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The root of Bupleurum marginatum var. stenophyllum (H. Wolff) Shan & Yin Li (Apiaceae), a new substitution for the popular Chinese medicinal material, Bupleuri Radix (Chai hu), is not easily distinguishable via traditional methods. The complete chloroplast genome sequence of B. marginatum var. stenophyllum was characterized using next-generation sequencing and the de novo assembly method. The complete genome was 155,576 bp in length and contained two inverted repeat (IR) regions of 26,311 bp, a large single-copy (LSC) region of 85,351 bp, and a small single-copy (SSC) region of 17,603 bp. It encoded 113 unique genes consisting of 79 protein-coding genes (PCGs), 30 transfer RNA genes, and four ribosomal RNA genes. Importantly, three genes (petB, petD and rps16) with small exon, and one trans-splicing gene (rps12) were correctly annotated. The overall GC content of the B. marginatum var. stenophyllum chloroplast genome is 37.7%. The phylogenetic analyses indicated that B. marginatum var. stenophyllum was closely related to B. marginatum. Moreover, many genetic information sites were available for distinguishing B. marginatum var. stenophyllum from the official ‘Chai hu’ plant sources, B. scorzonerifolium Willd. and B. chinense DC.

With the widespread use of traditional medicine around the world, the safety and efficacy of traditional herbal patent medicine have become an increasing concern to the public. However, it is difficult to supervise the authenticity of herbal materials in mixed herbal products according to the current quality standards, especially for traditional herbal patent medicine, with a distinct variance in the dosage of herbal materials. This study utilized the shotgun metabarcoding approach to analyze the biological ingredients of Fuke Desheng Wan (FKDSW), which is an effective traditional herbal product for the treatment of dysmenorrhea. Six herbal materials were collected, and a lab-made mock FKDSW sample was produced to establish a method for the authentication assessment of biological ingredients in traditional herbal patent medicine based on shotgun metabarcoding. Furthermore, four commercial FKDSW samples were collected to verify the practicality of the shotgun metabarcoding approach. Then, a total of 52.16 Gb raw data for 174 million paired-end reads was generated using the Illumina NovaSeq sequencing platform. Meanwhile, 228, 23, and 14 operational taxonomic units (OTUs) were obtained for the ITS2, matK, and rbcL regions, respectively, after bioinformatic analysis. Moreover, no differences were evident between the assembly sequences obtained via shotgun metabarcoding and their corresponding reference sequences of the same species obtained via Sanger sequencing, except for part of the ITS2 and matK assembly sequences of Paeonia lactiflora Pall., Saussurea costus (Falc.) Lipsch. and Bupleurum chinense DC. with 1–6 different bases. The identification results showed that all six prescribed ingredients were successfully detected and that the non-authentic ingredient of Bupleuri Radix (Chaihu, Bupleurum chinense DC. or Bupleurum scorzonerifolium Willd.) was found in all the commercial samples, namely Bupleurum falcatum L. Here, 25 weed species representing 16 genera of ten families were detected. Moreover, 26 fungal genera belonging to 17 families were found in both lab-made and commercial FKDSW samples. This study demonstrated that the shotgun metabarcoding approach could overcome the biased PCR amplification and authenticate the biological ingredients of traditional herbal patent medicine with a distinct variance in the dosage of the herbal materials. Therefore, this provides an appropriate evaluation method for improving the safety and efficacy of traditional herbal patent medicine.

As one of the high-incidence diseases in the world, pharyngitis seriously affects the lives of those with the condition. Qingguo Wan is a herbal medicine used for treating pharyngitis, and its quality evaluation is currently only accomplished via traditional identification. However, precise identification becomes challenging with fake products on the market or fungal contamination during the production process. This study used the Illumina NovaSeq platform for targeting the ITS2, psbA-trnH, matK, and rbcL sequences to survey the species composition of lab-made and commercial samples. The results showed that a total of 34.56 Gb of raw data that was obtained represented more than 0.23 billion reads. After assembly, annotation, and operational taxonomic unit clustering, 103, 12, 10, and 12 OTUs were obtained, which belonged to the ITS2, psbA-trnH, matK, and rbcL sequences of the mock lab-made and commercial samples. The analytical results indicated that the sequences of all the prescription ingredients were successfully obtained in the two lab-made samples. The positive control medicinal Panax quinquefolius L. sequence was obtained in HSZY175, while Scutellaria baicalensis Georgi, Lonicera japonica Thunb. Menispermum dauricum DC. and Paeonia lactiflora Pall. were detected in the three commercial samples. The detection results of the other four herbs in different fragments were not all the same. In addition, a total of 28 fungi OTUs, representing 19 families and 20 genera, were obtained from both the commercial and mock lab-made samples. Aspergillus, Cladosporium, and Penicillium dominated among the 20 genera. This study demonstrated that the shotgun metabarcoding method is a powerful tool for the molecular identification of the biological ingredients in Qingguo Wan. It can be used to effectively supplement traditional methods while providing a new technique for the quality evaluation of Qingguo Wan.

Traditional herbal patent medicine typically consists of multiple ingredients, making it challenging to supervise contamination by impurities and the improper use of raw materials. This study employed shotgun metabarcoding for the species identification of biological ingredients in traditional herbal patent medicine, Wuhu San. The five prescribed herbal materials found in Wuhu San were collected, and their reference sequences were obtained by traditional DNA barcoding using Sanger sequencing. Two lab-made and three commercial Wuhu San samples were collected, and a total of 37.14 Gb of shotgun sequencing data was obtained for these five samples using the Illumina sequencing platform. A total of 1,421,013 paired-end reads were enriched for the Internal Transcribed Spacer 2 (ITS2), psbA and trnH intergenic spacer region (psbA-trnH), maturase k (matK), and ribulose-1, 5-bisphosphate carboxylase (rbcL) regions. Furthermore, 80, 11, 9, and 8 operational taxonomic units were obtained for the ITS2, psbA-trnH, matK, and rbcL regions, respectively, after metagenomic assembly, annotation, and chimeric detection. In the two lab-made mock samples, all labeled ingredients in the Wuhu San prescription were successfully detected, and the positive control, Panax quinquefolius L., was detected in the HSZY172 mock sample. Three species, namely Angelica sinensis (Oliv.) Diels, Saposhnikovia divaricata (Turcz. ex Ledeb.) Schischk., and Carthamus tinctorius L., belonging to three labeled ingredients, Angelicae Sinensis Radix (Danggui), Saposhnikoviae Radix (Fangfeng), and Carthami Flos (Honghua), were detected in the three commercial samples. Angelica dahurica (Hoffm.) Benth. & Hook. f. ex Franch. & Sav., the original Angelicae Dahuricae Radix (Baizhi) species, was only detected in WHS003. Arisaema erubescens (Wall.) Schott, Arisaema heterophyllum Blume, or Arisaema amurense Maxim., the original Arisaematis Rhizoma (Tiannanxing) species, were not detected in any of the commercial samples, which could be attributed to the fact that this medicinal material underwent extensive processing. In addition, the Saposhnikovia divaricata adulterant was detected in all the commercial samples, while 24 fungal genera, including Aspergillus, were identified in both the lab-made and commercial samples. This study showed that shotgun metabarcoding provided alternative strategy and technical means for identifying prescribed ingredients in traditional herbal patent medicine and displayed the potential to effectively complement traditional methods.

Increased public awareness of nutritional and health issues has resulted in the increasing consumption of food and herbal products made from the root of Pueraria montana var. lobata (Willd.) Maesen & S. M. Almeida ex Sanjappa & Predeep (kudzu vine) and P. montana var. thomsonii (Benth.) M. R. Almeida. The famous herbal medicine Yufeng Ningxin, which is used to treat cardiovascular diseases, can be legally produced only using P. montana var. lobata. However, precise identification at the subspecies level is usually challenging when these products’ ingredients lose their morphological characteristics after deep processing. Here, six herbarium specimens, 21 expert-identified original plant samples, 30 raw material samples, 10 food products and 12 herbal products were collected to test the subspecies-level authentication abilities of ITS2 sequences. The results showed that ITS2 sequences can distinguish P. montana var. lobata from P. montana var. thomsonii with stable single nucleotide polymorphism (SNP) sites. A total of 93.3% of raw material samples were consistent with the markings on their labels, but only 50% of Gegen Powder samples were made from P. montana var. lobata. High-quality ITS2 sequences were successfully obtained from nine of the 12 herbal products using Sanger sequencing. Substitution and fungal contamination were detected in 3 herbal products by further DNA metabarcoding, even though thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) tests verified that the products met existing quality standards. This study demonstrated that DNA barcoding is a powerful tool for the identification of P. montana var. lobata and P. montana var. thomsonii at the subspecies level, and we conclude that DNA barcodes can be broadly applied to trace the raw materials of food and herbal products.

The efficacy and safety of traditional Chinese medicine are global concerns. Here, we selected a traditional medicinal powder Ruyi Jinhuang San as a research subject and employed DNA metabarcoding technology to identify its ingredients. First, 161 specimens constituting the ingredients of Ruyi Jinhuang San and their adulterants were collected. In addition, 154 ITS2 sequences belonging to the closed related species of these ingredients were extracted from the published literatures. First, a DNA barcode reference library of Ruyi Jinhuang San, comprising 119 species belonging to 10 genera, was constructed. Second, three different batches of Ruyi Jinhuang San were collected, and ITS2 was selected as a DNA barcode. PCR products were sequenced with the Illumina HiSeq sequencing platform using PE250 libraries. Taxonomy assignments were completed with the help of the above-mentioned DNA barcode reference library. The results indicated that all ingredients of Ruyi Jinhuang San can be successfully detected except Houppo. The medicine Cangzu had been contaminated with Atractylodes koreana . The medicine Tiannanxing had been substituted with Pinellia pedatisecta . The contamination and substitution of medicinal ingredients are potential threats to consumer safety and medicinal effectiveness. The authentication results of different samples are repeatable and stable. In conclusion, DNA metabarcoding is a valuable technology to validate the efficacy and safety of traditional Chinese medicine and has the potential to be a new method for identifying traditional Chinese medicine ingredients.

Seed is not only the main reproductive organ of most of herbal plants but also an important part of Traditional Chinese Medicine (TCM). Seed TCMs possess important medicinal properties and have been widely used as components of pharmaceutical products. In parallel with the increasing popularity and accessibility of seeds as medicinal products in recent years, numerous substitutes and adulterants have also appeared on the market. Due to the small volume and similar appearances of many seed TCMs, they are very difficult to accurately identify the constituent plant species through organoleptic methods. Usage of the wrong herb may be ineffective or may worsen the condition and even cause death. Correct identification of seed herbal medicines is therefore essential for their safe use. Here, we acquired 177 ITS2 sequences and 15 psbA-trnH sequences from 51 kinds of seed TCMs belonging to 64 species that have been described in the Chinese Pharmacopoeia. Tree-building analysis showed that the ITS2 sequences of 48 seed TCMs can be differentiated from each other, and they formed distinct, non-overlapping groups in the maximum-likelihood tree. Furthermore, all of the sequences acquired in this study have been submitted to the public DNA barcoding system for herbal medicine, and this integrated database was used to identify 400 seed TCM samples purchased from medicinal markets, drug stores, and the Internet, enabling the identification of 7.5% of the samples as containing non-declared species. This study provides a brief operating procedure for the identification of seed TCMs found in herbal medicine. In the future, researchers and traditional herbal medicine enterprises can use this system to test their herbal materials.

Herbal drug authentication is an important task in traditional medicine; however, it is challenged by the limitations of traditional authentication methods and the lack of trained experts. DNA barcoding is conspicuous in almost all areas of the biological sciences and has already been added to the British pharmacopeia and Chinese pharmacopeia for routine herbal drug authentication. However, DNA barcoding for the Korean pharmacopeia still requires significant improvements. Here, we present a DNA barcode reference library for herbal drugs in the Korean pharmacopeia and developed a species identification engine named KP-IDE to facilitate the adoption of this DNA reference library for the herbal drug authentication. Using taxonomy records, specimen records, sequence records, and reference records, KP-IDE can identify an unknown specimen. Currently, there are 6,777 taxonomy records, 1,054 specimen records, 30,744 sequence records (ITS2 and psbA-trnH) and 285 reference records. Moreover, 27 herbal drug materials were collected from the Seoul Yangnyeongsi herbal medicine market to give an example for real herbal drugs authentications. Our study demonstrates the prospects of the DNA barcode reference library for the Korean pharmacopeia and provides future directions for the use of DNA barcoding for authenticating herbal drugs listed in other modern pharmacopeias.

Kampo is the general designation for traditional Japanese herbal medicines, which are recognized as official medicines and listed in the Japanese pharmacopoeia (JP). In most cases, it is difficult to identify the crude drug materials to species level using only traditional identification methods. We report the first online DNA barcode identification system, which includes standard barcode sequences from approximately 95% of the species recorded in the JP (16th edition). This tool provides users with basic information on each crude drug recorded in the JP, DNA barcoding identification of herbal material, and the standard operating procedure (SOP) from sampling to data analysis. ITS2 sequences (psbA-trnH was an alternative when ITS2 could not be amplified) were generated from a total of 576 samples to establish the database. An additional 100 samples (from different medicinal parts, from both single origin and multiple origins and from both retailers and the planting base) were identified using the system. A total of 78% of the test samples were identified as the species listed on their label. This system establishes a model platform for other pharmacopeias from countries like China, Korea, the US and the European Union, for the safe and effective utilization of traditional herbal medicines.

Rheum tanguticum, a source plant for the traditional Chinese medicine Dahuang (rhubarb), is an endangered species. In this study, we first assessed genetic diversity using 102 internal transcribed spacer (ITS) sequences cloned from one population of R. tanguticum on the Qinghai-Tibet Plateau. In total, 1773 polymorphic sites were identified, including 707 sites in the ITS1 region, 773 sites in the ITS2 region, and 293 sites on the 5.8S gene. Overall, transitions were the most common type of mutation and represented 77% of all base substitutions found within the ITS regions of R. tanguticum. We found that the average intra-specific genetic distance was 0.1260±0.0755 and ranged from 0 to 0.3618. Furthermore, a neighbor-joining phylogenetic tree constructed using these sequences strongly supported the conclusion that abundant intra-specific variation exists within the ITS regions of R. tanguticum. Interestingly, we found that eightyseven clones of R. tanguticum had high similarity to the ITS sequence of R. rhabarbarum and ten clones had high similarity to the ITS sequence of R. officinale; however, five clones of R. tanguticum had similarity to Fagopyrum esculentum. Therefore, we observed a significant amount of ITS sequence variation at both the intra-genomic and intra-specific levels in R. tanguticum. Based on these results, we suggest that in situ conservation is preferable for R. tanguticum protection.

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.

DNA barcoding is a novel molecular identification method that aids in identifying traditional Chinese materia medica using traditional identification techniques. However, further study is needed to assess the stability and accuracy of DNA barcoding. Flos Lonicerae Japonicae, a typical medicinal flower, is widely used in China, Korea, and other Southeast Asian countries. However, Flos Lonicerae Japonicae and its closely related species have been misused and traded at varying for a wide range of prices. Therefore, Flos Lonicerae Japonicae must be accurately identified. In this study, the ITS2 andpsbA-trnHregions were amplified by polymerase chain reaction (PCR). Sequence assembly was performed using CodonCode Aligner V 3.5.4. The intra- versus inter-specific variations were assessed using six metrics and “barcoding gaps.” Species identification was conducted using BLAST1 and neighbor-joining (NJ) trees. Results reveal that ITS2 andpsbA-trnHexhibited an average intraspecific divergence of 0.001 and 0, respectively, as well as an average inter-specific divergence of 0.0331 and 0.0161. The identification efficiency of ITS2 andpsbA-trnHevaluated using BLAST1 was 100%. Flos Lonicerae Japonicae was formed into one clade through the NJ trees. Therefore, Flos Lonicerae Japonicae can be stably and accurately identified through the ITS2 andpsbA-trnHregions, respectively.

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 potential DNA barcode, ITS2, was studied to discriminate herbal materials to confirm their identities and ensure their safe application in pharmaceuticals. Here, a total of 4385 samples of 2431 species were collected, and these samples are from 61 commonly used herbs and their closely related species or adulterants. Based on assessments of the extent of genetic divergence, the DNA barcoding gap and the ability for species discrimination, our results suggest that ITS2 is a powerful tool for distinguishing herbs. For the first dataset including 61 herbs, ITS2 correctly identified 100 % of them. For the second dataset containing 51 herbs and their 2382 closely related species, ITS2 could discriminate correctly 48 herbs from their closely related species. For the third dataset comprising 34 herbs and their 111 adulterants, ITS2 could distinguish successfully all the herbs from their adulterants. In conclusion, the ITS2 region is an efficient marker for the authentication of herbal materials, and our study will accelerate the process of the application of the DNA barcoding technique in differentiating herbs.