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.     

Phylogenetic relationship in the genus Panax: inferred from chloroplast trnK gene and nuclear 18S rRNA gene sequences

Chloroplast trnK gene and nuclear 18S rRNA gene sequences of 13 Panax taxa, collected mainly from Sino-Japanese floristic region, were investigated in order to construct phylogenetic relationship and to assist taxonomic delimitation within this genus. The length of trnK gene sequence varied from 2537 bp to 2573 bp according to the taxa, whereas matK gene sequences, embedded in the intron of trnK gene, were of 1512 bp in all taxa. Species-specific trnK/ matK sequence provided much insight into phylogeny and taxonomy of this genus. 18S rRNA gene sequences were of 1808 or 1809 bps in length, only 9 types of 18S rRNA sequences were observed among 13 taxa. Parsimony and neighbor-joining analyses of the combined data sets of trnK-18S rRNA gene sequences yielded a well-resolved phylogeny within genus Panax, where three main clades were indicated. P. pseudoginseng and P. stipuleanatus formed a sister group located at a basal position in the phylogenetic tree, which suggested the relatively primitive position of these two species. Monophyly of P. ginseng, P. japonicus (Japan) and P. quinquefolius, which are distributed in northern parts of Asia or America, was well supported (Northern Clade). The remaining taxa distributed in southern parts of Asia formed a relatively large clade (Southern Clade). The taxonomic debated taxa traditionally treated as subspecies or varieties of P. japonicus or P. pseudoginseng showed various nucleotide sequences, but all fell into one cluster. It might suggest these taxa are differentiated from a common ancestor and are in a period of high variation, which is revealed not only on morphological appearance, but also on molecular divergence. By comparing trnK and 18S rRNA gene sequences among 13 Panax taxa, a set of valuable molecular evidences for identification of Ginseng drugs was obtained.     

广藿香与土藿香的DNA序列分析及其分子鉴别

目前市场上藿香类商品药材有两种 ,一种为唇形科刺蕊草属 (Ｐｏｇｏｓｔｅｍｏｎ)植物广藿香Ｐｏｇｏｓｔｅｍｏｎｃａｂｌｉｎ (Ｂｌａｎｃｏ)Ｂｅｎｔｈ.的干燥地上部分 ,主产广东、海南 ,习称“广藿香”,均为栽培品 ,有芳香化浊、开胃止呕、发表解暑的功效 ,是中成药“藿香正气水”的主要原料。据我们分析广州市郊黄村产“石牌广藿香”药材茎枝挥发油成分 ,其中 71 %为广藿香酮(ｐｏｇｏｓｔｏｎｅ) [1 ] ;另一种来源于同科另一属 ,即藿香属(Ａｇａｓｔａｃｈｅ)植物藿香Ａｇａｓｔａｃｈｅｒｕｇｏｓａ (Ｆｉｓｃｈ.ｅｔＭｅｙ.)Ｏ .Ｋ…     

广藿香的基因序列与挥发油化学型的相关性分析

目的探讨“南药”广藿香Pogostemon cablin (Blanco) Benth.不同产地间的叶绿体和核基因组的基因型与挥发油化学型的关系，为广藿香道地性品质评价、规范化种植提供分子依据。方法用PCR直接测序技术对广藿香6个产地样本的叶绿体matK基因和核18S rRNA基因核苷酸序列进行测序分析研究。结果广藿香6个样本的matK基因序列长均为1 245 bp，编码415个氨基酸成熟酶。18S rRNA基因序列长为1 803～1 805 bp。根据排序比较，广藿香6个样本间的matK基因序列存在47个变异位点，18S rRNA基因存在17个变异位点，非加权组平均法构建的系统分支树表明广藿香基因序列分化与其产地、所含挥发油化学变异类型呈良好的相关性。结论结合挥发油分析数据，基因测序分析技术可作为广藿香道地性品质评价方法这一以及规范化种植过程关键技术“物种鉴定”的强有力工具。     

Rapid detection of Panax ginseng by loop-mediated isothermal amplification and its application to authentication of Ginseng

We have developed a novel method called loop-mediated isothermal amplification (LAMP) to detect Panax ginseng, the botanical source of Ginseng (Ginseng Radix), and to distinguish P. ginseng from Panax japonicus. Six allele-specific primers (two outer primers, two inner primers, and two loop primers) were designed based on the 18S ribosomal RNA gene sequence of P. ginseng, and LAMP was performed using those primers and total DNA extracted from P. ginseng as template. Amplifications were observed from approximately 30 min onwards at DNA concentrations of 0.5 to 10.0 ng. The presence of loop primers shortened the reaction time considerably. In contrast, in the reactions using total DNA from P. japonicus as template, no amplifications were observed. LAMP also enabled us to distinguish Ginseng from Japanese Ginseng (Panacis Japonici Rhizoma). LAMP was proven to be a rapid, highly sensitive, and specific method for the detection of P. ginseng and Ginseng.     

Molecular analysis of medicinally-used Chinese and Japanese Curcuma based on 18S rRNA gene and trnK gene sequences.

Curcuma drugs have been used discriminatingly for invigorating blood circulation, promoting digestion, and as a cholagogic in China. However, there is confusion about the drug's botanical origins and clinical uses because of morphological similarity of Curcuma plants and drugs. In order to develop an ultimate identification, molecular analysis based on 18S rRNA gene and trnK gene sequences were performed on 6 Curcuma species used medicinally in China and Japan. The 18S rRNA gene sequences were found to be of 1810 bps in length. In comparison with the common sequence of C. longa, C. phaeocaulis, C. wenyujin and C. aromatica, that of C. kwangsiensis had one base substitution, and the same base difference was observed between the Chinese and the Japanese populations of C. zedoaria. The trnK gene sequences were found to span 2698-2705 bps. There were base substitutions, small deletions or insertions at some sites between the trnK coding region and matK region among each species. Based on the base substitutions, C. zedoaria and C. kwangsiensis specimens were divided into two groups, respectively. An identical sequence was detected in C. phaeocaulis and in the Chinese population of C. zedoaria, as well as in the Japanese population of C. zedoaria and in one group of C. kwangsiensis with a purple-colored band in leaves. New taxonomic information to be used for authenticating Curcuma drugs was obtained.     

光谱成像结合偏最小二乘判别法快速鉴别西洋参

目的：应用荧光光谱成像技术对不同市售来源的西洋参饮片及其伪品进行检测,结合偏最小二乘判别法区分其真伪,以期实现西洋参饮片的快速无损鉴别。方法：采用凝视式荧光光谱成像装置,对西洋参、人参、桔梗各30份样品进行了荧光光谱成像,提取了其特征光谱曲线。分别采用全光谱偏最小二乘判别（PLS-DA）与联合区间偏最小二乘判别（siPLS-DA）,对3种药材的光谱数据进行了建模分析。结果：相比于PLS-DA方法,siPLS-DA方法提高了模型质量和精度,其在训练集和验证集的识别率分别达到98.33%和96.67%。结论：所建立的模型精度高、预测性能优异,可实现西洋参饮片的快速无损鉴别。     

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。     

Species identification from Ginseng drugs by multiplex amplification refractory mutation system (MARMS)

The multiplex amplification refractory mutation system (MARMS) was applied to the identification of 5 Panax species ( P. ginseng, P. japonicus, P. quinquefolius, P. notoginseng and P. vietnamensis). A set of specific primers, including 2-pair primers on chloroplast trnK gene and nuclear 18S rRNA gene regions, respectively, was designed and synthesized for each species on the basis of species-specific sequences of the 2 genes. By using 5 sets of specific primers, in turn, PCR amplifications were performed with total DNA extracted from 5 Panax species as template under appropriate condition, and each resulting product was detected by agarose gel electrophoresis. The results showed that two expected fragments, one from trnK gene and another from 18S rRNA gene regions, were observed simultaneously only when the set of species-specific primers encountered template DNA of the corresponding species. This assay could give more reliable results for identification of not only 5 Panax species but also corresponding Ginseng drugs by simultaneous detection of 4-site nucleotide differences on 2 completely different genes.     

美国等五国西洋参人参药材中重金属残留量分析

目的建立原子吸收分光光度法测定五国四种人参药材中的铅、镉、砷、汞、铜重金属含量。方法采用原子吸收法在324.8nm波长检测铜含量,在283.3nm波长检测铅含量,在228.8nm波长检测镉含量,另外采用原子荧光光谱法测定砷、汞的含量。结果 24批五国四种人参药材中的重金属残留量西洋参为10.581～12.025mg/kg;红参（高丽参）为12.719～14.002mg/kg。结论五国四种西洋参人参药材样品中重金属残留量均符合2005年版《中国药典》（一部）及香港特区《中成药注册申请手册》的有关重金属标准规定。     

Molecular authentication of Radix Puerariae Lobatae and Radix Puerariae Thomsonii by ITS and 5S rRNA spacer sequencing

In the present study, we examined nuclear DNA sequences in an attempt to reveal the relationships between Pueraria lobata (Willd). Ohwi, P. thomsonii Benth., and P. montana (Lour.) Merr. We found that internal transcribed spacer (ITS) sequences of nuclear ribosomal DNA are highly divergent in P. lobata and P. thomsonii, and four types of ITS with different length are found in the two species. On the other hand, DNA sequences of 5S rRNA gene spacer are highly conserved across multiple copies in P. lobata and P. thomsonii, they could be used to identify P. lobata, P. thomsonii, and P. montana of this complex, and may serve as a useful tool in medical authentication of Radix Puerariae Lobatae and Radix Puerariae Thomsonii.     

基于COⅠ与16S rRNA基因对广地龙的DNA分子鉴定研究

目的:建立一种快速、准确和标准化的广地龙DNA分子标记鉴别方法。方法:测定了5个不同居群广地龙的线粒体细胞色素酶亚单位(CO)Ⅰ和16S rRNA基因序列,采用CodonCode Aligner进行序列拼接,通过下载GenBank地龙原动物的COⅠ与16S rRNA序列,采用MEGA4.1计算广地龙及其伪品地龙的种内、种间的K2P遗传距离,并基于K2P模型构建NJ和MP树。结果:COⅠ变异位点、信息位点均高于16SrRNA,COⅠ基因无插入和缺失,16S rRNA存在4个插入和缺失。COⅠ和16S rRNA序列种间遗传距离均明显大于种内,COⅠ和16S rRNA基因均能将广地龙从其他地龙或蚯蚓物种鉴别开来。结论:获得的广地龙COⅠ和16S rRNA序列可为动物性中药材地龙的分子水平鉴定提供参考,为动物性中药材DNA条形码数据库积累了相关信息数据。     

Detection of genetic homogeneity of Panax notoginseng cultivars by sequencing nuclear 18S rRNA and plastid matK genes

The nuclear 18S rRNA and chloroplast MATK genes of 18 samples of Panax notoginseng and its processed material Sanqi (Radix Notoginseng) were analyzed. The two genes, regardless of cultivar origin, were found to be identical to genotype R1 and M1, respectively, of the published sequences (GenBank accession no. D85171 and AB027526). This phenomenon implies that the species is highly conserved, which is probably caused by the use of the same strain in cultivation and the lack of active mutation in these two genes.     

中日产川芎的matK、ITS基因序列及其物种间的亲缘关系

目的分析中国产川芎Ligusticum chuanxiong Hort.及日本产川芎Cnidium officinale Makino的核基因组ITS和叶绿体基因组matK序列,为探讨中日产川芎物种间的亲缘关系提供分子依据。方法采用PCR直接测序技术测定川芎和日本川芎的ITS基因和matK基因核苷酸序列并作序列变异分析。结果川芎和日本川芎的matK序列长度均为1268 bp,编码422个氨基酸。ITS1-5.8S-ITS2序列长度均为699 bp,其中18S rRNA基因3′端序列54 bp,ITS1序列215 bp,5.8S rRNA基因序列162 bp,ITS2序列222 bp,26S rRNA基因5′端序列46 bp。根据排序比较,川芎原植物与其商品药材间的matK基因和ITS基因序列完全相同,而川芎与日本川芎间matK基因则仅有1个变异位点,即在上游959 nt处1个转换替代(T→C),反映在氨基酸序列则发生一个非同义取代V(GTG)→A(GCG);ITS基因也仅有1个变异位点,即在ITS1上游54 nt处1个转换替代(T→C)。结论通过进化速率较快的基因序列同源性分析,基本可以认为中日所产川芎基原一致,日本川芎学名似应改为Ligusticum chuanxiong Hort.。     

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.     

应用HPLC／ELSD法测定西洋参中拟人参皂苷F11的含量

目的：测定西洋参中拟人参皂苷Ｆ１１的含量。方法：应用高效液相色谱法－蒸发光散射检测器（ＨＰＬＣ／ＥＬＳＤ）,ＰｌａｔｉｎｕｍＣ１８色谱柱,乙腈－水（３０∶７０）为流动相,并优化选择了蒸发光散射检测器参数,测定了西洋参中拟人参皂苷Ｆ１１的含量。结果：拟人参皂苷Ｆ１１在２１９２～１０９６μｇ范围内呈良好线性（ｒ＝０９９９２,ｎ＝５）,回收率为（１０２６±４１）％。结论：本方法精密度、重现性良好,适用于人参皂苷类成分的分析测定。     

DNA sequencing analysis of ITS and 28S rRNA of Poria cocos

We determined the DNA sequences of the internal transcribed spacer 1 and 2 (ITS 1 and 2), the 5.8S rRNA gene and most of the 28S rRNA gene of Poria cocos for the first time, and conducted analysis of 20 samples including cultured mycelias and crude drug materials obtained from various localities and markets. Direct sequencing of the ITS 1 and 2 regions of the samples, except for four wild samples, showed that they had identical DNA sequences for ITS 1 and 2 with nucleotide lengths of 997 bps and 460 bps, respectively. By cloning, the four wild samples were found to have combined sequences of common ITS sequences with 1 or 2-base-pair insertions. Altogether both ITS 1 and 2 sequences were substantially longer than those of other fungal crude drugs such as Ganoderma lucidum and Polyporus umbellatus. Thus, Poria cocos could be distinguished from these crude drugs and fakes by comparing the nucleotide length of PCR products of ITS 1 and 2. Contrary to the basic homogeneity in ITS 1 and 2, three types (Group 1, 2, 3) of the 28S rRNA gene with distinctive differences in length and sequence were found. Furthermore, Group 1 could be divided into three subgroups depending on differences at nucleotide position 690. Products with different types of 28S rRNA gene were found in crude drugs from Yunnan and Anhui Provinces as well as the Korean Peninsula, suggesting that the locality of the crude drugs does not guarantee genetic uniformity. The result of DNA typing of Poria cocos may help discrimination of the quality of the crude drug by genotype.     

Simultaneous determination of ten ginsenosides in panacis quinquefolii radix by ultra performance liquid chromatography and quality evaluation based on chemometric methods

A rapid, sensitive and reliable method based on ultra performance liquid chromatography coupled with a photodiode array detector (UPLC-PAD) was developed for both the quantitative analysis of ten bioactive ginsenosides and a chemical-fingerprint analysis. The chromatography was performed on an ACQUITY UPLC BEH C18 column using a gradient elution with acetonitrile/water as the mobile phase. To compare the UPLC fingerprints and to evaluate their quality, chemometric methods including similarity analysis (SA) and hierarchical-clustering analysis (HCA) were implemented when classifying the Panacis Quinquefolii Radix samples. The Panacis Quinquefolii Radix samples were successfully grouped in accordance with their geographic origins.     

Sequence analysis of Chinese and Japanese Curcuma drugs on the 18S rRNA gene and trnK gene and the application of amplification-refractory mutation system analysis for their authentication

The botanical origins of Chinese and Japanese Curcuma drugs were determined to be Curcuma longa, C. phaeocaulis, the Japanese population of C. zedoaria, C. kwangsiensis, C. wenyujin, and C. aromatica based on a comparison of their 18S rRNA gene and trnK gene sequences with those of six Curcuma species reported previously. Moreover, to develop a more convenient identification method, amplification-refractory mutation system (ARMS) analysis of both gene regions was performed on plants. The ARMS method for the 18S rRNA gene was established using two types of forward primers designed based on the nucleotide difference at position 234. When DNAs of four Curcuma species were used as templates, PCR amplification with either of the two primers only generated a fragment of 912 base pairs (bp). However, when DNAs of the purple-cloud type of C. kwangsiensis and C. wenyujin were used, PCR amplifications with both primers unexpectedly generated the fragment, suggesting that these two were heterozygotes. The ARMS method for the trnK gene was also established using a mixture of four types of specific reverse primers designed on the basis of base substitutions and indels among six species, and common reverse and forward primers. C. phaeocaulis or the Chinese population of C. zedoaria, the Japanese population of C. zedoaria or the purple-cloud type of C. kwangsiensis, the pubescent type of C. kwangsiensis or C. wenyujin, and C. aromatica were found to show specific fragments of 730, 185, 527 or 528, and 641 or 642 bp, respectively. All species including C. longa also showed a common fragment of 897-904 bp. Using both ARMS methods, together with information on producing areas, the identification of Curcuma plants was achieved. Moreover, the ARMS method for the trnK gene was also useful for authentication of Curcuma drugs.     

Molecular analysis of Rheum species used as Rhei Rhizoma based on the chloroplast matK gene sequence and its application for identification

Rhei Rhizoma (Dahuang in Chinese) is widely known as a purgative and antiinflammatory agent. In the Japanese Pharmacopoeia, Rhei Rhizoma is prescribed for four Rheum species, Rheum palmatum, R. tanguticum, R. officinale, and R. coreanum, while the first three species are prescribed for Dahuang in the Chinese Pharmacopoeia. Due to the morphologic similarity of the aerial parts and frequent occurrence of intermediate forms, the taxonomy of this genus and the correct identification of Rheum species and their derivative drugs are very difficult. To resolve taxonomic problems of the genus Rheum and develop an ultimate identification method for plants and drugs, molecular analysis of the chloroplast matK gene and nuclear 18S ribosomal RNA gene were performed on nine species. The sequence comparison of the matK gene revealed that most species had variable sequences not only inter- but also intraspecies. However, the specimens of the same species belonged to the same subclade in the phylogenetic tree constructed based on matK gene sequences, except for R. palmatum, in which specimens belonged to three subclades related to their production areas. The nucleotide differences at positions 587, 707, and 838 distinguished official species from others, while specific nucleotides at positions 367 and 937 became identification markers for R. palmatum, R. tanguticum, and R. officinale (or R. coreanum). Moreover, three groups of R. palmatum, each belonging to three subclades, were characterized by the nucleotides at positions 619, 769, 883, and 1061. By detecting marker nucleotides, the botanical origins of Rhei Rhizoma were determined.