论中药分子鉴定的方法和原则

中药的准确鉴定涉及到人民生命安全和切身利益。传统的鉴定方法,即感官评价、显微鉴定和理化鉴定均存在不同程度的局限性,而分子鉴定则为中药的快速和准确鉴定带来了新的契机。为了使中药的分子鉴定得到更加广泛和有效的应用,应该高度重视相关标准、规范的制订。本文提出了中药分子鉴定的一些主要方法：1)真伪品鉴定：特异聚合酶链式反应法;2)正品和替代品鉴定：DNA条形码鉴定法;3)多基源鉴定：群体遗传学分析法;4)产地鉴别：分子谱系地理学分析法。经上述方法仍无法鉴别的贵重药材可进一步应用人类亲子鉴定的方法,开发特异微卫星标记进行进一步的鉴定。     

Identification of herbal medicinal materials using DNA barcodes

Herbal medicinal materials have been used worldwide for centuries to maintain health and to treat disease. However, adulteration of herbal medicines remains a major concern of users and industry for reasons of safety and efficacy. Identification of herbal medicinal materials by DNA technology has been widely applied,started from the mid-1990s. In recent years, DNA barcoding of global plant species using four standard barcodes (rbcL, matK, trnH-psbA and ITS) has been a major focus in the fields of biodiversity and conservation. These DNA barcodes can also be used as reliable tools to facilitate the identification of herbal medicinal materials for the safe use of herbs, quality control, and forensic investigation. Many studies have applied these DNA barcodes for the identification of herbal medicinal species and their adulterants. The present article reviews efforts in the identification of herbal medicinal materials using the standard DNA barcodes and other DNA sequence-based markers.     

The Investigation on Utilization of Wild Medicinal Plants in Dietary Culture of Quanzhou,Southern Fujian

An ethnobotanical survey on the medicinal plant species marketed in Quanzhou, southern Fujian, was conducted in order to document traditional medicinal knowledge and application of medicinal plants in dietary. In addition to literature review, data was obtained using ethnobotanical tools and methods, including personal interviews during field trips, collecting herbarium specimens, and species identification by cross referencing. The results showed that: 1) There are 148 wild medicinal species, belonging to 56 plant families and 117 genera present in Quanzhou, southern Fujian. Among them, 46 species are edible wild herbs, 37 species are medicinal plants, 38 species are used as tea substituting plants or herbal tea,22 species are wild fruits, 4 species are used as spices and 1 species is used to make bean jelly; 2) The plant families which comprise a major part of the pool are Compositae, Lamiaceae, Rosaceae, Leguminosae, Rubiaceae, Liliaceae, Orchidaceae and Moraceae; 3) This study explored the utilization of the medicinal plants in Quanzhou area, and proposed methods to inherit the culture of using wild plants in dietary, as well as methods to conserve the edible medicinal wild plant resources.     

Correlation between the different therapeutic properties of Chinese medicinal herbs and delayed luminescence

In the practice and principle of Chinese medicine, herbal materials are classified according to their therapeutic properties. ‘Cold’ and ‘heat’ are the most important classes of Chinese medicinal herbs according to the theory of traditional Chinese medicine (TCM). In this work, delayed luminescence (DL) was measured for different samples of Chinese medicinal herbs using a sensitive photon multiplier detection system. A comparison of DL parameters, including mean intensity and statistic entropy, was undertaken to discriminate between the ‘cold’ and ‘heat’ properties of Chinese medicinal herbs. The results suggest that there are significant differences in mean intensity and statistic entropy and using this method combined with statistical analysis may provide novel parameters for the characterization of Chinese medicinal herbs in relation to their energetic properties. Copyright © 2015 John Wiley & Sons, Ltd.     

Separation and isolation methods for analysis of the active principles of Sho-saiko-to (SST) oriental medicine

Sho-saiko-to (SST) was introduced into Japan as an oriental classical medicine from China approximately 1500 years ago, and it is currently the most representative Kampo medicine (traditional Japanese medicine). SST is manufactured in Japan as an ethical drug on a modern industrial scale in which the quality of ingredients is standardized with Good Manufacturing Practices (GMP) regulation. SST is widely used for the treatment of chronic hepatitis. Experimental and clinical studies including multi-center, placebo-controlled, double-blind studies have demonstrated the various pharmacological effects of SST. SST is prepared from the hot water extraction of seven raw materials, therefore many kinds of constituents are included. Three-dimensional (3D) HPLC analysis is useful for obtaining many kinds of constituents, especially low molecular ultraviolet (UV) quenching compounds, contained in SST as well as its fractions. Fingerprint pattern provided by 3D HPLC analysis makes possible to identify the overall-viewing of SST. Databases of UV spectra of the components of medicinal herbs obtained by reversed-phase (RP) HPLC using a photodiode array (PDA) and fingerprint patterns of crude drugs made by 3D HPLC analysis facilitate the identification, analysis and quality of herbal drugs. Studies using both PDA HPLC and an amino acid analysis with a fluorometric detector have found that SST contains fifteen major low molecular compounds (i.e. baicalin, wogonin-7-O-glucuronide, liquiritin, their three aglycons, liquiritin apioside, glycyrrhizin, saikosaponin b1, saikosaponin b2, ginsenoside Rg1, ginsenoside Rb1, (6)-gingerol, (6)-shogaol and arginine). These compounds have various pharmacological actions, and are assumed to be responsible, at least partly, for the pharmacological effects of SST. Although there have only been a few investigations on high molecular compounds with pharmacological actions contained in SST, several kinds of polysaccharides have been isolated from constituent herbs of SST. This review paper summarizes analytical methods of separation, isolation and identification of compounds with biological activities from SST, which is a mixture drug of medicinal herbs. Accordingly, this paper would not focus on methods of separation, isolation and analysis of particular compounds from each constituent herb of SST.     

Molecular-networking-guided discovery of species-specific markers for discriminating five medicinal Paris herbs

BackgroundParidis Rhizoma (PR) is a famous traditional herbal medicine. Apart from two officially recorded species, viz. Paris polyphylla Smith var. yunnanensis (Franch.) Hand. - Mazz. (PPY) and P. polyphylla Smith var. chinensis (Franch.) Hara (PPC), there are still many other species used as folk medicine. It is necessary to understand the metabolic differences among Paris species.PurposeTo establish a strategy that can discover species-specific steroidal saponin markers to distinguish closely-related Paris herbs for quality and safety control.MethodsA new strategy of molecular-networking-guided discovery of species-specific markers was proposed. Firstly, the ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) was applied to obtain the MS and MS/MS data of all samples. Then, molecular networking (MN) was created using MS/MS data to prescreen the steroidal saponins for subsequent analysis. Next, the principal component analysis (PCA) and orthogonal partial least square discriminant analysis (OPLS-DA) models were established to discover potential markers. Finally, the verification, identification and distribution of chemical markers were performed.ResultsA total of 126 steroidal saponins were screened out from five species using MN. Five species were classified successfully by OPLS-DA model, and 18 species-specific markers were discovered combining the variable importance in the projection (VIP) value, P value (one-way ANOVA) and their relative abundance. These markers could predict the species of Paris herbs correctly.ConclusionThese results revealed that this new strategy could be an efficient way for chemical discrimination of medicinal herbs with close genetic relationship.     

PCR–RFLP to distinguish three Phyllanthus sp., commonly used in herbal medicines

The herbs Phyllanthus amarus, P. fraternus and P. urinaria, are known for their use in preparation of herbal medicines. These herbs grow together and are morphologically indistinguishable. Hence, collections of these herbs for raw drug trade, frequently contain mixture of the three species. Such mixing can result in preparation of herbal drugs with altered efficacy. To prevent this, it is essential to develop reliable methods/markers for accurate identification of species. Keeping this in view, PCR–RFLP (Polymerase Chain Reaction–Restriction Fragment Length Polymorphism) of chloroplast genome regions was carried out, using six pairs of universal primers of chloroplast DNA for amplification, followed by restriction with HinfI and TaqI in P. amarus, P. fraternus and P. urinaria. Distinct restriction patterns for the three species were obtained in all primer pair-restriction enzyme combinations. Since the primers used are conserved over a wide range of plant taxa, PCR–RFLP technique using universal cpDNA primers may be applied to other medicinal/economically important plants, posed with similar problems of identification.     

Medicinal Plants Recommended by the World Health Organization: DNA Barcode Identification Associated with Chemical Analyses Guarantees Their Quality

Medicinal plants are used throughout the world, and the regulations defining their proper use, such as identification of the correct species and verification of the presence, purity and concentration of the required chemical compounds, are widely recognized. Herbal medicines are made from vegetal drugs, the processed products of medicinal species. These processed materials present a number of challenges in terms of botanical identification, and according to the World Health Organization (WHO), the use of incorrect species is a threat to consumer safety. The samples used in this study consisted of the dried leaves, flowers and roots of 257 samples from 8 distinct species approved by the WHO for the production of medicinal herbs and sold in Brazilian markets. Identification of the samples in this study using DNA barcoding (matK, rbcL and ITS2 regions) revealed that the level of substitutions may be as high as 71%. Using qualitative and quantitative chemical analyses, this study identified situations in which the correct species was being sold, but the chemical compounds were not present. Even more troubling, some samples identified as substitutions using DNA barcoding contained the chemical compounds from the correct species at the minimum required concentration. This last situation may lead to the use of unknown species or species whose safety for human consumption remains unknown. This study concludes that DNA barcoding should be used in a complementary manner for species identification with chemical analyses to detect and quantify the required chemical compounds, thus improving the quality of this class of medicines.     

江苏宝华山区药用维管植物资源组成分析

为全面了解江苏宝华山区药用维管植物资源现状,依据“第四次全国中药资源普查”项目的要求,采用线路调查和样方调查相结合的方法,对该区域药用维管植物的种类组成、科属构成、生活型、濒危现状和中药学特征以及重点药用维管植物的资源状况进行了调查和分析。结果显示：宝华山区共有药用维管植物164科620属1089种(含种下等级),分别占江苏省药用维管植物科、属、种数的88.2%、87.3%、81.0%,其中,蕨类植物25科33属50种、裸子植物7科13属16种、被子植物132科574属1023种;在科属构成上均以包含单种和寡种的小科、小属为主。从该区域药用维管植物的生活型看,以草本最为丰富,有734种;乔木和灌木(含木质藤本)较少,分别有125和230种。从所在的植被类型看,阔叶林数量最多,其后依次为针阔混交林、灌草丛、人工植被,针叶林数量最少。从地理分布看,与同区域种子植物的地理分布特征基本吻合,具有亚热带向温带过渡的特点。从珍稀濒危状况看,该区域现存中国特有属14属、国家级珍稀濒危药用维管植物18种、江苏省省级保护药用维管植物32种。从中药材上看,有道地药材17种和重点药用维管植物130种,但各种类的数量总体上较少。从该区域药用维管植物的中药学特征看,在药用部位上以全草(株)类、根和根茎类较多,分别占总种数的44.2%和24.0%;在药性上以平性最多,占30.9%;在药味上以苦味最多,占40.0%;有毒的药用植物占12.9%,其中小毒种类最多。综合分析结果显示：宝华山区药用维管植物种类丰富,其中,道地药材占有一定比例,但重点药用维管植物数量有限且分布频度总体较低。根据上述调查结果,对宝华山区药用维管植物资源的保护和利用提出了一些建议。     

药用植物DNA条形码鉴定研究进展

DNA条形码是利用相对较短的标准DNA片段对物种进行快速准确鉴定的一门技术。DNA条形码技术可以从分子水平弥补传统鉴定方法的一些不足。该技术具有良好的通用性,使得物种鉴定过程更加快速,已经广泛应用于动物物种的鉴定研究中。近年来,随着药用植物DNA条形码鉴定研究的快速发展,逐渐形成了药用植物和植物源中药材鉴定的完善体系。本文综述了DNA条形码技术鉴定药用植物的原理,介绍了中草药传统鉴定方法及其缺陷、使用DNA条形码技术鉴定植物源药材的意义以及DNA条形码在药用植物鉴定中的应用,对其应用前景进行了展望。     

Classification of Chinese herbs based on the cluster analysis of delayed luminescence

Traditional Chinese material medica are an important component of the Chinese pharmacopeia. According to the traditional Chinese medicinal concept, Chinese herbal medicines are classified into different categories based on their therapeutic effects, however, the bioactive principles cannot be solely explained by chemical analysis. The aim of this study is to classify different Chinese herbs based on their therapeutic effects by using delayed luminescence (DL). The DL of 56 Chinese herbs was measured using an ultra‐sensitive luminescence detection system. The different DL parameters were used to classify Chinese herbs according to a hierarchical cluster analysis. The samples were divided into two groups based on their DL kinetic parameters. Interestingly, the DL classification results were quite consistent with classification according to the Chinese medicinal concepts of ‘cold’ and ‘heat’ properties. In this paper, we show for the first time that by using DL technology, it is possible to classify Chinese herbs according to the Chinese medicinal concept and it may even be possible to predict their therapeutic properties. Copyright © 2015 John Wiley & Sons, Ltd.     

Evaluation of rapid molecular diagnostics for differentiating medicinal Kaempferia species from its adulterants

Accurate detection of unique herbs is crucial for herbal medicine preparation. Zingiberaceae species, which are important in Ayurvedic medicine of India, are often misidentified in Northeast (NE) Indian herbal markets. Kaempferia galanga (Zingiberaceae) is one of the major components of popular Ayurvedic drugs used for rheumatic diseases (i.e.,“Gandha Thailam” and “Rasnairandadi Kashayam”), contusions, fractures, and sprains. In NE India, herbal healers often misidentify plants from the Marantaceae family (e.g., Calathea bachemiana and Maranta leuconeura) as Kaempferia, which leads to adulteration of the medicinal herb. This misidentification of herbs occurs in NE India because Zingiberaceae plant barcoding information is inadequate. As a consequence, herbal medicine is not only therapeutically less effective but may also cause adverse reactions that range from mild to life-threatening. In this study, we used eight barcoding loci to develop “fingerprints” for four Kaempferia species and two species frequently mistaken for Kaempferia. The PCR and sequencing success of the loci matK, rbcL and trnH-psbA were found to be 100%;the combination of matK, rbcL, and trnH-psbA proved to be the ideal locus for discriminating the Kaempferia species from their adulterants because the combined loci showed greater variability than individual loci. This reliable tool was therefore developed in the current study for accurate identification of Kaempferia plants which can effectively resolve identification issues for herbal healers.     

Chemical Composition of Selected Commercial Herbal Remedies in Relation to Geographical Origin and Inter-Species Diversity

Infusions prepared from medicinal herbs that are rich in flavonoids are very popular herbal remedies in societies of Eastern Europe. Therefore, the content of essential elements together with total flavonoids was analyzed in 65 commercially available samples of herbal drugs originating from Ukraine, Romania, and Belarus. The results showed that metallic elements (in mg kg^?1 d.w.) have occurred in the following order: Fe > Mn > Zn > Cu, both for total and water-extractable species. Total flavonoids were determined in the range from 10.0 to 191.8 mg g^?1 d.w. Several significant correlations have been found between the analytes, especially among water-extractable Fe with other metals, and total flavonoids and Fe, Zn, and Mn. Analysis of variance has revealed significant differences among studied samples due to their origin from different countries, especially between Belarussian samples and others. Differences owing to belonging to various plant species were also found, as it was noticed in the case of Polygoni aviculare herba in comparison with other botanical plant species. Moreover, multivariate statistical techniques, such as cluster analysis (CA) and principal component analysis (PCA) were used to gather herbal drugs based on similarity of chemical composition. CA grouped the samples into clusters with similar level of elements and total flavonoid contents, and PCA has indicated Hyperici herba, Tiliae flores, and Crataegi fructus as herbal remedies with close concentration of studied elements and flavonoids.     

The Safety Assessment of Toxic Metals in Commonly Used Pharmaceutical Herbal Products and Traditional Herbs for Infants in Jordanian Market

The objective of this study was to assess the levels of contamination by toxic metals (Pb, Al, Ni, Cd and As) that may be present in 25 infant pharmaceutical herbal products and 15 traditional herbs in Jordan. Both products and medicinal herbs are currently prescribed by paediatricians. They are available as over-the-counter medicines and are sold the in herbal market, ensuring easy accessibility for parents. Inductively coupled plasma-optical emission spectroscopy (ICP-OES), with limit of detections (LODs) of 0.10, 1.00, 0.20, 0.15 and 2.00 mg.kg⁻¹ for Pb, Al, Ni, Cd and As respectively, was employed to measure the levels of toxic metals in the samples. Pb, Al and Ni were detected in 88, 76 and 4% of the analysed samples of pharmaceutical herbal products and in 93, 87 and 13% of the analysed samples of traditional herbs, respectively. Neither Cd or As were detected in all analysed samples. The data obtained were subsequently compared by referral to the acceptable limits of toxic heavy metals according to World Health Organisation (WHO) standards. Largely, the results showed acceptable toxic metal levels in the finished pharmaceutical products and the traditional medicinal herbs for infants. One exception to this was Persian Thyme (Satureja thymbra) with Pb content of 41.18 mg.kg⁻¹. Also, the daily intake of detected metals through pharmaceutical herbal products was found to be lower than the daily tolerable intake limit set by the regulatory bodies, except of 8% of products that exceeded the tolerable daily intake of Pb set by US FDA, as compared to traditional medicinal herbs, where the tolerable daily intake for Pb, Al and Ni were exceeded in 40, 60 and 8% of the analysed herbs, respectively. The results obtained revealed that the excessive use of medicinal plants as alternative medicine should be used with caution keeping in mind the safety factor in infants.

     

福建省福州市马尾区药用植物资源研究

依托第四次全国中药资源普查工作,摸清马尾区药用植物资源种类、分布、重点中药材品种以及药材栽培等情况。根据第四次全国中药资源普查要求,通过实地调查、走访以及栽培基地调查等方法对马尾区药用植物资源进行调查,查阅相关资料确定野生药用植物的药用部位、生活型等。马尾区野生药用植物种类共计506种,隶属于132科360属,含2～5种的寡种科占绝对优势地位;草本为主要生活型,药用部位集中在根类和全草类;有福建省重点药材50种、特色药材22种。由于长期受人为干扰,马尾区原生植被破坏严重,药用植物种类较为贫乏。因此,可考虑林下种植中药改善土壤环境,实现中药资源可持续发展。     

Carbon isotope composition as a tool to control the quality of herbs and medicinal plants

Isotope screening is a simple test for determining the photosynthetic pathway used by plants. The scope of this work was to classify the photosynthetic type of some herbs and medicinal plants through studies of the carbon isotope composition (δ¹³C). Also, we propose the use of carbon isotope composition as a tool to control the quality of herbs and medicinal plants. For studies of δ¹³C, δ¹³C‰ = [R (sample)/R (standard) − 1] × 10⁻³, dry leaves powdered in cryogenic mill were analyzed in a mass spectrometer coupled with an elemental analyzer for determining the ratio R = ¹³CO₂/¹²CO₂. In investigation of δ¹³C of 55 species, 23 botanical families, and 44 species possessed a C₃ photosynthetic type. Six species found among the botanical families Euphorbiaceae and Poaceae were C₄ plants, and 5 species found among the botanical families Agavaceae, Euphorbiaceae, and Liliaceae possessed CAM-type photosynthesis. Carbon isotope composition of plants can be used as quality control of herbs and medicinal plants, allowing the identification of frauds or contaminations. Also, the information about the photosynthetic type found for these plants can help in introducing and cultivating exotic and wild herbs and medicinal plants.     

PCR-based rapid diagnostic tools for the authentication of medicinal mistletoe species

BackgroundTaxilli Herba (TH) and Visci Herba (VH), defined as the leaves and branches of the mistletoe species Taxillus chinensis and Viscum coloratum, respectively, are popular herbal medicines in East Asia. However, commercial TH and VH products are frequently adulterated with related inauthentic mistletoe species, posing efficacy and safety concerns. Accurate species identification of herbal medicinal products is a prerequisite for quality control, but traditional morphological identification methods are hampered by difficulties in discriminating among closely related species and in identifying the source materials in processed products.PurposeThis study aimed to develop sequence-characterized amplified region (SCAR) markers and a multiplex-SCAR assay for rapid and accurate identification of authentic TH and VH.MethodsThe matK region was sequenced in a total of 20 samples from five mistletoe species, namely T. chinensis and V. coloratum, and three species often found in adulterated herbal medicines, T. sutchuenensis, V. articulatum, and Macrosolen tricolor. Species-specific nucleotide polymorphisms were identified and short regions (21–22 bp) containing at least two species-specific nucleotides close to the 3ʹ end were incorporated into SCAR primers that produced uniquely sized PCR amplicons for each species. The five SCAR primer sets were also combined into a multiplex-SCAR assay.ResultsThe SCAR primers successfully generated amplicons of the expected size for each target species even with low-DNA templates or with templates containing DNA from multiple samples. No amplification was observed in non-target species. The SCAR markers and the multiplex-SCAR assay successfully identified commercial TH and VH products that were counterfeit or adulterated in both dried and processed products.ConclusionThis is the first report to illustrate discrimination of genuine medicinal mistletoe species with DNA-based marker assays, enabling rapid and accurate species identification. The SCAR assays developed in this study will facilitate the standardization of commercial mistletoe products.     

云南常见药用石斛DNA条形码筛选

选择云南省内常见的12种药用石斛，采用分子生物学鉴定技术筛选其适用的DNA条形码序列。以12种药用石斛共36个样品为材料，提取样品总DNA，对核基因片段ITS和ITS2、叶绿体基因片段psbA-trnH和matK序列进行扩增、测序，结合GenBank下载部分石斛序列；利用生物信息学软件进行序列分析和系统发育分析。结果表明，4条序列扩增和测序成功率均为100%；4条序列没有明显的Barcoding Gap，但ITS序列与ITS2序列的种内和种间重叠部分较少，有偏向两端的趋势；系统发育树显示，ITS和psbA-trnH序列能成功区分12种云南常见的药用石斛，ITS2序列未能区分长距石斛(Dendrobium longicornu)和矮石斛(D. bellatulum)，matK序列仅区分6种石斛。建议以ITS和psbA-trnH序列作为云南药用石斛鉴定序列，为药用石斛的种源鉴定提供理论依据。     

甘草药材上的污染真菌类群及其产毒素特性

对药材市场上霉变甘草样品的污染真菌进行分析,共得到4属7种真菌,包括Penicillium、Aspergillus、Fusarium、Mucor属,其中Penicillium polonicum、Aspergillus parasiticus以及P.crustosum是优势真菌。采用高效液相色谱-质谱联用技术对优势菌菌株产黄曲霉毒素及赭曲霉毒素A的特性进行检测。结果表明A.parasiticus主要产生黄曲霉毒素(AFG2、AFG1、AFB2、AFB1)和赭曲霉毒素A(OTA);而Penicillium polonicum主要产生赭曲霉毒素A(OTA)。     

Ethnomedicinal assessment of Irula tribes of Walayar valley of Southern Western Ghats,India

The present study was aimed to explore the traditional knowledge of Irula tribal people who are practicing herbal medicine in Walayar valley, the Southern Western Ghats, India. A total number of 146 species of plants distributed in 122 genera belonging to 58 families were identified as commonly used ethnomedicinal plants by them. Interestingly, 26 new claims were also made in the present study. Through the data obtained from Irula tribal healers, the herbs were mostly used for medicine (40.4%) followed by trees (26.7%) and climbers (18.5%). In addition leaves were highly used for medicinal purposes, collected from 55 species (38%) followed by multiple parts from 18 species (12%). Acorus calamus is the species of higher use value (1.80) assessed to be prescribed most commonly for the treatment of cough. High informant consensus factor (1.0) obtained for insecticidal uses and cooling agent indicates that the usage of Canarium strictum and Melia dubia, and Mimosa pudica and Sesamum indicum respectively for that purposes had obtained high degree of agreement among the healers in using these species for the respective purposes. The most commonly used method of preparation was decoction (63%) followed by raw form (23%), paste (12%) and powder (2%). Therefore, it is suggested to take-up pharmacological and phytochemical studies to evaluate the species to confirm the traditional knowledge of Irulas on medicinal plants.