Establishment of a PCR Assay for the Detection and Discrimination of Authentic Cordyceps and Adulterant Species in Food and Herbal Medicines.

Byeong Moon,Wook Kim,Gi-Ho Sung,Inkyu Park,Pureum Noh

doi:10.3390/molecules23081932

Abstract

Accurate detection and differentiation of adulterants in food ingredients and herbal medicines are crucial for the safety and basic quality control of these products. Ophiocordyceps sinensis is described as the only fungal source for the authentic medicinal ingredient used in the herbal medicine “Cordyceps”, and two other fungal species, Cordyceps militaris and Isaria tenuipes, are the authentic fungal sources for food ingredients in Korea. However, substitution of these three species, and adulteration of herbal material and dietary supplements originating from Cordyceps pruinosa or Isaria cicadae, seriously affects the safety and reduces the therapeutic efficacy of these products. Distinguishing between these species based on their morphological features is very difficult, especially in commercially processed products. In this study, we employed DNA barcode-based species-specific sequence characterized amplified region (SCAR) markers to discriminate authentic herbal Cordyceps medicines and Cordyceps-derived dietary supplements from related but inauthentic species. The reliable authentication tool exploited the internal transcribed spacer (ITS) region of a nuclear ribosomal RNA gene (nrDNA). We used comparative nrDNA-ITS sequence analysis of the five fungal species to design two sets of SCAR markers. Furthermore, we used a set of species-specific SCAR markers to establish a real-time polymerase chain reaction (PCR) assay for the detection of species, contamination, and degree of adulteration. We confirmed the discriminability and reproducibility of the SCAR marker analysis and the real-time PCR assay using commercially processed food ingredients and herbal medicines. The developed SCAR markers may be used to efficiently differentiate authentic material from their related adulterants on a species level. The ITS-based SCAR markers and the real-time PCR assay constitute a useful genetic tool for preventing the adulteration of Cordyceps and Cordyceps-related dietary supplements.

Highlights

IntroductionOphiocordyceps sinensis (Berk.) G.H. Sung, J.M. Sung, Hywel-Jones and Spatafora
We further confirmed the species of the fungal materials by an analysis of phylogenetic trees using 31 samples of nrDNA-internal transcribed spacer (ITS) sequences obtained in the current study, and 13 that were retrieved from NCBI GenBank (Table 1, Figure S1, and Materials and Methods)
The assay might be employed for the purity assessment of herbal medicines and diverse food ingredients related to Cordyceps. These results indicated that a real-time polymerase chain reaction (PCR) assay with sequence characterized amplified region (SCAR) markers was able to distinguish between the five fungal species with high sensitivity

Summary

Introduction

Ophiocordyceps sinensis (Berk.) G.H. Sung, J.M. Sung, Hywel-Jones and Spatafora Cordyceps sinensis (Berk.) Sacc.), a constituent of the traditional herbal medicine Cordyceps, is an entomogenous and well-known medicinal fungus that belongs to the family Ophiocordycipitaceae According to the Korean and Chinese Pharmacopoeia, the fungal source of the herbal medicine ‘Cordyceps’, namely Dong Chung Ha Cho in Korean and Dong Chong Xia Cao in Molecules 2018, 23, 1932; doi:10.3390/molecules23081932 www.mdpi.com/journal/molecules. Chinese, is described as originating only from the species O. sinensis [2]. Several other fungal species, Isaria cicadae Miq. Cordyceps cicadae (Miq.) Massee or Paecilomyces cicadae), Cordyceps militaris (L.) Fr., Cordyceps pruinosa Petch., Cordyceps gunnii Berk., Ophiocordyceps robertsii (Hook.) Berk., and Ophiocordyceps ophioglossoides (J.F. Gmel.) Fr., are repeatedly used as adulterants of authentic Cordyceps [1,3]. Only two species, C. militaris and Isaria tenuipes Peck

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Conclusion