The complete chloroplast genome of Euphorbia hirta (Euphorbiaceae), a commonly used medicinal plant in China.

Plants in the genus Euphorbia have been widely used as herbal medicine, and for ornamental horticulture and biofuel production. In this study, we report the complete chloroplast genome of Euphorbia hirta which is known as the 'asthma-plant' due to its medicinal use. The chloroplast genome of this species is 164,340 bp in length, including a pair of inverted repeat regions (IRs) (27,354 bp) that are divided by a large single-copy region (LSC) (91,373 bp) and a small single-copy region (SSC) (18,259 bp). The chloroplast genome of E. hirta contains 111 unique genes (77 protein-coding, 30 tRNA, and four rRNA), 19 of which are duplicated in the IR regions. The overall GC content is 35.4%. Phylogenetic analysis fully resolved E. hirta groups with other species of Euphorbia. The complete chloroplast genome of E. hirta provides useful information that can be used to distinguish related species and reconstruct evolutionary relationships.

Species Quantification in Complex Herbal Formulas-Vector Control Quantitative Analysis as a New Method.

Product mislabeling and/or species fraud in Traditional Chinese Medicine (TCM) not only decrease TCM quality, but also pose a potential health issue to the end user. Up to now, methods to control TCM quality have been developed to detect specific metabolites or identify the original species. However, species quantification in complex herbal formulas is rarely concerned. Here, we reported a simple Vector Control Quantitative Analysis (VCQA) method for flexible and accurate multiplex species quantification in traditional Chinese herbal formulas. We developed PCR-based strategy to quickly generate the integrated DNA fragments from multiple targeted species, which can be assembled into the quantitative vector in one round of cloning by Golden Gate ligation and Gateway recombination technique. With this method, we recruited the nuclear ribosomal DNA Internal Transcribed Spacer (ITS) region for the quantification of Ligusticum sinense "Chuanxiong," Angelica dahurica (Hoffm.) Benth. & Hook.f. ex Franch. & Sav., Notopterygium incisum K. C. Ting ex H. T. Chang, Asarum sieboldii Miq., Saposhnikovia divaricata (Turcz.) Schischk., Nepeta cataria L., Mentha canadensis L., and Glycyrrhiza uralensis Fisch. ex DC. in ChuanXiong ChaTiao Wan, a classic Chinese herbal formula with very long historical background. We found that, firstly, VCQA method could eliminate the factors affecting such as the variations in DNA extracts when in combination with the use of universal and species-specific primers. Secondly, this method detected the limit of quantification of A. sieboldii Miq. in formula products down to 1%. Thirdly, the stability of quality of ChuanXiong ChaTiao Wan formula varies significantly among different manufacturers. In conclusion, VCQA method has the potential power and can be used as an alternative method for species quantification of complex TCM formulas.

High frequency plant regeneration from leaf culture of Neolamarckia cadamba.

Neolamarckia cadamba is a miracle tree species with considerable economic potential uses as a timber wood, woody forage and traditional medicine resource. The present study aimed to establish a highly efficient and robust protocol of plant regeneration for N. cadamba. Greenish callus was induced from very young leaf explants of sterile in vitro plantlets cultured on Murashige and Skoog's (MS) medium supplemented with 3 mg l-1 thidiazuron (TDZ), 0.1 mg l-1 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.05 mg l-1 α-naphthaleneacetic acid (NAA). The callus could differentiate into nodular embryogenic structures or adventitious shoots, and these two regeneration pathways often occurred in the same callus clumps. The micro-shoots developed roots in MS supplemented with 0.05 mg l-1 NAA and 0.05 mg l-1 indole-3-butyric acid (IBA), while the nodular embryogenic structures germinated directly and developed into plantlets on induction medium contained with 0.5 mg l-1 (or 1 mg l-1) 6-benzyladenine (6-BA) and 0.05 mg l-1 NAA. The rooted plantlets could be successfully acclimatized to a greenhouse with more than 92.0% survival. This regeneration protocol can be used in large scale cultivation needs and may be useful for future genetic modifications of N. cadamba.

Identifying the Species of Seeds in Traditional Chinese Medicine Using DNA Barcoding.

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.

Using SSR-HRM to Identify Closely Related Species in Herbal Medicine Products: A Case Study on Licorice.

Traditional herbal medicines have played important roles in the ways of life of people around the world since ancient times. Despite the advanced medical technology of the modern world, herbal medicines are still used as popular alternatives to synthetic drugs. Due to the increasing demand for herbal medicines, plant species identification has become an important tool to prevent substitution and adulteration. Here we propose a method for biological assessment of the quality of prescribed species in the Chinese Pharmacopoeia by use of high resolution melting (HRM) analysis of microsatellite loci. We tested this method on licorice, a traditional herbal medicine with a long history. Results showed that nine simple sequence repeat (SSR) markers produced distinct melting curve profiles for the five licorice species investigated using HRM analysis. These results were validated by capillary electrophoresis. We applied this protocol to commercially available licorice products, thus enabling the consistent identification of 11 labels with non-declared Glycyrrhiza species. This novel strategy may thus facilitate DNA barcoding as a method of identification of closely related species in herbal medicine products. Based on this study, a brief operating procedure for using the SSR-HRM protocol for herbal authentication is provided.

DNA barcoding based identification of Hippophae species and authentication of commercial products by high resolution melting analysis.

Sea buckthorn (Hippophae), an ancient crop with modern virtues, is increasingly consumed in source of foods and nutraceuticals. The growing demand leads to the adulteration of commercial sea buckthorn products, which is a common form of food fraud. Herein, a high resolution melting assay, targeting a DNA barcoding region of the internal transcribed spacer 2 (ITS2) (Bar-HRM) was developed to identify the seven native Chinese Hippophae species, and to authenticate commercial sea buckthorn products. Melting data from the HRM assay demonstrated that all Hippophae species could be clearly distinguished. Then, application to commercial sea buckthorn products indicated the existence of adulterants or contamination, further confirmed using Sanger sequencing results for PCR products from HRM. The Bar-HRM technique proposed in this work could provide a method for regulatory agencies, promoting consumers trust, and raise the quality and safety of sea buckthorn products.

Metabolic discrimination of sea buckthorn from different Hippophaë species by 1H NMR based metabolomics.

Sea buckthorn (Hippophaë; Elaeagnaceae) berries are widely consumed in traditional folk medicines, nutraceuticals, and as a source of food. The growing demand of sea buckthorn berries and morphological similarity of Hippophaë species leads to confusions, which might cause misidentification of plants used in natural products. Detailed information and comparison of the complete set of metabolites of different Hippophaë species are critical for their objective identification and quality control. Herein, the variation among seven species and seven subspecies of Hippophaë was studied using proton nuclear magnetic resonance (1H NMR) metabolomics combined with multivariate data analysis, and the important metabolites were quantified by quantitative 1H NMR (qNMR) method. The results showed that different Hippophaë species can be clearly discriminated and the important interspecific discriminators, including organic acids, L-quebrachitol, and carbohydrates were identified. Statistical differences were found among most of the Hippophaë species and subspecies at the content levels of the aforementioned interspecific discriminators via qNMR and one-way analysis of variance (ANOVA) test. These findings demonstrated that 1H NMR-based metabolomics is an applicable and effective approach for simultaneous metabolic profiling, species differentiation and quality assessment.

Study of Commercially Available Lobelia chinensis Products Using Bar-HRM Technology.

There is an unmet need for herbal medicine identification using a fast, sensitive, and easy-to-use method that does not require complex infrastructure and well-trained technicians. For instance, the detection of adulterants in Lobelia chinensis herbal product has been challenging, since current detection technologies are not effective due to their own limits. High Resolution Melting (HRM) has emerged as a powerful new technology for clinical diagnosis, research in the food industry and in plant molecular biology, and this method has already highlighted the complexity of species identification. In this study, we developed a method of species specific detection of L. chinensis using HRM analysis combined with internal transcribed spacer 2. We then applied this method to commercial products purporting to contain L. chinensis. Our results demonstrated that HRM can differentiate L. chinensis from six common adulterants. HRM was proven to be a fast and accurate technique for testing the authenticity of L. chinensis in herbal products. Based on these results, a HRM approach for herbal authentication is provided.

Applying high-resolution melting (HRM) technology to identify five commonly used Artemisia species.

Many members of the genus Artemisia are important for medicinal purposes with multiple pharmacological properties. Often, these herbal plants sold on the markets are in processed forms so it is difficult to authenticate. Routine testing and identification of these herbal materials should be performed to ensure that the raw materials used in pharmaceutical products are suitable for their intended use. In this study, five commonly used Artemisia species included Artemisia argyi, Artemisia annua, Artemisia lavandulaefolia, Artemisia indica, and Artemisia atrovirens were analyzed using high resolution melting (HRM) analysis based on the internal transcribed spacer 2 (ITS2) sequences. The melting profiles of the ITS2 amplicons of the five closely related herbal species are clearly separated so that they can be differentiated by HRM method. The method was further applied to authenticate commercial products in powdered. HRM curves of all the commercial samples tested are similar to the botanical species as labeled. These congeneric medicinal products were also clearly separated using the neighbor-joining (NJ) tree. Therefore, HRM method could provide an efficient and reliable authentication system to distinguish these commonly used Artemisia herbal products on the markets and offer a technical reference for medicines quality control in the drug supply chain.

Development of 18 novel microsatellite primers for Begonia fimbristipula (Begoniaceae), an endangered medicinal plant in China.

PREMISE OF THE STUDY: Begonia fimbristipula (Begoniaceae) is a medicinal herb distributed in the Chinese provinces of Fujian, Guangdong, Guangxi, Hainan, Hunan, Jiangxi, and Zhejiang, and it is on the verge of extinction due to habitat destruction and deterioration of its ecosystem. Here we developed a set of highly polymorphic microsatellite markers for population genetic and conservation studies of this endangered medicinal plant. METHODS AND RESULTS: Using the Fast Isolation by AFLP of Sequences COntaining repeats (FIASCO) protocol, 18 polymorphic microsatellite markers were identified within 48 individuals from two geographic locations. The observed and expected heterozygosities ranged from 0.208 to 1.000 and from 0.291 to 0.812, respectively. These microsatellite markers were cross-amplified in five related Begonia species, and six loci were successfully amplified in all species. CONCLUSIONS: These 18 markers will be useful for better conservation and utilization of wild resources of B. fimbristipula and other Begonia species in the future.

The Potential Power of Bar-HRM Technology in Herbal Medicine Identification.

The substitution of low-cost or adulterated herbal products for high-priced herbs makes it important to be able to identify and trace herbal plant species and their processed products in the drug supply chain. PCR-based methods play an increasing role in monitoring the safety of herbal medicines by detecting adulteration. Recent studies have shown the potential of DNA barcoding combined with high resolution melting (Bar-HRM) analysis in herbal medicine identification. This method involves precisely monitoring the change in fluorescence caused by the release of an intercalating DNA dye from a DNA duplex as it is denatured by a gradual increase in temperature. Since the melting profile depends on the GC content, length, and strand complementarity of the amplification product, Bar-HRM analysis opens up the possibility of detecting single-base variants or species-specific differences in a short region of DNA. This review summarizes key factors affecting Bar-HRM analysis and describes how Bar-HRM is performed. We then discuss advances in Bar-HRM analysis of medicinal plant ingredients (herbal materia medica) as a contribution toward safe and effective herbal medicines.

[Application of high resolution melting curve to identification of Cimicifugae Rhizoma].

High-resolution-melting analysis (HRM) is a new technology derived from q PCR and is widely used in the study of polymorphism, genotyping, and single nucleotide mutation. Advantages of HRM include cost-effectiveness and time-efficiency over PCR-based genotyping. However, the application of HRM in the authentication of herbal products is still limited with few studies on the classification and identification of herbal products. In this study, Cimicifugae Rhizoma was used as an example to verify the stability and accuracy of HRM technique in identification of Chinese materia medica. HRM assay was established for identification based on ITS2 region of Cimicifugae Rhizomas and its adulterants(including 41 samples). Our findings showed that HRM allows not only the identification of adulteration but also the quantification of the most common admixture. This study is significant for better quality in the verification of the authenticity of herbal medicine. The method is promising for future identification of traditional Chinese medicinal materials.

Loop-Mediated Isothermal Amplification (LAMP): Emergence As an Alternative Technology for Herbal Medicine Identification.

Correct identification of medicinal plant ingredients is essential for their safe use and for the regulation of herbal drug supply chain. Loop-mediated isothermal amplification (LAMP) is a recently developed approach to identify herbal medicine species. This novel molecular biology technique enables timely and accurate testing, especially in settings where infrastructures to support polymerase chain reaction facilities are lacking. Studies that used this method have altered our view on the extent and complexity of herbal medicine identification. In this review, we give an introduction into LAMP analysis, covers the basic principles and important aspects in the development of LAMP analysis method. Then we presented a critical review of the application of LAMP-based methods in detecting and identifying raw medicinal plant materials and their processed products. We also provide a practical standard operating procedure (SOP) for the utilization of the LAMP protocol in herbal authentication, and consider the prospects of LAMP technology in the future developments of herbal medicine identification and the challenges associated with its application.

Porous biomimetic membranes: fabrication, properties and future applications.

A cell membrane is a flexible lipid bilayer with sophisticated functions which dominate the exchange of material, energy and information between the outside and the inside of the cell. In order to understand and imitate these structures and functions, scientists had already developed a variety of mimic membranes which are alike in form based on lipid bilayer and organic channel-molecules. With the rise of nanotechnology, a large number of synthetic nano-devices are widely used to construct porous biomimetic membranes which are alike in spirit instead of the conventional lipid bilayer membranes. This perspective will first introduce several typical methods to fabricate porous biomimetic membranes, and then discuss the "smart" properties and future applications of these membranes in materials transport, energy transformation and signal transduction aspects.