Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and amplification refractory mutation system (ARMS) analyses of medicinally used Rheum species and their application for identification of Rhei Rhizoma

Previously, we have determined marker nucleotides on the chloroplast matK gene to identify Rheum palmatum, R. tanguticum and R. officinale used as Rhei Rhizoma officially. In the present study, we further developed a convenient and efficient identification method on the basis of marker nucleotides with Amplification Refractory Mutation System analysis. On the basis of the nucleotide substitutions at positions 367 and 937 among the three species on the matK gene, at each position two kinds of reverse primers with complementary 3'-terminal nucleotides were designed. Upon PCR amplification using three sets of primers and template DNA from each species, one or two fragments (202 bp or/and 770 bp) were detected. As the resultant three fragment profiles were species-specific, the procedure enabled us to classify the botanic origins of 22 drug samples of Rhei Rhizoma.     

大黄药理作用研究及临床应用概况

<正>大黄为蓼科多年生草本植物掌叶大黄Rheum palmatum L、唐古特大黄R．tanguticum Maxim．exbalf、药用大黄R．offcinale Baill的干燥根及根茎。掌叶大黄和唐古特大黄称北大黄,主产于我国青海、甘肃、四川等地；药用大黄称南大黄,     

Molecular identification of "Chuanxiong" by nucleotide sequence and multiplex single base extension analysis on chloroplast trnK gene

Chloroplast trnK gene sequences of Cnidium officinale and Ligusticum chuanxiong were determined to establish an effective method for identifying Japanese Senkyu and Chinese Chuanxiong, the two which have the same drug name in Chinese characters, similar external feature, but different botanical origins. Three sites of nucleotide differences were found between these 2 species at positions 767,924 and 964 from upstream in trnK gene sequence, allowing molecular identification of the two plants and crude drugs. Further, three kinds of specific primers of 14 mer, 23 mer and 30 mer long were designed to detect these 3 sites of marker nucleotides. By using multiplex single base extension (MSBE) analysis with the 3 specific primers, C. officinale and L. chuanxiong could be distinguished clearly by the electrophoretograms, where 3 peaks with different color of ddTMP, ddCMP and ddTMP were observed in case of C. officinale and those of ddGMP, ddAMP and ddGMP in L. chuanxiong. Moreover, trnK gene sequence of "Dongxiong," a kind of Chuanxiong cultivated in Northeast China, suggested that its botanical origin was C. officinale.     

Phylogenetic analysis based on 18S rRNA gene and matK gene sequences of Panax vietnamensis and five related species

Panax vietnamensis was discovered recently in Vietnam. Its bamboo-like rhizomes, called Vietnamese Ginseng, have attracted considerable attention because of their specific pharmacological activities. In order to define the taxonomic position of this new species and include it in the molecular authentication of Ginseng drugs, the 18S ribosomal RNA gene and matK gene sequences of P. vietnamensis were determined and compared with those of its related taxa, P. japonicus var. major and P. pseudo-ginseng subsp. himalaicus, besides previously reported P. ginseng, P. japonicus and P. quinquefolius. The 18S rRNA gene sequences were found to be 1809 bps in length. The sequence of P. vietnamensis was identical to that of P. quinquefolius, and presented one base substitution from those of both P. japonicus var. major and P. pseudo-ginseng subsp. himalaicus. The matK gene sequences of 6 taxa were found to be 1509 bps in length. The sequence of P. vietnamensis differed from those of P. japonicus var. major, P. pseudo-ginseng subsp. himalaicus, P. ginseng, P. japonicus and P. quinquefolius at 4, 5, 9, 9 and 10 nucleotide positions, respectively. The phylogenetic tree reconstructed by the combined 18S rRNA-matK gene analysis using the maximum parsimony method showed that P. vietnamensis was sympatric with other Panax species and had a close relationship with P. japonicus var. major and P. pseudo-ginseng subsp. himalaicus.     

Genetic variation and identification of cultivated Fallopia multiflora and its wild relatives by using chloroplast matK and 18S rRNA gene sequences

FALLOPIA MULTIFLORA (Thunb.) Harald . has been widely and discriminatingly used in China for the study and treatment of anemia, swirl, deobstruent, pyrosis, insomnia, amnesia, atheroma and also for regulating immune functions. However, there is still confusion about the herbal drug's botanical origins and the phylogenetic relationship between the cultivars and the wild relatives. In order to develop an efficient method for identification, a molecular analysis was performed based on 18 S rRNA gene and partial MATK gene sequences. The 18 S rRNA gene sequences of F. MULTIFLORA were 1809 bp in length and were highly conserved, indicating that the cultivars and the wild F. MULTIFLORA have the same botanical origin. Based on our 18 S rRNA gene sequences analysis, F. MULTIFLORA could be easily distinguished at the DNA level from adulterants and some herbs with similar components. The MATK gene partial sequences were found to span 1271 bp. The phylogenetic relation of F. MULTIFLORA based on the MATK gene showed that all samples in this paper were divided into four clades. The sequences of the partial MATK gene had many permutations, which were related to the geographical distributions of the samples. MATK gene sequences provided valuable information for the identification of F. MULTIFLORA. New taxonomic information could be obtained to authenticate the botanical origin of the F. MULTIFLORA, the species and the medicines made of it.     

基于454FLX高通量技术的厚朴叶绿体全基因组测序及应用研究

 叶绿体基因组序列在中药DNA条形码鉴定及基因工程生物制药方面具有广泛的应用前景。本文应用454高通量测序技术对厚朴进行了叶绿体全基因组测序, 建立了标准测序流程。生物信息学分析共获得有效序列重叠群 (contig) 14个, 测序覆盖度达到99.99%。厚朴叶绿体基因组大小为160 183 bp, 大 (LSC)、小 (SSC) 单拷贝区大小分别为88 210 bp和18 843 bp, 反向互补重复区 (IR) 大小为26 565 bp, 共注释叶绿体基因126个, 其中每个IR区17个。厚朴与木兰亚纲其他物种的叶绿体编码基因在种类、排列顺序及结构大小上基本一致。采用叶绿体81个蛋白编码基因对厚朴及同属5个种9个样本进行了分子鉴定, 鉴定效率100%, 并且可以成功区分不同产地的凹叶厚朴。以上结果表明, 本研究建立的标准测序流程适用于叶绿体基因组测序, 叶绿体基因组序列可有效区分厚朴及近缘物种。     

Sequence analysis of chloroplast chlB gene of medicinal Ephedra species and its application to authentication of Ephedra Herb

Chloroplast chlB gene encoding subunit B of light-independent protochlorophyllide reductase was amplified from herbarium and crude drug specimens of Ephedra sinica, E. intermedia, E. equisetina, and E. przewalskii. Sequence comparison of the chlB gene indicated that all the E. sinica specimens have the same sequence type (Type S) distinctive from other species, while there are two sequence types (Type E1 and Type E2) in E. equisetina. E. intermedia and E. prezewalskii revealed an identical sequence type (Type IP). E. sinica was also identified by digesting the chlB fragment with Bcl I. A novel method for DNA authentication of Ephedra Herb based on the sequences of the chloroplast chlB gene and internal transcribed spacer of nuclear rRNA genes was developed and successfully applied for identification of the crude drugs obtained in the Chinese market.     

基于网络分析的大黄毒性作用机制

目的采用网络毒理学的方法预测并分析大黄中有毒物质的相关毒性作用机制。方法将在中药系统药理数据库和分析平台(TCMSP)可以查询到已知的大黄的化合物成分,与在比较毒物基因组学数据库(CTD)中查询到的大黄化合物成分的毒物信息进行比对,筛选出大黄中9种有毒成分,并用Swiss Target Prediction服务器等查询有毒成分的靶点信息,进而使用Cytoscape软件构建了毒性成分-靶点互作网络以及靶点之间的互作网络,发现了其中Degree值最高的几种靶点蛋白。使用DAVID生物信息平台,进行基因本体(GO)分析以及京都基因和基因组百科全书(KEGG)通路富集分析,得出了大黄中有毒物质可能通过哪些通路对人体产生危害。结果大黄中毒性成分可能通过p53信号通路、钙离子信号通路、Toll样受体信号通路、Wnt信号通路引起毒性;还可能通过细胞凋亡调节引起毒性及其他自身免疫系统疾病。结论初步探究了大黄的毒理机制,并预测了大黄可能存在的毒性,并为预测中药成分的毒性以及探究毒性机制提供了新思路。     

DNA barcoding for identification of agarwood source species using trnL-trnF and matK DNA sequences

Journal of Natural Medicines - Agarwood is a type of resinous wood found in the trunks of Aquilaria, Gonystylus, and Gyrinops species [1]. High-quality agarwood is extraordinarily expensive and...     

基于rbcL序列鉴别十大功劳叶及其伪品

目的：利用叶绿体基因rbcL序列探讨十大功劳叶及其伪品枸骨叶的分子鉴定方法。方法：分别提取十大功劳叶的植物基因组DNA,经PCR扩增后测序,利用相关软件对序列进行分析。结果：所得各样品的rbcL序列长度为655bp,共存在碱基差异49bp,从构建的系统分枝树可知,各物种可明显分开。结论：rbcL序列可用于鉴定十大功劳叶及其伪品。     

Differentiation of Dendrobium species used as "Huangcao Shihu" by rDNA ITS sequence analysis

The genus Dendrobium Sw. is composed of 74 species and two varieties in China, and 32 species carry the name "Huangcao Shihu" on the herbal medicine market, making the identification of the origin of "Huangcao Shihu" difficult for consumers. Here, the ITS regions were sequenced and evaluated to differentiate the 18 Dendrobium species used as "Huangcao Shihu". Diversity in DNA sequences among various species was found with the inter-specific sequence divergence ranging from 3.2% to 37.9% in ITS1 and 5.0% to 26.6% in ITS2. Moreover, the variations within species were very low, ranging in sequence divergence from 0 to 3.0% in ITS1 and 0 to 4.0% in ITS2. Therefore, these species could be easily distinguished at the DNA level. Furthermore, based on the divergent ITS regions, five pairs of species-specific primers were designed and used for the rapid PCR identification of five Dendrobium species listed in the Chinese Pharmacopoeia.     

Identification of dendrobium species used for herbal medicines based on ribosomal DNA internal transcribed spacer sequence

Stems of genus Dendrobium (Orchidaceae) have been traditionally used as an herbal medicine (Dendrobii Herba) in Eastern Asia. Although demand for Dendrobium is increasing rapidly, wild resources are decreasing due to over-collection. This study aimed to identify plant sources of Dendrobii Herba on the market based on sequences of the internal transcribed spacer (ITS) regions of nuclear ribosomal DNA. We constructed an ITS1-5.8S-ITS2 sequence database of 196 Dendrobium species, and the database was employed to identify 21 herbal samples. We found that 13 Dendrobium species (D. catenatum, D. cucullatum, D. denudans, D. devonianum, D. eriiflorum, D. hancockii, D. linawianum, D. lituiflorum, D. loddigesii, D. polyanthum, D. primulinum, D. regium, and D. transparens) were possibly used as plant sources of Dendrobii Herba, and unidentified species allied to D. denudans, D. eriiflorum, D. gregulus, or D. hemimelanoglossum were also used as sources. Furthermore, it is clear that D. catenatum is one of the most important sources of Dendrobii Herba (5 out of 21 samples).     

Molecular phylogenetic analysis of Phyllanthus species in Thailand and the application of polymerase chain reaction-restriction fragment length polymorphism for Phyllanthus amarus identification

The genus Phyllanthus (Phyllanthaceae) is distributed in tropical and subtropical regions, and its members are widely used as medicinal plants in many countries. We analyzed the nucleotide sequences of the internal transcribed spacers of ribosomal DNA of 56 plant samples covering 23 Phyllanthus species collected from various habitats in Thailand. Based on the sequence alignment, we constructed phylogenetic trees of all Phyllanthus species distributed in Thailand. Furthermore, a simple protocol to discriminate three important medicinal Phyllanthus species, P. amarus, P. debilis, and P. urinaria, was developed using a Polymerase Chain Reaction-Restriction Fragment Length Polymorphism method and successfully applied to the crude drug samples obtained in Thai markets.     

Molecular analysis of the genus Mitragyna existing in Thailand based on rDNA ITS sequences and its application to identify a narcotic species: Mitragyna speciosa

In Thailand, there are four Mitragyna species; M. speciosa, M. hirsuta, M. diversifolia, and M. rotundifolia. One, M. speciosa, is a narcotic plant and has medicinal importance for its opium-like effect. Since the use of M. speciosa has been forbidden in Thailand, the leaves of M. diversifolia or others are frequently used as substitutes but are not considered as effective. Therefore, accurate authentication of M. speciosa is essential for both medicinal and forensic purposes. The nucleotide sequences of internal transcribed spacers (ITS) and the 5.8S coding region of nuclear ribosomal DNA (rDNA) of the Mitragyna species were analyzed. The whole length of ITS1-5.8S-ITS2 region was 608 bp in M. speciosa, 607 bp in the other species. Nineteen sites of nucleotide substitutions and 3 sites of 1-bp indels were observed, and M. speciosa showed specific sequence differed from the others. Based on the ITS sequences, a distinctive site recognized by a restriction enzyme XmaI in M. speciosa was found and then PCR-restriction fragment length polymorphism (RFLP) analysis was established to differentiate M. speciosa from the others. By the method, a 409-bp PCR fragment of ITS1-5.8S (partial) rDNA region from M. speciosa was cleaved into two fragments of 119 bp and 290 bp while the other species remained undigested. This method provides an effective and accurate identification of M. speciosa.     

Molecular identification of LOX-1 and analysis of its pathophysiological role

Recent progress in the study of atherosclerosis revealed that the proatherogenic property of LDL is attributable to oxidized LDL. Macrophages recruited to vascular wall phagocytose oxidized LDL and transformed into foam cells, which is a hallmark of atheroma. Endothelial cells also binds oxidized LDL and changes its phenotype to the status of "endothelial dysfunction." We successfully cloned the endothelial receptor for oxidized LDL, designated LOX-1. LOX-1-mediated action of oxidized LDL induces the decrease in NO release and the increased expression of adhesion molecules, which are typical changes in endothelial dysfunction. The expression of LOX-1 is quite inducible. Proinflammatory cytokines, etc. induce the expression of LOX-1 in vitro; and proatherogenic conditions, e.g., hypertension, hyperlipidemia, and diabetes, induce the expression of LOX-1 in vivo. This manner of expression suggests the importance of LOX-1 in pathological settings. LOX-1 binds not only oxidized LDL, but also binds apoptotic cells and activated platelets through the interaction with anionic phospholipids. This property might bridge atherosclerosis and thrombosis. A novel system to detect LOX-1 ligand in plasma detected the increased level of LOX-1 ligand in hypercholesterolemic rabbits compared with normal ones. This system might be useful to predict the status of endothelial function and the risk of ischemic heart disease.     

三七的18S rRNA,matK基因序列和HPLC化学指纹图谱分析研究

目的分析中药三七Panaxnotoginseng的18SrRNA和matK基因的分子特征和三七的化学指纹特征,为三七的正品药材基原鉴定提供分子和化学依据。方法采用PCR直接测序技术测定三七及其7种伪品的18SrRNA和matK基因部分核苷酸序列以及不同产地三七的DNA分子特征。利用HPLC的化学分析技术,明确产地对三七化学成分的影响,以及三七不同部位的化学指纹特征。结果(1)三七及其7种常见伪品的核糖体18SrRNA基因序列存在很大的差异。(2)不同产地的三七的核糖体18SrRNA和叶绿体matK基因序列特征完全一致,分别与GenBank上已报道的R1型(D85171)和M1型(AB027526)序列吻合。(3)不同产地的三七HPLC指纹图谱相似。(4)三七不同部位均具有其相对稳定的HPLC指纹特征,其中花、叶具有特有的指纹区,根、须根、剪口、筋条等不同商品规格的HPLC指纹图谱比较相似。结论基因序列标记能从分子水平定性分辨三七及其伪品的遗传背景差异,为中药品种标准化提供了先进可行、稳定可靠的分子标准;HPLC指纹图谱分析可以直观地为三七的化学成分定性,三七不同商品规格的特征性指纹有望成为以其为原材料的各种产品的质控标准,而三七不同部位(尤其是花和叶)的HPLC指纹图谱将有望成为制定三七花、三七叶新药用资源质控标准的依据。     

基于高效液相色谱法定性定量跟踪的大黄结合型与游离型蒽醌分离及纯化初步研究

目的：考察大黄中结合型与游离型蒽醌分离及纯化方法。方法：采用高效液相色谱（HPLC）法对大黄药材中8个结合型蒽醌及5个游离型蒽醌含量进行测定,采用Triple-Q-TOF/MS技术对大黄药材色谱图中主要色谱峰成分进行鉴定,95%乙醇提取大黄药材中的蒽醌类成分,提取液回收乙醇后加水沉淀,分离为结合型蒽醌和游离型蒽醌2个部位,分别采用碱性醇沉法和氯仿-酸水双相萃取法对结合型蒽醌和游离型蒽醌进行初步纯化,并结合HPLC-DAD法在280 nm和430 nm 2个波长下进行定性定量跟踪。结果：大黄药材中检测到的43个主要成分全部转移到了提取液中,经纯化处理后,结合型蒽醌部位杂质明显减少,游离型部位纯度从19.98%提高到92.97%。结论：考察的方法可用于大黄结合型与游离型蒽醌初步分离及纯化。     

Sequencing analysis of the medicinal plant Stemona tuberosa and five related species existing in Thailand based on trnH-psbA chloroplast DNA

In Thailand, Stemona tuberosa Lour. , S. phyllantha Gagnep. , S. collinsae Craib , S. burkillii Prain , S. aphylla Craib and S. sp. are found. The identification based on morphological characters alone is difficult and can lead to confusion regarding chemical constituents and biological activities. The tuberous roots of S. tuberosa have long been used for treatment of respiratory diseases and as anthelmintics. However, accurate identification of S. tuberosa is needed to ensure efficacy. Sequence comparison indicated that these Stemona spp. could be identified from the sequence of the trnH- psbA locus. As a result of different sequence lengths, the PCR products generated from newly designed primers could be used to preliminarily group the two species, S. tuberosa and S. phyllantha, apart from others. However, these products could be further sequenced to discriminate among Stemona spp.     

Genetic identification of cinnamon (Cinnamomum spp.) based on the trnL-trnF chloroplast DNA

Genetic identification among cinnamon species was studied by analyzing nucleotide sequences of chloroplast DNA from four species (Cinnamomum cassia, C. zeylanicum, C. burmannii and C. sieboldii). The two regions studied were the intergenic spacer region between the trnL 3'exon and trnF exon (trnL -trnF IGS) and the trnL intron region. We found nucleotide variation at one site in the trnL-trnF IGS, and at three sites in the trnL intron. With the sequence data from analysis of these regions, the four Cinnamomum species used in this study were correctly identified. Furthermore, single-strand conformation polymorphism (SSCP) analysis of PCR products from the trnL-trnF IGS and the trnL intron resulted in different SSCP band patterns among C. cassia, C. zeylanicum and C. burmannii.