used to move from pilot experiments at identifying plants that use small numbers of unique specimens of the same species to much larger sample sizes. Morphologically similar or identical plants that are functionally the same species can show variability in DNA due to genetic drift, so the reliability of chosen genetic markers must be rigorously evaluated. This is especially true when considering the full range of geographic variation and hybrid zones that occur within a species. She noted that to validate a new method, it must first be compared against a known sample that was morphologically identified. It must then be shown that to work reliably when implemented correctly by appropriately trained individuals.

Hans Wohlmuth, PhD, Integria Healthcare/MediHerb, reported on research conducted by an Australian research consortium that included the Australian Genome Research Facility, the Medicinal Plant Herbarium at Southern Cross University, and the University of Queensland. ³ The group employed four widely used DNA barcoding methods to analyze 61 samples, including raw plant material, botanical extracts, and tablets containing the extract. Their approach using the DNA barcoding method was able to identify correctly just over 50% of the raw material. Processing of the raw material to produce both botanical extract and tablets reduced the ability of the DNA barcoding method to identify the plant species from which the extracts or tablets were made. This result was primarily due to the degradation of DNA by the processing method rather than to a lack of uniqueness of the DNA sequences investigated. The same samples were also evaluated using classical phytochemical methods such as thin-layer chromatography. These tests indicated that both the chemical profiles and the active compounds were present in extracts and tablets, even if the DNA was degraded. Dr. Wohlmuth stopped short of saying that DNA techniques were useless. He emphasized that uncritical selection of “off-the-shelf” techniques could result in incorrect results.

Two published papers revealed that using specific methods that are fit for purpose, rather than the more general barcoding methods such as those used above, can be constructed. These methods both focused on two different DNA target sequences within the plant of interest that were used to identify ginkgo (Gingko biloba) ⁴ and saw palmetto (Serenoa ripens). ⁵ Ferri et al. also found that when compared with a single set of genetic markers, the use of multiple genetic markers improved the authentication accuracy of medicinal plant material from 40–55% to 80–95%, depending on the combination of markers used. ⁶

It helps to understand a bit more of how the methods deployed create problems that have yet to be solved. DNA methods target genetic markers, a specific short sequence of nucleotides within the genome of the target organism. To find the specific sequence of DNA unique to a plant species, a technique called polymerase chain reaction (PCR) is used slice up the organism's DNA strands into small pieces and then to amplify the specific fragment of DNA so that it may be measured. Since DNA is easily degraded by processing of plant material to produce herbal supplements, reliable amplification using PCR evaluates mini-barcodes (sequences) of fewer than 200 base pairs rather than the entire DNA sequence. This approach was shown by Little ⁴ and Little and Jeanson ⁵ to work for identification of ginkgo and saw palmetto, respectively. Using complete sequence DNA barcoding methods, Harnly et al. were only able to authenticate 10% of crude raw materials, and no finished products made with extracted herbal ingredients. ⁷ This compared with authentication of 90% and 60%, respectively, using DNA methods specific to the plant material tested.

Gingko is a special circumstance, since it is the only member of its family and its genus and is phylogenetically isolated. Non-ginkgo specimens were correctly identified to taxonomic level of order, but the method was unable to discriminate finer levels of plant identity (family, genus, and species). The method was unable to distinguish between samples that did not include ginkgo DNA and gingko samples where processing had degraded the DNA beyond the point where intact sequences were available for PCR amplification. Results were somewhat better with saw palmetto. The application of characteristic mini barcodes in saw palmetto distinguished Serenoa repens from several closely related species.

There are additional limitations to plant DNA methods as a quality assurance or regulatory tool. For instance, even when the technique correctly identifies the plant, DNA testing cannot identify the plant part used to make the product. It cannot tell us whether the plant was grown under the right environmental conditions to produce the appropriate phytochemical profile. And because many of the herbs in supplements are botanical extracts, not intact plants, it may not be able to identify the plant ingredients. The method is best applied to non-extracted plant materials and as a complement to other chemical tests. Even identifying plants in unprocessed herbal material can be a challenge.

The difficulty of using DNA methods to identify a raw material includes many potential sources of error: selection of the right primer for the DNA sequence; lack of variability in the chosen sequence; multiple species with identical sequences; amplification of non-specific sequences; PCR inhibiting compounds isolated along with DNA; contamination from outside samples such as houseplants in an office or human DNA; DNA introduced by normal GMP-compliant manufacturing processes; and many more. The FDA requires validation studies be conducted and compared to establish whether the method is fit for the purpose. Currently DNA-based methods lack the necessary supportive data.

Validated chemical methods have been the mainstay of industry approaches for quality assurance purposes. Harnly et al. looked at flow injection mass spectrometry and nuclear magnetic resonance spectrometry, and compared the results to DNA sequencing specific to the plant material. ⁸ They verified that DNA from black cohosh (Actaea racemosa) root samples was adulterant DNA from another related plant, but they were unable to verify herbal supplements that lacked DNA because of extraction processes. DNA methods were also unable to characterize inert inorganic material accurately for which appropriate macroscopic, microscopic, or standard chemical tests were successful. Although able to detect fungal contaminants, such as common soil fungi from non-sterile samples, the selective sensitivity of the methods lead to amplification of fungal DNA and overestimation of the microbial load.

What is lacking is the accumulated proof of validity for DNA technology for the purpose of differentiating multiple plant genera and species, and for differentiating plant material from excipients, adulterating plant genera and species and from microbial contaminants. Not enough cross-characterization of the technique in relation to validated chemical and morphological methods has occurred to date. In addition, it is worth noting that public faith in the test may lead to a new form of product adulteration that adds small amounts of DNA-containing material to non-DNA containing finished products. This practice could introduce lower quality, less safe products into the market.

This review suggests that the initial use of DNA techniques might be effective at the front end of the herbal product development process, namely verifying and characterizing raw plant material in the global supply chain. Three important issues remain in developing a viable quality-assurance tool: (1) finding appropriate sequences; (2) developing methods that are fit for purpose; and (3) validating plant-specific DNA methods with existing validated chemical analytical methods.