Variations of the fungal microbiome in Corydalis Rhizoma with different collection areas, processing methods, and storage conditions.

Corydalis Rhizoma (CR, Yanhusuo in Chinese) has been widely used as an analgesic in herbal medicine and functional food. Cases of fungal and mycotoxin contamination in CR have been reported. In this study, the composition and diversity of fungal microbiome in CR samples from four herbal markets and two processing methods were investigated by DNA metabarcoding. Variations of the fungal microbiome in CR during cold and conventional storage were monitored. Results showed that Aspergillus was the dominant genus and saprotroph was the dominant trophic mode. Six potential toxigenic fungi, namely, Aspergillus fumigatus, Aspergillus ostianus, Aspergillus terreus, Penicillium citrinum, Penicillium oxalicum, and Trichothecium roseum, were detected. Differences in fungal composition and diversity among various groups based on collection areas and processing methods were also observed. Moreover, the relative abundance of dominant genera in CR samples stored at different temperatures was significantly different and changed with storage time. This study is the first to reveal the influence of collection areas, processing methods, and storage conditions on the fungal microbiome in CR, which was expected to provide a basis for control strategies of fungal contamination in the industrial chain of CR.

DNA Metabarcoding Reveals the Fungal Community on the Surface of Lonicerae Japonicae Flos, an Edible and Medicinal Herb.

Lonicerae Japonicae Flos (LJF) has been globally applied as an herbal medicine and tea. A number of reports recently revealed fungal and mycotoxin contamination in medicinal herbs. It is essential to analyze the fungal community in LJF to provide an early warning for supervision. In this study, the fungal community in LJF samples was identified through DNA metabarcoding. A total of 18 LJF samples were collected and divided based on the collection areas and processing methods. The results indicated that Ascomycota was the dominant phylum. At the genus level, Rhizopus was the most abundant, followed by Erysiphe and Fusarium. Ten pathogenic fungi were detected among the 41 identified species. Moreover, Rhizopus, Fusarium, and Aspergillus had lower relative abundances in LJF samples under oven drying than under other processing methods. This work is expected to provide comprehensive knowledge of the fungal community in LJF and a theoretical reference for enhanced processing methods in practical manufacturing.

Characterization of the Fungal Community in Fritillariae Cirrhosae Bulbus through DNA Metabarcoding.

Fritillariae Cirrhosae Bulbus (FCB) is a well-known and precious traditional Chinese medicine with a medicinal history spanning thousands of years. In recent years, it has been reported that fungal and mycotoxin contamination influenced the safety and quality of FCB. It is essential to systematically study the fungal community for the early warning of fungal and mycotoxin contamination in this herb. A total of 15 FCB samples were collected from five provinces in China, and the fungal communities in the FCB samples were analyzed via amplifying the internal transcribed spacer 2 region through the Illumina Miseq PE300 platform. Furthermore, we compared the differences in fungal community in five groups based on collection areas. Results showed that Ascomycota (41.58-99.66%) and Mucoromycota (0-57.42%) were dominant at the phylum level. Eurotiomycetes (8.49-63.93%), Eurotiales (8.49-63.53%), and Aspergillaceae (8.49-63.51%) were the most abundant at the class, order, and family levels. Aspergillus (8.49-63.41%), Rhizopus (0-57.42%), Fusarium (0-22.81%), Cladosporium (0.16-9.14%), and Alternaria (0.06-17.95%) were the main genera in FCB samples. A total of 34 fungal taxa were identified at the species level, including five potentially toxigenic fungi namely Penicillium brevicompactum, P. citrinum, P. oxalicum, Trichothecium roseum, and Aspergillus restrictus. The differences in fungal community between the five groups were observed. Our findings provide references for the safe utilization and quality improvement of FCB.

Analysis of Fungal Microbiomes in Edible Medicinal Morindae Officinalis Radix and Alpiniae Oxyphyllae Fructus Using DNA Metabarcoding.

Morindae Officinalis Radix (MOR) and Alpiniae Oxyphyllae Fructus (AOF) have been widely used as dietary supplements and traditional herbal medicines for centuries. Fungal and mycotoxin contamination in MOR and AOF has been reported recently. In this study, fungi in MOR and AOF are first investigated using DNA metabarcoding, and the differences in fungal microbiome between moldy and non-moldy samples are analyzed. The results show that Ascomycota is the most prevailing fungus at the phylum level in MOR and AOF with relative abundances of 49.53-94.32% and 14.81-81.85%, respectively. Penicillium (1.86-76.14%), Cladosporium (1.82-56.65%), and Trichoderma (0.12-19.71%) are the dominant genera in MOR. Penicillium (0.27-56.06%), Papiliotrema (0.04-51.71%), and Cladosporium (3.08-44.41%) are the dominant genera in AOF. Two potential toxigenic fungi were detected, namely, Trichoderma atroviride and Fusarium equiseti. Moreover, the differences in fungal communities between moldy and non-moldy samples were monitored. In conclusion, DNA metabarcoding can be used to assess the fungal microbiome in edible medicinal herbs, thereby providing a basis for ensuring food safety and drug efficacy.

Detection of Adulteration and Pesticide Residues in Chinese Patent Medicine Qipi Pill Using KASP Technology and GC-MS/MS.

Chinese patent medicines (CPMs) are of great value for the prevention and treatment of diseases. However, adulterants and pesticide residues in CPMs have become the "bottleneck" impeding the globalization of traditional Chinese medicine. In this study, 12 batches of commercially available Qipi pill (a famous CPM recorded in Chinese Pharmacopeia) from different manufacturers were investigated to evaluate their authenticity and quality safety. Considering the severely degraded DNA in CPMs, kompetitive allele specific PCR (KASP) technology combined with DNA mini-barcodes was proposed for the quality regulation of a large number of products in CPM market. The residues of four kinds of pesticides including pentachloronitrobenzene (PCNB), hexachlorocyclohexane (HCH), aldrin, and dichlorodiphenyltrichloroethane (DDT) were quantified using gas chromatography and tandem mass spectrometry (GC-MS/MS). The results indicated that in two of the 12 batches of Qipi pill, the main herbal ingredient Panax ginseng was completely substituted by P. quinquefolius, and one sample was partially adulterated with P. quinquefolius. The PCNB residue was detected in 11 batches of Qipi pill, ranging from 0.11 to 0.46 mg/kg, and the prohibited pesticide HCH was present in four samples. Both adulteration and banned pesticides were found in two CPMs. This study suggests that KASP technology combined with DNA mini-barcodes can be used for the quality supervision of large sample size CPMs with higher efficiency but lower cost. Our findings also provide the insight that pesticide residues in CPMs should be paid more attention in the future.

Assessment of the Microbiome and Potential Aflatoxin Associated With the Medicinal Herb Platycladus orientalis.

Platycladi Semen, which is derived from the dried ripe seed of Platycladus orientalis, has been used for the treatment of insomnia and constipation in China for 2000 years. However, it is susceptible to fungal and aflatoxin contamination under proper humidity and temperature during storage. Although aflatoxin contamination in Platycladi Semen has been reported preliminarily, few studies have been conducted on fungal infection and aflatoxin contamination simultaneously. Thus, this work aims to provide an in-depth understanding of fungal contamination in Platycladi Semen, and information on aflatoxin contamination. We focused on a comparison of the difference in fungal diversity between aflatoxin-contaminated and aflatoxin-free Platycladi Semen samples. First, aflatoxin levels in 11 Platycladi Semen samples, which were collected from local herbal markets in Shandong, Anhui, and Hebei provinces throughout China, were determined by IAC-HPLC-FLD, and positive confirmation of detected samples was performed by LC-MS/MS. The samples were divided into two groups, based on production or non-production of aflatoxin. We then used the Illumina MiSeq PE250 platform, and targeted the internal transcribed spacer two sequences to analyze the diversity and composition of the fungal microbiome, as well as to assess the presence of potential mycotoxin-producing fungi. Results showed that five samples were contaminated with aflatoxins, one of which exceeded the legal limits of Chinese Pharmacopeia Commission (2015). At the phylum level, the Ascomycota was the most dominant in all tested samples, with a relative abundance of 83.04-99.46%. Aspergillus (27.88-97.28%), Xerochrysium (0-28.49%), and Xeromyces (0-22.24%) were the three predominant genera. Furthermore, differences in fungal composition between the aflatoxin-contaminated and aflatoxin-free groups, as well as between different provinces were observed. A total of 74 species were identified, and four potential mycotoxin-producing fungi were detected in all samples, namely Aspergillus flavus, Aspergillus fumigatus, Fusarium poae, and Penicillium steckii. In conclusion, we report the great diversity of fungi associated with Platycladi Semen, highlight the risk to consumers of ingesting potent aflatoxin, and provide a reference for the safe application and quality improvement of Platycladi Semen.

Investigating the authenticity of Ophiopogonis Radix and its Chinese patent medicines by using a nucleotide signature.

ETHNOPHARMACOLOGICAL RELEVANCE: Ophiopogonis Radix (Maidong), derived from the dried root tuber of Ophiopogon japonicus (Thunb.) Ker Gawl., has been widely used in the treatment of chronic inflammatory and cardiovascular diseases. However, Ophiopogonis Radix is often adulterated with some species because of morphological similarities. Adulterants circulating in herbal markets are a latent threat to the clinical safety and consumers' interest. AIM OF THE STUDY: We aimed to develop a nucleotide signature for identification of Ophiopogonis Radix and its Chinese patent medicines. MATERIALS AND METHODS: A total of 255 ITS2 sequences representing 39 species and 4 varieties were used to develop a nucleotide signature of Ophiopogonis Radix. The nucleotide signature was used to investigate 17 commercial crude drugs and eight batches of Chinese patent medicines. RESULTS: A 69 bp nucleotide signature unique to Ophiopogonis Radix was found. The survey revealed that 2 of 17 crude drug samples were adulterants detected as Liriopes Radix (Shanmaidong). Fortunately, no adulterants were detected in the eight batches of Chinese patent medicines. CONCLUSIONS: The newly developed nucleotide signature could be efficiently applied to identify Ophiopogonis Radix and its Chinese patent medicines, aiding in the authentication, quality control, and supervision of processed products in herbal markets.

Characterizing fungal communities in medicinal and edible Cassiae Semen using high-throughput sequencing.

Cassiae Semen (CS) has been widely used as roasted tea and traditional Chinese medicine for decades. However, CS is easily contaminated by fungi and mycotoxins during pre-harvest and post-harvest process, thus posing a potential threat to consumer health. In this study, we used the Illumina MiSeq PE300 platform and targeted the internal transcribed spacer 2 sequences to survey the occurrence of fungi in raw and roasted CS samples. Results showed the fungal contamination in all 12 test samples. Ascomycota was the prevailing fungus at the phylum level, with the relative abundance of 66.50%-99.42%. At the genus level, Aspergillus, Cladosporium, and Penicillium were the most dominant genera, accounting for 0.66%-85.51%, 0.20%-29.11%, and 0.11%-32.92% of the fungal reads, respectively. A total of 68 species were identified, among which six potential toxigenic fungi belonging to Aspergillus, Penicillium, Candida, and Schizophyllum genera were detected. Moreover, differences in fungal communities were observed in raw and roasted CS samples. In conclusion, amplicon sequencing is feasible for analyzing fungal communities in CS samples, which provides a new approach to investigate the fungal contamination in edible-medicinal herb, thereby ensuring food safety and drug efficacy.

Detecting Schisandrae Chinensis Fructus and Its Chinese Patent Medicines with a Nucleotide Signature.

Schisandrae Chinensis Fructus (Wuweizi) is often adulterated with Schisandrae Sphenantherae Fructus (Nanwuweizi) in the herbal market. This adulteration is a threat to clinical treatment and safety. In this study, we aimed to develop a nucleotide signature for the identification of Wuweizi and its Chinese patent medicines based on the mini-DNA barcoding technique. We collected 49 samples to obtain internal transcribed spacer 2 (ITS2) sequences and developed a 26-bp nucleotide signature (5'-CGCTTTGCGACGCTCCCCTCCCTCCC-3') on the basis of a single nucleotide polymorphism (SNP) site within the ITS2 region that is unique to Wuweizi. Then, using the nucleotide signature, we investigated 27 batches of commercial crude drug samples labeled as Wuweizi and eight batches of Chinese patent medicines containing Wuweizi. Results showed that eight commercial crude drug samples were adulterants and one of the Chinese patent medicines contained adulterants. The nucleotide signature can serve as an effective tool for identifying Wuweizi and its Chinese patent medicines and can thus be used to ensure clinical drug safety.

Analysis of the Fungal Community in Ziziphi Spinosae Semen through High-Throughput Sequencing.

Ziziphi Spinosae Semen (ZSS) has been widely used in traditional Chinese medicine system for decades. Under proper humidity and temperature, ZSS is easily contaminated by fungi and mycotoxins during harvest, storage, and transport, thereby posing a considerable threat to consumer health. In this study, we first used the Illumina MiSeq PE250 platform and targeted the internal transcribed spacer 2 sequences to investigate the presence of fungi in moldy and normal ZSS samples collected from five producing areas in China. Results showed that all 14 samples tested were contaminated by fungi. Ascomycota was the dominant fungus at the phylum level, accounting for 64.36⁻99.74% of the fungal reads. At the genus level, Aspergillus, Candida, and Wallemia were the most predominant genera, with the relative abundances of 13.52⁻87.87%, 0.42⁻64.56%, and 0.06⁻34.31%, respectively. Meanwhile, 70 fungal taxa were identified at the species level. Among these taxa, three potential mycotoxin-producing fungi, namely, Aspergillusflavus, A. fumigatus, and Penicillium citrinum that account for 0.30⁻36.29%, 0.04⁻7.37%, and 0.01⁻0.80% of the fungal reads, respectively, were detected in all ZSS samples. Moreover, significant differences in fungal communities were observed in the moldy and normal ZSS samples. In conclusion, our results indicated that amplicon sequencing is feasible for the detection and analysis of the fungal community in the ZSS samples. This study used a new approach to survey the fungal contamination in herbal materials. This new approach can provide early warning for mycotoxin contamination in herbal materials, thereby ensuring drug efficacy and safety.

Inspecting the True Identity of Herbal Materials from Cynanchum Using ITS2 Barcode.

Cynanchum is a large genus with some important medicinal species in China. The medicinal species in Cynanchum are easily confused, leading to potential safety risks. In this study, the internal transcribed spacer 2 (ITS2) barcode was used to discriminate the medicinal plants in Cynanchum. The identifying capability of ITS2 was assessed using the specific genetic divergence, BLAST1, neighbor-joining (NJ) tree, maximum-likelihood (ML) tree, and single-nucleotide polymorphism (SNP) methods. Results indicated that the intra-specific genetic divergences of Cynanchum species were lower than their inter-specific genetic divergences. Of the 87 samples from 17 species, ITS2 showed a high identification efficiency of 90.8 and 87.4% at the species level through BLAST1 and the nearest distance methods. NJ tree and ML tree also demonstrated the suitability of ITS2 to differentiate Cynanchum species. Meanwhile, a stable SNP was found, and it could accurately authenticate Cynanchum paniculatum and Cynanchum atratum. Furthermore, we collected 64 commercial samples from three commonly used herbal medicines and evaluated the capability of ITS2 to survey their authentication. Of these samples, Cynanchi Atrati Radix et Rhizoma (Baiwei) showed a potential safety problem, and all the 11 test samples were adulterants. In conclusion, ITS2 can distinguish medicinal species in Cynanchum effectively, and its application could greatly improve the identification efficiency and accuracy of commercial herbal medicines in this genus.

A Nucleotide Signature for the Identification of American Ginseng and Its Products.

American ginseng (derived from Panax quinquefolius) is one of the most widely used medicinal herbs in the world. Because of its high price and increasing demand, there are many adulterants on the market. The proposed internal transcribed spacer 2 (ITS2) has been used to identify raw medicinal materials, but it is not suitable for the identification of Chinese patent medicine ingredients. Therefore, a short barcode for the identification of processed American ginseng and its corresponding Chinese patent medicines would be profitable. In this study, 94 samples of American ginseng and Asian ginseng were collected from all over the world. The ITS2 region was sequenced, and a nucleotide signature was developed based on one single nucleotide polymorphism (SNP) site unique to American ginseng. The nucleotide signature (atcactcctt tgcgggagtc gaggcgg) consists of 27 bases over the length of the ITS2 sequence (420 bp). Furthermore, we also designed primer pairs to amplify the nucleotide signature; the specific primer pair 4F/4R has been found to be unique to the ginseng species and capable of amplifying the nucleotide signatures from Chinese patent medicines and decoctions. We used the nucleotide signature method to inspect ginseng products in Chinese patent medicines; 24 batches of Chinese patent medicine from stores in Beijing were amplified and sequenced successfully. Using the double peaks at the SNP sites of the nucleotide signature, 5 batches were found to be counterfeits, and 2 batches were found to contain adulterants. Thus, this nucleotide signature, with only 27 bp, has broadened the application of DNA barcoding in identification of decoctions, Chinese patent medicines and other ginseng products with degraded DNA. This method can rapidly identify ginseng products and could also be developed as an on-site detection method.

An authenticity survey of herbal medicines from markets in China using DNA barcoding.

Adulterant herbal materials are a threat to consumer safety. In this study, we used DNA barcoding to investigate the proportions and varieties of adulterant species in traditional Chinese medicine (TCM) markets. We used a DNA barcode database of TCM (TCMD) that was established by our group to investigate 1436 samples representing 295 medicinal species from 7 primary TCM markets in China. The results indicate that ITS2 barcodes could be generated for most of the samples (87.7%) using a standard protocol. Of the 1260 samples, approximately 4.2% were identified as adulterants. The adulterant focused on medicinal species such as Ginseng Radix et Rhizoma (Renshen), Radix Rubi Parvifolii (Maomeigen), Dalbergiae odoriferae Lignum (Jiangxiang), Acori Tatarinowii Rhizoma (Shichangpu), Inulae Flos (Xuanfuhua), Lonicerae Japonicae Flos (Jinyinhua), Acanthopanacis Cortex (Wujiapi) and Bupleuri Radix (Chaihu). The survey revealed that adulterant species are present in the Chinese market, and these adulterants pose a risk to consumer health. Thus, regulatory measures should be adopted immediately. We suggest that a traceable platform based on DNA barcode sequences be established for TCM market supervision.

Internal transcribed spacer 2 barcode: a good tool for identifying Acanthopanacis cortex.

Acanthopanacis cortex has been used in clinical applications for a long time. Considering some historical and geographical factors, Acanthopanacis cortex is easily confused with other herbs in medicine markets, thereby causing potential safety issues. In this study, we used the internal transcribed spacer 2 (ITS2) barcode to identify 69 samples belonging to six species, including Acanthopanacis cortex and its adulterants. The nearest distance, single-nucleotide polymorphisms (SNPs), and neighbor-joining (NJ) tree methods were used to evaluate the identification ability of the ITS2 barcode. According to the kimura-2-parameter model, the intraspecific distance of Eleutherococcus nodiflorus ITS2 sequences ranged from 0 to 0.0132. The minimum interspecific distance between E. nodiflorus and E. giraldii was 0.0221, which was larger than the maximum intraspecific distance of E. nodiflorus. Three stable SNPs in ITS2 can be used to distinguish Acanthopanacis cortex and its closely related species. The NJ tree indicated that the Acanthopanacis cortex samples clustered into one clade, which can be distinguished clearly from the adulterants of this herb. A secondary structure of ITS2 provided another dimensionality to identify species. In conclusion, the ITS2 barcode effectively identifies Acanthopanacis cortex, and DNA barcoding is a convenient tool for medicine market supervision.

[Identification of Bupleurum chinense and B. longiradiatum based on ITS2 barcode].

In this study, ITS2 barcode was used to identify Bupleurum chinense and B. longiradiatum. The ITS2 regions of 48 samples were amplified and sequenced. The sequences obtained above were aligned and the K2P distances were calculated. We used three methods, BLAST1, nearest distance and phylogenetic tree (NJ-tree), to test the identification ability. The results showed that the maximum intraspecific genetic distance of B. chinense was 0.013, and the minimum interspecific genetic distance between B. chinense and B. longiradiatum was 0.049. The NJ-tree can easily identify B. chinense and B. longiradiatum. Therefore, the ITS2 barcode is suitable to identify B. chinense and B. longiradiatum.

[Identification of albiziae cortex, albiziae flos and their adulterants using ITS2 barcoding].

The ITS2 barcode was used to accurately identify Albiziae Cortex, Albiziae Flos and their adulterants in this study. A total of46 samples from Albiziae Cortex, Albiziae Flos and their adulterants were collected. The ITS2 regions were amplified and sequenced. Sequences were assembled using the CodonCode Aligner. The genetic distances of ITS2 region were calculated using MEGA 5.0. BLAST1, nearest distance and phylogenetic tree (NJ-tree) methods were used to assess the identification efficiency of the ITS2 barcode. The results revealed that the intraspecific genetic distances of Albizia julibrissin were lower than the interspecific genetic distances between A. julibrissin and its adulterants. The identification efficiency of ITS2 barcode using BLAST1 was 100%. The NJ-tree showed that A. julibrissin and their adulterants can be easily differentiated according to their monophyly. The ITS2 barcode is suitable to be as a barcode to identify Albiziae Cortex, Albiziae Flos and their adulterants.

A renaissance in herbal medicine identification: from morphology to DNA.

Numerous adverse reactions have arisen following the use of inaccurately identified medicinal plant ingredients, resulting in conditions such as aristolochic acid nephropathy and herb-induced poisoning. This problem has prompted increased global concern over the safety of herbal medicines. DNA barcoding, a technique aiming at detecting species-specific differences in a short region of DNA, provides a powerful new tool for addressing this problem. A preliminary system for DNA barcoding herbal materials has been established based on a two-locus combination of ITS2+psbA-trnH barcodes. There are 78,847 sequences belonging to 23,262 species in the system, which include more than 95% of crude herbal drugs in pharmacopeia, such as those of China, Japan, Korea, India, USA, and Europe. The system has been widely used in traditional herbal medicine enterprises. This review summarizes recent key advances in the DNA barcoding of medicinal plant ingredients (herbal materia medica) as a contribution towards safe and efficacious herbal medicines.

A fast SNP identification and analysis of intraspecific variation in the medicinal Panax species based on DNA barcoding.

Medicinal plants of the Panax genus belonging to Araliaceae family are well-known, rare plants used as tonics in traditional Chinese medicine and have been described in the Chinese Pharmacopoeia. Because of the high price and the huge human demand, these commercial products often contain adulterants. In this study, 377 sequences from four species were analyzed. Single nucleotide polymorphisms (SNPs) were detected and patterns of intragenomic variation in internal transcribed spacer 2 (ITS2) from the four Panax species were studied. Intraspecific variations were analyzed based on three typical DNA barcodings (ITS2, matK and psbA-trnH). Results from this study revealed that intraspecific genetic distances in Panax ginseng and Panax quinquefolius were quite low (0-0.002) and the multi-copy ITS2 could be considered a single locus in the genomes of these two species. Five stable SNPs were detected in ITS2 region to identify the Panax medicinal species. Considering the mixed powder of P. ginseng and P. quinquefolius, double peaks could be clearly examined at SNP positions and the height of the peaks could indicate the mixed ratio roughly. Our findings indicate that SNP-based molecular barcodes could be developed as a routine method for the identification of the Panax genus with closely related species and the mixed powder P. ginseng and P. quinquefolius.

Molecular identification of Corni Fructus and its adulterants by ITS/ITS2 sequences.

UNLABELLED: The DNA barcoding method was used to accurately and rapidly identify Corni Fructus and its adulterants. METHODS: Genomic DNA extracted from Corni Fructus and its adulterants were used as templates. The ITS (internal trascribed spacer) regions were amplified using polymerase chain reaction. Sequence assembly was performed using CodonCode Aligner V 3.5.4. Genetic distances were computed using MEGA V 5.0. Species identification was conducted using neighbor-joining (NJ) trees. RESULTS: The ITS sequence length of Corni Fructus was 659 bp. The average intra-specific genetic distance of Corni Fructus was 0.005, markedly lower than the inter-specific genetic distance between Corni Fructus and its adulterants (0.357). The ITS2 sequence length of Corni Fructus was 250 bp. No variation was found among the different samples. The interspecific genetic distance of ITS2 between Corni Fructus and its adulterants was 0.571. NJ trees and BLAST results indicated that Corni Fructus and its adulterants can be easily differentiated with monophyly. CONCLUSION: ITS/ITS2 regions can accurately and efficiently distinguish Corni Fructus and its adulterants. In addition, the results not only established the foundation for the clinical safety in the utilization of Corni Fructus, but also provided reference for molecular identification of other Chinese herbal medicine and Chinese herbal pieces.

[Principles for molecular identification of traditional Chinese materia medica using DNA barcoding].

Since the research of molecular identification of Chinese Materia Medica (CMM) using DNA barcode is rapidly developing and popularizing, the principle of this method is approved to be listed in the Supplement of the Pharmacopoeia of the People's Republic of China. Based on the study on comprehensive samples, the DNA barcoding systems have been established to identify CMM, i.e. ITS2 as a core barcode and psbA-trnH as a complementary locus for identification of planta medica, and COI as a core barcode and ITS2 as a complementary locus for identification of animal medica. This article introduced the principle of molecular identification of CMM using DNA barcoding and its drafting instructions. Furthermore, its application perspective was discussed.