Byeong Cheol Moon 1,* ID , Wook Jin Kim 1, Inkyu Park 1 ID , Gi-Ho Sung 2,3 and Pureum Noh 1

　　1 Division of Herbal Medicine Research, Korea Institute of Oriental Medicine, Daejeon 34054, Korea;ukgene@kiom.re.kr (W.J.K.); pik6885@kiom.re.kr (I.P.); pureum322@kiom.re.kr (P.N.)

　　2 Institute for Bio-Medical Convergence, College of Medicine, Catholic Kwandong University, Incheon 22711,Korea; sung97330@gmail.com

　　3 Department of Microbiology, College of Medicine, Catholic Kwandong University, Gangneung 25601, Korea* Correspondence: bcmoon@kiom.re.kr; Tel.: +82-42-868-9530 

Received: 20 June 2018; Accepted: 31 July 2018; Published: 2 August 2018

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.

Keywords: Ophiocordyceps sinensis; Cordyceps militaris; sequence characterized amplified region (SCAR) marker; real-time PCR; molecular authentication

1. Introduction

　　Ophiocordyceps sinensis (Berk.) G.H. Sung, J.M. Sung, Hywel-Jones and Spatafora (syn. 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 (division Ascomycota) [1]. 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 Chinese, is described as originating only from the species O. sinensis [2]. However, several other fungal species, Isaria cicadae Miq. (syn. 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]. Furthermore, only two species, C. militaris and Isaria tenuipes Peck (syn.Cordyceps polyarthra or Paecilomyces tenuipes), are described as authentic sources of food ingredients in the Korean market [4]. Hence, only O. sinensis-derived material can be used for medicinal purposes in Korean traditional medicine, whereas only the material derived from C. militaris or I. tenuipes can be used as food in Korea [2,4]. However, Cordyceps-related species are frequently substituted for one another in herbal and food material because of similar morphological features and confusing homonymous names; furthermore, Cordyceps derived from O. sinensis is very expensive (ca. 54 USD per g) [5]. C. pruinosa Petch. and I. cicadae Miq. are used as substitutes for C. militaris and I. tenuipes,respectively, because of their morphological similarities, including the color and shape of their fruiting bodies, and their host insect species. Thus, C. militaris, C. pruinosa, I. tenuipes, and I. cicadae are used to adulterate the herbal medicine Cordyceps (O. sinensis); C. pruinosa and I. cicadae are used as substitutes of C. militaris and I. tenuipes, respectively, in food products, and they are also used to adulterate the herbal medicine Cordyceps [3,6–8]. The various counterfeited products derived from these inauthentic species are consistently produced, and they adversely affect the safety, quality, and therapeutic efficacy of herbal medicines and dietary supplements [9].

　　Morphology-based identification of the authenticity and taxonomic origin of the source material in Cordyceps and related commercial products is usually impossible because the source material is typically used dried and powdered [9,10]. In addition, the relevant fungal species have distinct pharmacological properties, as summarized previously [1]. For example, O. sinensis is used for the tonification of the kidneys and lungs, to settle coughs and wheezing, and to inhibit sweating; on the other hand, C. pruinosa is used to treat stomach disorders, endotoxin shock, and inflammatory disease [1]. Therefore, reliable authentication tools for easy and quick identification of the taxonomic origins of Cordyceps and its related materials are consistently recommended in order to ascertain product safety, to regulate basic quality control, and as a counter-adulteration measure [6,11].

　　Diverse analytical methods are available for the quality standardization and identification of economically motivated adulterants in herbal and food materials. They are based on the assessment of taxonomic authenticity, including comparisons of morphological features, physiochemical properties,and DNA sequences [9]. Among these, DNA-based methods, such as DNA barcoding, random amplified polymorphic DNA, amplified fragment-length polymorphisms, and sequence characterized amplified region (SCAR) markers, are crucial, and these are widely used for the identification of the taxonomicorigins of various herbal and food materials to the species level [12–16]. A DNA barcoding method based on universal DNA barcodes and a large sequence database plays a key role in the molecular discrimination of species, and the assessment of authenticity for herbal medicines and food supplements.This method is commonly used for sequence analysis of the internal transcribed spacer of the ribosomal DNA(nrDNA-ITS) in the fungal nuclear genome, thematK and rbcL genes in the plant chloroplast genome,and the COI gene in the animal mitochondrial genome [12]. For over 10 years, numerous molecular genetic analyses involving nrDNA-ITS, the universal fungal DNA barcode, have been performed for the study of the phylogeny of Cordyceps and its related taxa, and for the identification of authentic Cordyceps [6]. However, these methods are limited to a simple and quick clarification of Cordyceps taxonomic origin, because the DNA barcoding method is complicated, and it involves techniques such as polymerase chain reaction (PCR) amplification, electrophoresis, gel isolation, sub-cloning (sometimes), sequencing, and database analysis. [6,17]. To overcome these limitations, a SCARmethod combining DNA barcodes and SCAR marker development is attracting attention as an intuitive, simple, and reproducible molecular tool for determining the authenticity of the taxonomic origins of diverse herbal and food materials [9,18,19]. This SCAR method is widely used for the detection of substitutes and adulterants,and/or to assess the authenticity of precious herbal medicines and food supplements, such as Panax,saffron, and Cordyceps [6,20–23]. Lam et al. [6] used random amplified polymorphic DNA sequences to develop SCAR markers for a rapid identification of O. sinensis, and compared the specificity and experimental duration of this method with that of ITS DNA barcoding. However, the authors designed the SCAR marker only for O. sinensis, which does not address the issue of the market distribution of Cordyceps and related species in Korea. Real-time PCR is used in conjunction with SCAR markers in diverse fields for the identification of the taxonomic origins of pathogens, food ingredients and products,and herbal medicines, because it is fast, easy, and quantitative [24,25]. Real-time PCR is widely employed,and it is considered to be very useful for the detection of adulterants in food products and herbal medicines, such as pork, fish, ginseng, etc. [26–29].

　　Some genetic methods are currently available for distinguishing authentic Cordyceps species,specifically O. sinensis, from adulterants. Several O. sinensis-specific genetic markers that solely detect O. sinensis in host larvae and soil, were developed from the use of species-specific primers based onO. sinensis ITS sequences [6,30–32]. However, these O. sinensis-specific PCR assay methods could not identify the species of adulterants and contaminants. Liu et al. reported a set of species-specific primers that can identify six Cordyceps and related species, using O. sinensis, C. militaris, and I. cicadae,C. gunnii, C. liangshanensis, and O. nutants [3]. Although they developed species-specific markers for identifying the six species, including O. sinensis, C. militaris, and I. cicadae, the range of species that are distributed as inauthentic adulterants in the market are, however, different between Korea and China,and the detection limits of the adulterant species were not clarified [3]. Therefore, an additional marker is needed to discriminate between Cordyceps and its related species, including additional adulterant fungal species, and to improve the currently available genetic tools, in order for the markers to be used as simple and rapid assays of commercially processed products in Korea. In the current study, we analyzed the nrDNA-ITS sequences of Cordyceps, and its related medicinal and food fungal species, i.e.,O. sinensis, C. militaris, C. pruinosa, I. tenuipes, and I. cicadae. We designed species-specific SCARmarkers and used them for the development of conventional and real-time PCR assays. We also confirmed that these assays were able to successfully discriminate between each species in commercially processed products. The established assays will facilitate the quality control of Cordyceps and related products,providing a reliable tool for the prevention of product adulteration.