Hindawi Evidence-Based Complementary and Alternative Medicine Volume 2018, Article ID 5160254, 18 pages https://doi.org/10.1155/2018/5160254 Review Article DNA Barcoding for the Identification and Authentication of Animal Species in Traditional Medicine Fan Yang,1,2 Fei Ding,3 Hong Chen,3 Mingqi He,3 Shixin Zhu,3 Xin Ma,1,2 Li Jiang,1,2 and Haifeng Li 3 1 Institute of Forensic Science, Ministry of Public Security, Beijing 100038, China 2Beijing Engineering Research Center of Crime Scene Evidence Examination, Institute of Forensic Science, Beijing 100038, China 3Center for Bioresources & Drug Discovery and School of Biosciences & Biopharmaceutics, Guangdong Pharmaceutical University, Guangzhou, Guangdong 510006, China Correspondence should be addressed to Haifeng Li; [email protected] Received 19 December 2017; Accepted 11 March 2018; Published 22 April 2018 Academic Editor: Yoshiki Mukudai Copyright © 2018 Fan Yang et al. Tis is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Animal-based traditional medicine not only plays a signifcant role in therapeutic practices worldwide but also provides a potential compound library for drug discovery. However, persistent hunting and illegal trade markedly threaten numerous medicinal animal species, and increasing demand further provokes the emergence of various adulterants. As the conventional methods are difcult and time-consuming to detect processed products or identify animal species with similar morphology, developing novel authentication methods for animal-based traditional medicine represents an urgent need. During the last decade, DNA barcoding ofers an accurate and efcient strategy that can identify existing species and discover unknown species via analysis of sequence variation in a standardized region of DNA. Recent studies have shown that DNA barcoding as well as minibarcoding and metabarcoding is capable of identifying animal species and discriminating the authentics from the adulterants in various types of traditional medicines, including raw materials, processed products, and complex preparations. Tese techniques can also be used to detect the unlabelled and threatened animal species in traditional medicine. Here, we review the recent progress of DNA barcoding for the identifcation and authentication of animal species used in traditional medicine, which provides a reference for quality control and trade supervision of animal-based traditional medicine. 1. Introduction animal species have medicinal benefts according to the historical records in China, and approximately 77 kinds Traditional medicine (TM) has been widely used for the of medicinal animals and 50 kinds of medicinal materials prevention and treatment of various common ailments and derived from animal sources have been included in the complicatedillnessesinhumanhistory,andtheuseofani- Chinese Pharmacopoeia 2015 Edition [1, 10]. In Brazil and mals as medicine, which is known as zootherapy, has Latin America, at least 354 and 584 animal species have long been an essential part of traditional therapeutic prac- been reported to be used in TM, respectively [4, 5]. It is tices, such as Traditional Chinese Medicine (TCM), Kampo estimated that animal-derived TM has been increasingly medicine, Ayurvedic medicine, and American folk medicine used in many countries and its annual global trade accounts [1–6]. Besides the traditional usages, modern clinical studies for billions of dollars [2]. Moreover, recent studies have have demonstrated that animal-based TMs possess a num- revealed that animal-based TM contains a variety of bioactive ber of pharmacological efects, including anti-infammatory, compounds, which provides a valuable chemical library for antitumor, anti-infective, anticonvulsant, analgesic, and im- drug discovery [11], indicating the potential of medicinal munomodulatory activities [6–9]. Terefore, zootherapy animals in developing modern pharmaceuticals. continues to serve as an important complementary and alter- During recent centuries, a wide range of wild animal native therapy in modern societies. For example, over 1,500 species is known to be threatened with extinction; for 2 Evidence-Based Complementary and Alternative Medicine example, according to the latest statistics of the International 2. Animal Identification by DNA Barcoding Union for Conservation of Nature (IUCN) Red List of Treatened Species, the number of endangered and criti- 2.1. DNA Barcoding and Minibarcoding. Te accurate species cally endangered animal species has reached 7597 and 5101, identifcation by DNA barcoding relies on a suitable DNA respectively [12]. Such rapid loss of biodiversity is closely barcode, which refers to a standardized sequence (usually less associated with animal habitat loss and human overexploita- than 1,000 bp) of the genome [21]. Te barcode should have tion. For instance, despite some parts such as horns and universality so that it can be easily amplifed from diverse secretions, almost all types of tissues and organs used in TM species, and it should contain few insertions or deletions to require animal sacrifce, which inficts substantial stress to facilitate sequence alignment. Also, its mutation rate must medicinal animal resources [2, 13, 14]. To solve the species be sufcient to generate a barcoding gap, which means the crisis, the trade of threatened animals has been regulated maximum intraspecifc variation is less than the minimum under international legislation including the Convention on interspecifc distance [21]. For animal identifcation, the most International Trade in Endangered Species (CITES), and the broadly used barcode marker is mitochondrial cytochrome therapeutic application of such species has been forbidden in c oxidase subunit I (COI), which is highly conserved across many countries. However, some countries such as those in species employing oxidative phosphorylation for metabolism East and Southeast Asia still utilize threatened animals for [22]. Numerous studies have shown that COI-based DNA medicinal purposes [2, 15], and the illegal trade of medicinal barcoding can delimit diverse animal species, indicating the animals further impairs biodiversity [16, 17]. Moreover, a high rates of sequence change at species level and constraints variety of adulterants and counterfeits has emerged in the on intraspecifc divergence in COI sequence [21–24]. For ana- market for greater interests, which brings challenges to the lyzing fresh and well-preserved animal tissues, a full-length safety of animal-based TM. Terefore, identifcation and barcodesuchasa658-bpregionofCOIgeneisrecommended authentication of animal species is of vital importance for TM as its PCR amplifcation and sequencing can be feasible. trade supervision and quality control. However, animal tissues are usually processed before used as It is known that animal-based TMs are mainly derived TM materials, and some processing approaches, such as sun- from animal tissues, organs, or metabolites, and these parts drying, stir-frying, and boiling, can cause DNA degradation, are usually processed into diverse preparations such as slice, leading to difculties for PCR amplifcation of full-length powder, and tablet, resulting in difculties for morphology- barcodes [19]. Since the amplifcation feasibility improves based species identifcation. In addition, several commer- with reduced sequence length, a minibarcoding method, cial technologies used for species identifcation, including which utilizes a shorter region within the standard barcode, chromatography and immunoassay, are relatively costly and has received increasing attention [28]. Recent studies have expertise-dependent, which is not suitable for bulk detec- shownthatanarrayofminibarcodescanbeefectively tion of TM preparations and forensic specimens [18, 19]. amplifed and sequenced from various processed products Terefore, developing an accurate and efcient identifcation including TMs and foods and provide sufcient sequence method represents an urgent need for animal-based TM. information necessary for species identifcation [26, 29, 30]. In recent decade, advance in molecular techniques has promoted the application of simple and precise DNA analysis 2.2. DNA Metabarcoding. It is known that many TM prepa- in taxonomic feld. Among the prevailing genome-based rations are composed of multiple materials derived from approaches, DNA barcoding provides a robust strategy to diverse animal and/or plant species. In such mixtures, mul- identify existing species and discover unknown species via tiple barcode sequences will be coamplifed and the con- comparative analysis of sequence variation [19–21]. Exten- ventional sequencing will generate multiple or overlaying sive studies have shown that DNA barcoding is capable sequencing peaks, resulting in ambiguity or false sequence of identifying a wide range of animal species, including information [31]. Terefore, DNA metabarcoding has been mammals, birds, reptiles, amphibians, fshes, and crustaceans proposed to identify multiple species within complex samples [21–24]. Moreover, many international organizations such as using next-generation sequencing (NGS) technology [32, 33]. the Consortium for the Barcode of Life (CBOL) and the NGS can rapidly yield millions of DNA reads and obtain Barcode of Life Data System (BOLD) have been established all representative sequences presented in mixtures, which to