Detection of Japanese Sophora in Commercial Ginkgo Leaf Products Using Species-Specific DNA Sequences

Reviewed: Liu Y, Wang XY, Wei XM, Gao ZT, Han JP. Rapid authentication of Ginkgo biloba herbal products using the recombinase polymerase amplification assay. Sci Rep. 2018;8(1):8002.

Keywords: adulteration, ginkgo, Ginkgo biloba, Japanese sophora, recombinase polymerase amplification, RPA-LFS, Styphnolobium japonicum, Sophora japonica

As documented in the BAPP Botanical Adulterants Prevention Bulletin on ginkgo leaf extract, the addition of non-ginkgo flavonols and flavonol glycosides to ingredients labeled to contain solely Ginkgo biloba leaf extract as a means to obtain the 24% flavonol glycoside concentration required by pharmacopeial monographs has been reported by a number of scientists over the past 15 years.¹ A particularly rich source of such flavonols is Japanese sophora (Sophora japonica, syn. Styphnolobium japonicum, Fabaceae), which has been identified as a ginkgo leaf extract adulterant based on the presence of marker compounds such as genistein, genistein-4′-O-glucoside, and genistein-4′-O-neohesperidoside.^2-4 The present investigation set out to detect the presence of Japanese sophora in commercial ginkgo leaf teas and dietary supplements made with ginkgo leaf extracts.

Two sets of species-specific primers for short sequences of the rbcL and ITS2 regions were developed for ginkgo and Japanese sophora, respectively. After extraction, DNA was amplified using the recombinase polymerase amplification (RPA) assay, which reportedly is more sensitive, in particular with shorter (< 200 base pairs) sequences than regular PCR. The results were visualized using a lateral flow stick (LFS), and verified by comparing the RPA-LFS results for each of the commercial products with data obtained using the more widely used PCR amplification and further separation of the amplified sequences on an electrophoresis gel.

Commercial products in form of teas (n = 8) or dietary supplements (n = 28) were purchased online. Manufacturing countries included Australia, China, Germany, Japan, and the United States. While tea samples contained only ginkgo material, nine dietary supplements (32%) contained a mixture of ginkgo and Japanese sophora, as evidenced by the presence of the sophora ITS2 sequence obtained from these products. Contrary to RPA-LFS, regular amplification using PCR provided Japanese sophora sequences only for seven ginkgo dietary supplement products. Two ginkgo dietary supplements (7%) did not provide any useful genetic information, most likely due to the DNA being too highly fragmented or altogether absent.

Comment: The data provided is in agreement with several previously published papers reporting that adulteration of ginkgo extracts with purified flavonoid-rich materials from Japanese sophora.^2-4 To our knowledge, this is the first paper that provides genetic evidence for such adulteration. The use of species-specific primers targeting short DNA sequences is one of the strengths of this research project, allowing the detection of smaller DNA fragments that are often found in processed herbal materials such as extracts. The species-specific approach reduces the risk of false positive results, but it also limits the number of detectable adulterants to one species. Nevertheless, this is an example where a species-specific molecular testing may be a valuable addition to chemical methods that could potentially miss ginkgo leaf extract adulteration when following standard pharmacopoeial testing methodologies. The fact that 32% of the commercial dietary supplements were found to contain Japanese sophora suggests that this species is likely the most widely used adulterant of Ginkgo biloba leaf extracts and reconfirms the widespread adulteration of this important botanical ingredient.

References

1. Gafner S. Adulteration of Ginkgo biloba leaf extract – Botanical Adulterants Prevention Bulletin. ABC-AHP-NCNPR Botanical Adulterants Prevention Program. 2018;1-8.
2. Chandra A, Li Y, Rana J, Persons K, Hyun C, Shen S, Mulder T. Qualitative categorization of supplement grade Ginkgo biloba leaf extracts for authenticity. J Funct Foods. 2011;3(2):107-114.
3.  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.
4. Avula B, Sagi S, Gafner S, Upton R, Wang YH, Wang M, Khan IA. Identification of Ginkgo biloba supplements adulteration using high performance thin layer chromatography and ultra high performance liquid chromatography-diode array detector-quadrupole time of flight-mass spectrometry. Anal Bioanal Chem. 2015;407(25):7733-7746.