Nutritional Outlook

Nutritional Outlook, April 2018

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■ NUTRITIONAL OUTLOOK 50 Botanicals/Herbs APRIL 2018 animals, plants, fungi, and bacteria," Miller says. Indeed, adds Sudberg, when applied to herbs and botanicals, DNA barcoding can iden- tify precise gene segments of a given species in such a way that it "allows increased specif city when identifying closely related species." Yet, "DNA barcoding hasn't always been completely understood or used in the proper situations," Miller says. "In the New York at- torney general's case, inappropriate test methods were used on highly processed ma- terials, which led to inaccurate results." And that's no small matter. As Qi Jia, PhD, chief scientif c of cer, Unigen Inc. (Tacoma, WA), explains, DNA barcoding "relies on the existence and integrity of targeted DNA re- gions in the sample that can be extracted, amplif ed, and then sequenced." T e rigors of harvesting, processing, formulating, and storage, however, mean that a sample may not preserve those targeted sequences. In addition, the complex medium of a dietary supplement's f nished formulation may also interfere with DNA extraction. All of these obstacles can interfere with the otherwise proper application of DNA testing and can yield false negatives for the plant origin, for instance, or indicate an exaggerated pres- ence of small amounts of irrelevant biomass, like inert f ller or minor contaminants with their own intact genomes. "T e DNA method is very sensitive to all contaminants or minor coexists on the plant," Jia emphasizes, "and caution thus must be taken with f nal conclusions solely based on a DNA test." Consider that when his company tested one of its own ingredients, the origin of which was a wild-harvested tree bark, barcoding found not only the correct plant species but DNA f ngerprints of at least 24 others—algae, lichen, fungi, moss—all common to tree bark. "To prevent the algae or microbial growth on the bark of a tree in the wild can be a daunting task," Jia con- cedes, pointing out that "in the extraction and standardization process to make the ex- tract, those minor contaminants have been removed in washing, extracting, concentrat- ing, and drying." Naming of Parts Another gripe some have with DNA bar- coding as applied to botanicals: "No DNA technology is capable of identifying plant parts," says Miller, be it stem, leaves, seeds, etc. "T is is noteworthy," Miller says, "be- cause certain phytochemicals are only found in specif c plant parts." It's also noteworthy because compliance with cGMPs frequently requires identif ca- tion of the plant part used. "T is can be done with other, far better-established techniques and tools of botanical identif cation with a longer track record of accuracy," Sudberg says. He's talking about tools like plant morphol- ogy—both micro- and macro-morphology using industry-standard methods like high- performance thin-layer chromatography (HPTLC) and microscopy. "While DNA bar- coding does increase the ability to be more specif c for species identif cation of herbal supplement ingredients," Sudberg says, "it has inherent limitations that render it inadequate as a standalone test method for regulatory compliance." Miller agrees. "T is is why we recommend DNA testing to authenticate the species and identify the DNA of other plants that could cause potential safety or quality concerns," he says. "If appropriate, we then recom- mend following up with necessary chemical or morphological testing to determine the plant part present." Covering All Databases But even when DNA barcoding functions in concert with other methods in a com- prehensive testing toolbox, it still faces hur- dles to optimal utility. For one, "A DNA test method is only as good as its DNA sequence database—and not all DNA sequence data- bases are equal," says Miller. T e material used to create a reference standard must be validated, for example; for- tunately, NSF International maintains a da- tabase of validated DNA reference sequences for all relevant botanical ingredients and adulterant species of plants, fungi, and pro- biotic bacteria. Many of these reference se- quences came to the organization as herbar- ium specimens from its partnerships with leading institutions like Harvard University and the University of California, Berkeley. And that raises another potential problem with DNA databases: their variable transpar- ency. "As I've been saying for years," Sudberg notes, "there has been and still is a severe lack of transparency in the industry." T e reasons are understandable. "T ere's a lot of work to do to reach the point where DNA testing is as reliable as longer-established methods," Sudberg concedes. "T is needs a lot of funding and collaboration between indus- try, academic institutions, and public organi- zations. If the required resources come from the private sector, there's the tendency to keep f ndings secret to recoup the investment—yet unless we have transparency, there will always be dif culty duplicating those DNA tests, which sets the stage for huge problems in sup- ply chain and brand relationships." Now that FDA is working on a public ge- nome database for plants used in the supple- ment industry, and T e National Institute of Standards and Technology "is sending sam- ples to dif erent labs to understand DNA test- ing," Sudberg says, the transparency outlook is "f nally changing—slowly—for the better." One Piece of the Puzzle Cosimo Palumbo, marketing director, Indena (Milan, Italy), describes yet another hurdle, particularly as regards testing of plants: the lack of a universal DNA barcoding methodol- ogy for them. As a consequence, "the choice of a particular technique is often a compro- mise that depends on a number of factors. Each plant needs a dedicated method, devel- oped on its own genome," he says. "In this respect," Palumbo continues, "so- phisticated DNA sequencing–based tests are powerful technologies, but they're just one piece of the puzzle, as what really counts is gaining knowledge in plant genetic diversity." Miller thinks so, as well. "Combining DNA with other testing methodologies is always the best way to make sure a product is the safest it can be," he says. "Between DNA, chemical, microscopic, and microbiological testing, a full-service testing suite can pro- vide the information needed to make a deci- sion about a product sample." With this in mind, more industry orga- nizations are in fact working to make DNA methods better and more f t-for-purpose for the botanical industry. For example, NSF International and NSF AuthenTechnologies recently launched a verif cation program combining next-generation sequencing (NGS) technology and NSF's database of

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