A novel quantitative real-time PCR approach for authenticity assessment of herbal products with Ginkgo biloba

Plants have been widely used worldwide for medicinal purposes, leading to the increased consumption of herbal products, such as herbal infusions and plant food supplements. The growing demand of these products leads, inevitably, to the global market growing of herbal products and, consequently, to their adulteration. Ginkgo biloba is a Chinese tree whose leaves are extensively used in herbal preparations, being a very popular phytomedicine with high economic value. It has well-established therapeutic indications for the improvement of (age-associated) cognitive impairment and of quality of life in mild dementia, and treatment of problems associated with the peripheral circulation [1,2]. Ginkgo products have been adulteration targets through the addition of pure flavonols/flavonol glycosides or substitution with other botanical species [2]. Therefore, this work aims at proposing a new species-specific PCR approach to detect and quantify G. biloba to assess the authenticity of products thereof. Reference mixtures of known amounts (50% to 0.01%, w/w) of G. biloba leaves and Cammelia sinensis leaves were prepared. DNA was extracted from referencemixtures and other plant species using the Nucleospin Plant kit. Specific primers targeting the ITS1 region (Genbank: Y16892.1) were designed to amplify a 175 bp fragment of G. biloba. A species-specific PCR assay was developed and used to confirm the absence of cross-reactivity with other medicinal plant species. Afterwards, a quantitative real-time PCR assay with EvaGreen dye for G. biloba was developed and applied to binary mixtures of G. biloba in C. sinensis. A normalised calibration model was constructed based on the parallel amplification of the ITS1 region of G. biloba and the 18S rRNA gene (reference for eukaryotes). The normalised real-time PCR system allowed establishing a calibration curve covering the dynamic range of 50-0.1% (w/w) of ginkgo in C. sinensis. Additionally, a sensitivity down to 2 pg of ginkgo DNA was reached. The method exhibited adequate performance with values of correlation (0.996) and PCR efficiency (84.3%) in agreement with the acceptance criteria for these assays. The results showed that the proposed method could provide a species-specific quantitative tool for the authentication of herbal products containing ginkgo.This work was supported by UID/QUI/50006/2019 with funding from FCT/MCTES and NORTE-01-0145-FEDER-00001. L. Grazina acknowledges the PhD fellowship (SFRH/BD/132462/2017) funded by POPH-QREN (subsidised by FSE and MCTES).