[Research progress in original species identification in industry chain of Rhei Radix et Rhizoma].

Rhei Radix et Rhizoma is a kind of commonly used Chinese medicinal materials. Due to the overharvesting, the wild resource is endangering. Large market demand caused severely adulterant of commercial Rhei Radix et Rhizoma medicinal materials and decoction pieces. This manuscript reviewed the advances of the original species authentication in the industrial chain of Rhei Radix et Rhizoma during the latest decade, including characteristics and microscopic features, phytochemical analysis on anthraquinones, and molecular authentication based on DNA barcoding. Accordingly, an original species authentication route for the industrial chain of Rhei Radix et Rhizoma was summarized:(1)the identification of seeds and seedlings by DNA barcoding;(2) the selection of high variable sites based on the chloroplast genome;(3)biomonitoring of the Rhei Radix et Rhizoma medicinal materials and decoction pieces by two-dimensional DNA barcode;(4)traceability of Chinese patent medicines by third-generation sequencing. In conclusion, the combination of molecular identification and traditional identification methods provides a new idea for the identification of the original species of Rhei Radix et Rhizoma in the industrial chain and a essential guidance for the research of drug safety and efficacy of Rhei Radix et Rhizoma.

[Identification of water buffalo horn and its adulterants using COI barcode].

Bubali cornu (water buffalo horn) has been used as the substitute for Cornu rhinoceri asiatici (rhino horn) in clinical applications, and is the essential ingredient of Angong Niuhuang Wan. In recent years, there are a number of adulterants on the commercial herbal medicine markets. An efficient tool is required for species identification. In this study, 155 Bubali cornu samples have been taken from original animals and collected from commercial herbal medicine markets. 153 COI sequences have been successfully obtained from 155 samples through DNA extraction, PCR amplification, bidirectional sequencing and assembly. 93 COI sequences have been added to the DNA barcoding database of traditional Chinese animal medicine after validation using DNA barcoding GAP and tree-based methods. The species identification of the 62 commercial Bubali cornu medicines has been accomplished on the DNA barcoding system for identifying herbal medicine using the updated animal medicine database (www.tcmbarcode.cn). Except two samples failed to obtain COI sequences, 54.8% of the commercial Bubali cornu medicines were water buffalo horns and 29% were yak horns. Our results showed that yak horn was the major adulterant of Bubali cornu and the DNA barcoding method may accurately discriminate Bubali cornu and their adulterants. Therefore, we recommend that supervision on the herbal medicine markets should be strengthened with this new method to warren the effectiveness of herbal medicines.

[Dictamni Cortex powder-induced liver injury based on integrated evidence chain].

A typical clinical case of taking Dictamni Cortex(Baixianpi) powder was analyzed to study liver damage caused by Dictamni Cortex. Liver damage was diagnosed according to the integrated evidence chain method recommended by the Guideline for Diagnosis and Treatment of Herb-Induced Liver Injury. By analyzing clinical history and biochemistry and imaging examinations, underlying diseases, such as viral hepatitis, autoimmune liver disease and alcoholic liver disease, were excluded. Through the investigation of medication history, we made it clear that the patient only took Dictamni Cortex powder during the period, and thus suspected that the liver injury was induced by Dictamni Cortex. Furthermore, the quality of the drug was tested, and the results showed it was consistent with the quality standard of Chinese Pharmacopoeia. DNA barcoding showed that the drug was 100% similar with Dictamnus dasycarpus. Moreover, exogenous harmful substances and chemical drug additions were tested, and the results showed that the content of heavy metal, pesticide residues and microbial toxin were consistent with the required standards, and no chemical drug additions were found in Agilent Fake TCM-Drugs database. In summary, we confirmed that the clinical case of drug-induced liver injury was induced by D. dasycarpus with the dose of 15 g•d⁻¹, which exceeded the prescribed amount of Chinese Pharmacopoeia. According to the Guideline for Diagnosis and Treatment of Herb-Induced Liver Injury, the case of drug-induced liver injury induced by D. dasycarpus was confirmed, which provided a direct and reliable evidence for the study of risk of liver injury induced by D. dasycarpus and its relevant preparations.

[Identification of Bombyx Batryticatus based on DNA barcoding technology].

To identify the commercial medicinal materials of Bombyx Batryticatus, two-dimensional DNA barcode was used to construct the "Internet Plus" identification system for Chinese medicine, which should benefit the cross-platform communication of DNA barcode information. Bombyx Batryticatus contained Bombyx mori Linnaeus and Beauveria bassiana (Bals.) Vuillant. Both COI and ITS sequences were obtained via PCR amplification for total genomic DNA extracted from raw materials using the animal genomic DNA kit, while only ITS but no COI sequences was obtained when using the plant genomic DNA kit. The ITS sequences obtained using the animal genomic DNA kit were consistent with those using plant genomic DNA kit. The medicinal materials yielded COI sequences and identified as B. mori. According to analysis of ITS sequences, the main species of the medicinal materials were identified as B. bassiana and few were identified as other fungi. NJ trees analysis based on ITS sequences suggests that it can be easily distinguished from other fungi. Our results showed that total genomic DNA of B. mori and B. bassiana was extracted simultaneously using the animal genomic DNA kit, which could effectively solve the problem in species identification of animal and fungi mixture materials. COI and ITS regions as DNA barcodes can stably and accurately identify Bombyx Batryticatus. The "Internet Plus" two-dimensional DNA barcode system will promote the standardization and normalization of Chinese medicinal materials market.

[Herbgenomics].

Traditional Chinese medicine (TCM) has contributad greatly to improving human health. However, the biological characteristics and molecular mechanisms of TCM in the treatment of human diseases remain largely unknown. Genomics plays an important role in modern medicine and biology. Here, we introduce genomics and other related omics to the study of herbs to propose a new discipline, Herbgenomics, that aims to uncover the genetic information and regulatory networks of herbs and to clarify their molecular mechanisms in the prevention and treatment of human diseases. Herbgenomics includes herbal structural genomics, functional genomics, transcriptomics, proteomics, metabonomics, epigenomics and metagenomics. Genomic information, together with transcriptomic, proteomic, and metabolomic data, can therefore be used to predict secondary metabolite biosynthetic pathways and their regulation, triggering a revolution in discovery-based research aimed at understanding the genetics and biology of herbs. Herbgenomics provides an effective platform to support chemical and biological analyses of complex herbal products that may contain more than one active component. Herbgenomics is now being applied to many areas of herb related biological research to help understand the quality of traditional medicines and for molecular herb identification through the establishment of an herbal gene bank. Moreover, functional genomics can contribute to model herb research platforms, geoherbal research, genomics-assisted herb breeding, and herbal synthetic biology, all of which are important for securing the future of medicinal plants and their active compounds. In addition, Herbgenomics will facilitate the elucidation of the targets and mechanism of herbs in disease treatment and provide support for personalized precise medicine.Herbgenomics will accelerate the application of cutting-edge technologies in herbal research and provide an unprecedented opportunity to revolutionize the use and acceptance of traditional herbal medicines.

[Cloning and bioinformatics analysis of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase（PcHDR1）gene in Phlegmarirus carinatus].

The open reading frame of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase (HDR) was cloned from Phlegmarirus carinatus by RT-PCR method and the sequence was analyzed by bioinformatics tools. After searching the transcriptome dataset of P. carinatus, one unique sequence encoding 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase was discovered. The primers were designed according to the cDNA sequence of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase from the dataset. And then, the open reading frame (ORF) of 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase, named as PcHDR1 (GenBank Accession number：JQ957845), was cloned by RT-PCR strategy with the template of mixed RNA extracted from roots, stem and leaf of P. carinatus. The bioinformatic analysis of this gene and its corresponding protein was performed. The ORF of PcHDR1 consisted of 1 437 base pairs (bp), encoding one polypeptide with 478 amino acids. The sequence comparison showed that PcHDR1 is closest with GbHDR (Ginkgo biloba),and the sequence homology was up to 78%. Bioinformatics prediction and analysis indicated that PcHDR1 protein contained a conserved domain of LytB, without transmembrane region and signal peptides. This study cloned and analyzed 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate reductase from P. carinatus. The result will provide a foundation for exploring the function of PcHDR1 involved in terpene biosynthesis in P. carinatus plants.

[Identification of Notopterygium seeds using DNA barcoding method].

In order to guarantee the species correction of Notopterygium seeds, a molecular identification method with ITS2 as DNA barcode has been verified. In this study, 27 samples of Notopterygium seeds were collected from the main producing area of Notopterygium. The morphological characteristics of the Notopterygium seeds were firstly surveyed. Then the DNA extraction, PCR amplification, DNA sequencing and DNA assembly were carried out. The species identification for a Notopterygium seed was implemented through distance method, NJ-tree method and the DNA barcoding system for traditional Chinese medicine (www.tcmbarcode.cn). The results showed that the seeds of N. incisum and N. franchetii had similar morphological characteristics and were difficult to distinguish clearly based on morphological descriptions. With the results of molecular identification, 24 samples were genuine including 13 N. incisum seeds samples and 11 N. franchetii genuine seeds samples. In conclusion, DNA barcode technology can accurately and efficiently identify the species of Notopterygium seeds. Furthermore, this study will provide a new method for germplasm resources identification of medicinal materials and supplies some guidelines for establishing Chinese herbal seeds and seedlings quality standards.

[Identification of medicinal plant Dendrobium based on the chloroplast psbK-psbI intergenic spacer].

In this paper, the chloroplast psbK-psbI intergenic spacers of 18 species of Dendrobium and their adulterants were amplified and sequenced, and then the sequence characteristics were analyzed. The sequence lengths of chloroplast psbK-psbI regions of Dendrobium ranged from 474 to 513 bp and the GC contents were 25.4%-27.6%. The variable sites were 71 while the informative sites were 46. The inter-specific genetic distances calculated by Kimura 2-parameter (K2P) of Dendrobium were 0.006 1-0.058 1, with an average of 0.028 4. The K2P genetic distances between Dendrobium species and Bulbophyllum odoratissimum were 0.093 2-0.120 4. The NJ tree showed that the Dendrobium species can be easily differentiated from each other and 6 samples of the inspected Dendrobium species were identified successfully through sequencing the psbK-psbI intergenic spacer. Therefore, the chloroplast psbK-psbI intergenic spacer can be used as a candidate marker to identify Dendrobium species and its adulterants.

[Identification of antler powder components based on DNA barcoding technology].

In order to authenticate the components of antler powder in the market, DNA barcoding technology coupled with cloning method were used. Cytochrome c oxidase subunit I (COI) sequences were obtained according to the DNA barcoding standard operation procedure (SOP). For antler powder with possible mixed components, the cloning method was used to get each COI sequence. 65 COI sequences were successfully obtained from commercial antler powders via sequencing PCR products. The results indicates that only 38% of these samples were derived from Cervus nippon Temminck or Cervus elaphus Linnaeus which is recorded in the 2010 edition of "Chinese Pharmacopoeia", while 62% of them were derived from other species. Rangifer tarandus Linnaeus was the most frequent species among the adulterants. Further analysis showed that some samples collected from different regions, companies and prices, contained adulterants. Analysis of 36 COI sequences obtained by the cloning method showed that C. elaphus and C. nippon were main components. In addition, some samples were marked clearly as antler powder on the label, however, C. elaphus or R. tarandus were their main components. In summary, DNA barcoding can accurately and efficiently distinguish the exact content in the commercial antler powder, which provides a new technique to ensure clinical safety and improve quality control of Chinese traditional medicine

[Comparative studies on codon usage bias of Ganoderma lucidum based on analysis of genomic and transcriptomic data].

Codon usage bias is an important characteristic of genetic information transfer in organisms. Analysis of codon usage bias of different species is important for understanding the rules on genetic information transfer. The previous method for analysis of codon usage bias is mainly based on genomic data. However, this method is greatly limited, because the genome sequences of higher organisms are still not available up to now. In this study, we found that we could obtain the same optimal codons of Ganoderma lucidum (Curtis: Fr.) P. Karst based on its whole genomic data or large-scale transcriptomic data from its liquid-cultured hyphae, primordium and fruiting body, separately. This result indicated the feasibility to understand the codon usage bias based on the large-scale transcriptomic data. By calculating the proportion of rare codons of Escherichia coli and Saccharomyces cerevisiae in 26 terpene synthases (TS) of G. lucidum, we found that the rare codons of S. cerevisiae have a higher proportion in TS genes, while the rare codons of E. coli have relatively lower, suggesting that the TS genes of G. lucidum are possibly more difficult to be expressed in S. cerevisiae than in E. coli. Chemical synthesis of TS genes according to the yeast optimal codons will be an effective way to solve the problem on the mismatch of gene codon bias between the foreign genes and the host strain.

[RNA-Seq and genuine traditional Chinese medicine].

RNA-Sequencing (RNA-Seq) is a newly-developed method in transcriptome research, it can afford more accurate transcription information and be more quickly by using Next-generation Sequencing (NGS) technology. RNA-Seq has been widely used in various biological fields. Genuine traditional Chinese medicines (TCM), with good quality and therapeutic effect, were always praised highly and used by famous physicians. The geo-herbalism formation of TCM is based on the product of the gene expression at specific space and time. So it has been a research hotspot to analyze the mechanism of biosynthesis through RNA-Seq in the study on the secondary metabolism of medicinal plant. This article mainly illustrates the RNA-Seq and its advantages, it also discusses the potential application in genuine TCM, and it can provide useful information for other researchers.

[Progress in the study of Velvet and LaeA proteins and their relation to the development and bioactive compounds in medicinal fungi].

The medicinal fungi, which are of great importance in traditional medicine, are facing the problems of wild resources scarcity and low concentration of bioactive compounds. Velvet family and LaeA global regulator play a vital role in secondary metabolism and developmental programs, which are found in a wide variety of fungi ranging from Chytridiomycota to Basidiomycota. This review elaborates the structures and functions between Velvet family and LaeA protein. The Velvet family which shares the Velvet protein domain, including VeA (Velvet), VelB (Velvet like B), VosA (viability of spores A) and VelC (Velvet like C), acts on the regulation function is secondary metabolism and developmental programs such as asexual and sexual development. Furthermore, the function is affected by environmental factors such as light and temperature. LaeA protein which owns S-adenosylmethionine-dependent methyltransferase domain, coordinately regulates development and secondary metabolism by regulating and modifying the Velvet proteins. The regulation of LaeA is mediated by light receptor proteins. Therefore, clarifying the mechanism of Velvet and LaeA proteins in medicinal fungi will pave the way for nurturing medicinal fungi and improving production of bioactive compounds.

[Research progress of the regulation on active compound biosynthesis by the bHLH transcription factors in plants].

Transcription factor is one of the key factors in the regulation of gene expression at the transcriptional level. It plays an important role in plant growth, active components biosynthesis and response to environmental change. This paper summarized the structure and classification of bHLH transcription factors and elaborated the research progress of bHLH transcription factors which regulate the active components in plants, such as flavonoids, alkaloids, and terpenoids. In addition, the possibility of increasing the concentration of active substances by bHLH in medicinal plants was assessed. The paper emphasized great significance of model plants and multidisciplinary research fields including modern genomics, transcriptomics, metabolomics and bioinformatics, providing the contribution to improve the discovery and function characterization of bHLH transcription factors. Accelerating the research in the mechanism of bHLH transcription factors on the regulation of active components biosynthesis will promote the development of breeding and variety improvement of Chinese medicinal materials, also ease the pressure of resources exhaustion of traditional Chinese medicine home and abroad.

[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.

[DNA barcoding identification between arisaematis rhizoma and its adulterants based on ITS2 sequences].

Fifty-eight samples belonging to 7 species of Arisaematis Rhizoma and its adulterants were collected. The ITS2 locus was employed as a DNA barcode and amplified, sequenced and assembled for all of the collected samples. Then, ITS2 sequences have been annotated using HMM-based method. The intra- and inter-specific variations were calculated and NJ tree was constructed using MEGA 6.0 software. The results showed that inter-specific K2P distances were significantly larger than intra-specific distances for all of the three origin species of Arisaematis Rhizoma. Furthermore, three origin species, Arisaema amurense, A. erubescens and A. heterophyllum, can be respectively formed to be a single branch with high bootstrap values. It is concluded that ITS2 can be used to correctly identify Arisaematis Rhizoma from its adulterants and the application of ITS2 in the identification of traditional Chinese medicine has an important prospective.

[Molecular identification of aucklandiae radix, vladimiriae radix, inulae radix, aristolochiae radix and kadsurae radix using ITS2 barcode].

In order to identify Aucklandiae Radix, Vladimiriae Radix, Inulae Radix, Aristolochiae Radix and Kadsurae Radix using ITS2 barcodes, genomic DNA from sixty samples was extracted and the ITS2 (internal transcribed spacer) regions were amplified and sequenced. The genetic distances were computed using MEGA 5.0 in accordance with the kimura 2-parameter (K2P) model and the neighbor-joining (NJ) phylogenetic tree was constructed. The results indicated that for Aucklandiae Radix (Aucklandia lappa), Vladimiriae Radix (Vladimiria souliei and V. souliei var. cinerea), Inulae Radix (Inula helenium), Aristolochiae Radix (Aristolochia debilis) and Kadsurae Radix (Kadsura longipedunculata), the intra-specific variation was smaller than inter-specific one. There are 162 variable sites among 272 bp after alignment of all ITS2 sequence haplotypes. For each species, the intra-specific genetic distances were also smaller than inter-specific one. Furthermore, the NJ tree strongly supported that Aucklandiae Radix, Vladimiriae Radix, Inulae Radix, Aristolochiae Radix and Kadsurae Radix can be differentiated. At the same time, V. souliei (Dolomiaea souliei) and V. souliei var. cinerea( D. souliei var. cinerea) belonging to Vladimiriae Radix were clearly identified. In conclusion, ITS2 barcode could be used to identify Aucklandiae Radix, Vladimiriae Radix, Inulae Radix, Aristolochiae Radix and Kadsurae Radix. Our study may provide a scientific foundation for clinical safe use of the traditional Chinese medicines.

[Identification of peucedani radix, peucedani decursivi radix and its adulterants using ITS2 sequence].

In order to identify Peucedani Radix, Peucedani Decursivi Radix and their adulterants, the internal transcribed spacer 2 (ITS2) regions of Peucedani Radix, Peucedani Decursivi Radix and their adulterants were amplified and bidirectionally sequenced based on the Principles for Molecular Identification of Traditional Chinese Materia Medica Using DNA Barcoding, which has been promulgated by Chinese Pharmacopoeia Commission. Sequences were analyzed and assembled by Codon Code Aligner V3. 7.1. The relevant data were analyzed by MEGA 5. 0. Species identification analyses were performed by using the nearest distance methods and neighbor-joining (NJ) methods. The result showed that the ITS2 sequence lengths of Peucedani Radix were 229-230 bp and the average intra-specific genetic distances were 0.005. The ITS2 sequence lengths of Peucedani Decursivi Radix were 227 bp and the sequences contained no variation site. The average inter-specific K2P genetic distance of Peucedani Radix, Peucedani Decursivi Radix and their adulterants species were 0.044 and 0.065 respectively. The minimum inter-specific divergence is larger than the maximum intra-specific divergence of Peucedani Decursivi Radix. The nearest distance methods and NJ trees results indicated that Peucedani Radix, Peucedani Decursivi Radix and their adulterants species could be identification clearly. The ITS2 regions can stably and accurately distinguish Peucedani Radix, Peucedani Decursivi Radix and their adulterants.

[Study on traceability system of genuine medicinal materials].

Genuine medicinal materials with special characteristics of Traditional Chinese Medicine (TCM), is recognized as high quality medicine. Both ancient records and modern research considered that the origin is an important reason for the formation of genuine medicinal materials. However, blindly transplanting of genuine medicinal materials has led to the quality decline and counterfeit medicines appeared in production or sale progress, which may increase the risk of accidents in TCM. Frequent accidents emerged in Chinese herbal affects its export. What's more, it is a great threat to the medication safety in TCM clinical. There is an urgent need to implement traceability systems of TCM, which could provide convenient information record and traceability of TCM circulation. This paper reviews a variety of technical methods for genuine medicinal materials traceability, and proposed the establishment of genuine medicinal materials traceability system based on two-dimensional code and network database.

[Integrated DNA barcoding database for identifying Chinese animal medicine].

In order to construct an integrated DNA barcoding database for identifying Chinese animal medicine, the authors and their cooperators have completed a lot of researches for identifying Chinese animal medicines using DNA barcoding technology. Sequences from GenBank have been analyzed simultaneously. Three different methods, BLAST, barcoding gap and Tree building, have been used to confirm the reliabilities of barcode records in the database. The integrated DNA barcoding database for identifying Chinese animal medicine has been constructed using three different parts: specimen, sequence and literature information. This database contained about 800 animal medicines and the adulterants and closely related species. Unknown specimens can be identified by pasting their sequence record into the window on the ID page of species identification system for traditional Chinese medicine (www. tcmbarcode. cn). The integrated DNA barcoding database for identifying Chinese animal medicine is significantly important for animal species identification, rare and endangered species conservation and sustainable utilization of animal resources.

[Identification of Scolopendra subspinipes mutilans and its adulterants using DNA barcode].

In this study, the COI barcode was used to identify the Scolopendra medicinal materials and its adulterants in order to provide a new method for the identification of Scolopendra. Genomic DNA was extracted from the experimental samples. The COI sequences were amplified and sequenced bi-directionally. Sequence alignment and NJ tree construction was carried out by MEGA6.0 software. The results showed that the COI sequences can be obtained from all experimental samples. The average inter-specific K2P distance of Scolopendra was 0.222 and the minimum inter-specific distance was 0.190. All the Scolopendra subspinipes mutilans medicinal samples clustered into a clade in the NJ tree and can be distinguished from its adulterants. In a conclusion, COI can be used to correctly identify Scolopendra medicinal materials, and it will be a potential DNA barcode for identifying other animal medicinal materials.