Focus on Ginkgo biloba Adulteration

Part I: DNA Barcoding Confirms that Commercial Ginkgo Products Contain Ginkgo biloba

Reviewed: Little DP. Authentication of Ginkgo biloba herbal dietary supplements using DNA barcoding. Genome. 2014;57(9):513-516.

Consumer health products containing extracts of Ginkgo biloba (Ginkgoaceae) are among the most popular herbal products worldwide. The authors of this study used a DNA barcoding approach to identify G. biloba plant material unambiguously and to verify the presence of G. biloba in dietary supplements purchased in retail stores in the New York area and online. The 40 supplements consisted of dried and powdered G. biloba leaf (n=8) or G. biloba leaf extract (n=32). Since the DNA of the original plant found in extracts – if such DNA is present at all – is usually heavily fragmented, the authors designed a special 166 base-pair-long matK mini-barcode to ensure a more reliable DNA amplification.

The ginkgo mini-barcode sequences were compared to all gymnosperm matK sequences available in GenBank and to those obtained from the author’s collection of 12 related gymnosperm samples. The author was able to retrieve DNA of adequate quality for PCR amplification from 37 ginkgo supplements. In nine cases, digital PCR (dPCR, a method where the sample is divided into a larger number of subsamples that are all amplified, leading ideally to a direct counting of single DNA molecules) was used to separate the excipient/filler DNA from ginkgo DNA. Ginkgo DNA was recovered from 31 (84%) of the samples analyzed. The remaining six supplements contained fillers without any detectable G. biloba DNA. However, since the matK mini-barcode assay cannot distinguish between samples that do not contain any G. biloba and those that contain G. biloba using an extract manufacturing process that either eliminated the DNA or degraded it to a point where it can no longer be used for amplification by PCR, it is impossible to determine if the six samples (16%) without a trace of G. biloba DNA did not contain any G. biloba.

Comment: This paper is a good example of the strengths and limitations of DNA barcoding. The author has used a specific mini-barcode to identify ginkgo DNA in the commercial products, knowing that the processing used to make the extracts will leave – if any – only highly fragmented DNA behind, and, therefore, a general barcoding approach would likely be unsuccessful. While he succeeded in finding ginkgo DNA in 84% of the powdered ginkgo leaf and ginkgo extract supplements, the test is not suitable to assess the quality of the products beyond the presence/absence of ginkgo DNA. In particular, the admixture of purified flavonols like rutin, quercetin, or kaempferol, or the partial substitution of ginkgo with flavonoid-rich extracts from other plant sources, e.g., Japanese sophora (Sophora japonica syn. Styphnolobium japonicum, Fabaceae) or buckwheat (Fagopyrum esculentum, Polygonaceae), cannot be detected if these extracts are devoid of high-quality DNA. The addition of undeclared extraneous flavonols to ginkgo to achieve a 24% total flavonol content, which has been the industry standard based on the extensive chemical, pharmacological, toxicological, and clinical research performed with the EGb 761^® extract manufactured by Dr. Willmar Schwabe GmbH & Co. KG (Karlsruhe, Germany), has been reported by a number of research groups. It can be detected using HPLC fingerprinting methods prior to hydrolysis (hydrolysis and subsequent analysis of ginkgo extracts by HPLC-UV is described in compendial monographs as a standard test method for quantification of total flavonol glycosides).^1,2

References

1.  Wohlmuth H, Savage K, Dowell A, Mouatt P. Adulteration of Ginkgo biloba products and a simple method to improve its detection. Phytomedicine. 2014;21(6):912-918.

2.  Harnly JM, Luthria D, Chen P. Detection of adulterated Ginkgo biloba supplements using chromatographic and spectral fingerprints. J AOAC Int. 2012;95(6):1579-1587.