Studies on cultivated ephedra plants in inner mongolia autonomous region and ningxia hui autonomous region

Progression of the desertification in northern China has been causing damage to wild Ephedra plants on which we depend for most of supply of the traditional herbal medicine, "Ma huang." The Chinese government encourages the cultivation of Ephedra plants, and Ephedra fields have been reclaimed in the original Ephedra habitats in recent years. We surveyed 7 Ephedra fields that have been recently developed in the Inner Mongolia Autonomous Region and Ningxia Hui Autonomous Region to collect information on Ephedra plant cultivation, especially pertaining to crop species. Specimens taken from those Ephedra fields were genetically and morphologically analyzed, and their ephedrine alkaloid content was examined. DNA analyses of Ephedra specimens, including DNA sequencing of ITS (internal transcribing sequence of nuclear ribosomal DNA) and trn L/F (intron of trnL and intergenic spacer between the trnL and trnF of chloroplast DNA) region and species-specific amplification of trn L/F were conducted to identify Ephedra species. Based on the results of DNA sequencing and morphological determination, the crops grown in 6 fields ware identified as Ephedra sinica, while co-planting of E. sinica and E. intermedia was found in one field where a higher appearance rate of plants with varied morphology from wild Ephedra plants was observed. Furthermore, direct sequencing of the PCR product of the trn L/F region of some specimens from the field and their species-specific PCR showed ambivalent result. Cloning and sequencing of the PCR product of the trn L/F region of those specimens DNA suggested their heteroplasmy, containing both E. sinica- and E. intermedia-type chloroplasts. On the other hand, the profile of the ephedrine alkaloid content was clearly correlated with the result of direct sequencing of the trn L/F region; the specimens showing the E. sinica-type sequence contained more ephedrine than pseudoephedrine, and the specimens of the E. intermedia-type more pseudoephedrine.     

DNA barcodes for discriminating the medicinal plant Scutellaria baicalensis (Lamiaceae) and its adulterants

Scutellaria baicalensis GEORGI (Lamiaceae) is the botanical origin of the well-known traditional Chinese medicine "Huang Qin" (Radix Scutellariae). Due to overexploitation that had induced a decline in natural sources, the dried roots of its congeners, S. amoena, S. rehderiana, and S. viscidula, have been used to adulterate it in recent years. This practice may cause a series of inconsistent therapeutic effects and quality control problems in the herbal medicine industry. Hence, we sequenced and analyzed three candidate DNA barcodes, the ribosomal RNA maturase gene (matK), the ribulose-1,4-bisphosphate carboxylase large subunit gene (rbcL), and the psbA-trnH intergenic spacer (psbA-trnH), to discriminate S. baicalensis and its adulterants. All candidate DNA barcodes had been successfully amplified from leaf samples. Comparatively, only psbA-trnH had been yielded from commercially prepared crude drug samples. Based on the sequence divergence, rbcL can assign S. baicalensis and its adulterants into the correct family and genus, whereas, either matK or psbA-trnH can accurately discriminate S. baicalensis and its adulterants. We proposed the multilocus barcodes rbcL+psbA-trnH for the species identification of S. baicalensis and its adulterants, and the unique barcode psbA-trnH for the authentication of commercial Radix Scutellariae. The DNA barcoding technique could be applied to the quality control of "Huang Qin"-based medicinal preparations and to the management of medicinal herb trade in the markets.     

ITS2+psbA-trnH复合序列鉴定徐长卿、白薇和白前

目的：建立以ITS2+psbA-trnH复合序列鉴定徐长卿、白薇和白前及其同属近缘混伪品的DNA条形码鉴定方法。方法：搜集徐长卿、白薇、白前及其近源混伪品,采用改良的CTAB法提取DNA,通过实验分别获得ITS2和psbA-trnH序列,将同一样本的ITS2序列与psbA-trnH序列整合得到43条ITS2+psbA-trnH复合序列,从GenBank数据库中下载来源于同一样本的ITS2序列与psbA-trnH序列整合得到14条ITS2+psbA-trnH 网上复合序列,用MEGA 6.05软件分析徐长卿、白薇、白前及其近源混伪品的复合序列变异位点,计算种内、种间的K2P距离,并构建NJ系统聚类树。结果：徐长卿、白薇和白前药材ITS2+psbA-trnH复合序列比对后产生17个变异位点;徐长卿、白薇和白前基源物种种内最大K2P距离均小于其与混伪品的种间最小K2P距离;系统NJ树能准确将徐长卿白薇和白前及其近缘混伪品。结论：该研究建立了应用ITS2+psbA-trnH复合序列鉴定徐长卿、白薇、白前及其近缘混伪品的DNA条形码鉴定方法。     

Survey on resources of Ephedra plants in Xinjiang

The resources of wild Ephedra plants in the Xinjiang Uygur Autonomous Region were surveyed. Ephedra plants mainly grow on the fringes of the Taklimakan Desert and Gureban-tonggute Desert. We found six genotypes of Ephedra przewalskii growing widely in Xinjiang. Three genotypes of Ephedra intermedia were limited to the northern and eastern parts, and Ephedra regeliana scattered in the northern part of Xinjiang. These Ephedra specimens were analyzed for DNA sequences of nuclear ribosomal DNA, internal transcribed spacers 1 and 2, chloroplastic DNA, trnL intron and trnL-trnF intergenic spacer. Intraspecific variation of the nucleotide sequence in E. przewalskii was found in different habitats. Norephedrine, ephedrine, pseudoephedrine, and methylephedrine contents of the specimens were determined. Although Ephedra intermedia of all three genotypes contained ephedrine alkaloids, ephedrine alkaloids were not detected in E. regeliana and E. przewalskii.     

Species identification of medicinal pteridophytes by a DNA barcode marker, the chloroplast psbA-trnH intergenic region

Medicinal pteridophytes are an important group used in traditional Chinese medicine; however, there is no simple and universal way to differentiate various species of this group by morphological traits. A novel technology termed "DNA barcoding" could discriminate species by a standard DNA sequence with universal primers and sufficient variation. To determine whether DNA barcoding would be effective for differentiating pteridophyte species, we first analyzed five DNA sequence markers (psbA-trnH intergenic region, rbcL, rpoB, rpoC1, and matK) using six chloroplast genomic sequences from GeneBank and found psbA-trnH intergenic region the best candidate for availability of universal primers. Next, we amplified the psbA-trnH region from 79 samples of medicinal pteridophyte plants. These samples represented 51 species from 24 families, including all the authentic pteridophyte species listed in the Chinese pharmacopoeia (2005 version) and some commonly used adulterants. We found that the sequence of the psbA-trnH intergenic region can be determined with both high polymerase chain reaction (PCR) amplification efficiency (94.1%) and high direct sequencing success rate (81.3%). Combined with GeneBank data (54 species cross 12 pteridophyte families), species discriminative power analysis showed that 90.2% of species could be separated/identified successfully by the TaxonGap method in conjunction with the Basic Local Alignment Search Tool 1 (BLAST1) method. The TaxonGap method results further showed that, for 37 out of 39 separable species with at least two samples each, between-species variation was higher than the relevant within-species variation. Thus, the psbA-trnH intergenic region is a suitable DNA marker for species identification in medicinal pteridophytes.     

基于叶绿体psbA-trnH基因间区序列鉴定肉苁蓉属植物

肉苁蓉的干燥肉质茎是常用中药材, 药典收录基源植物有管花肉苁蓉和荒漠肉苁蓉两种, 但是市场上经常会与黄花列当、草苁蓉、盐生肉苁蓉及沙苁蓉等混用。本文对不同类群肉苁蓉及混淆品的叶绿体psbA-trnH基因间区进行PCR扩增并测序, 获得了该区间的完整序列, 用K-2P法建立了系统进化树, 聚类结果与形态分类相符。所得结果显示, psbA-trnH片段序列在肉苁蓉及混淆品种间存在丰富的变异, 肉苁蓉属各种的种间遗传距离从0.077% 到0.743%, 种内遗传距离从0% 到0.007%, 种内和种间存在明显的“barcoding gap”。肉苁蓉属各种与黄花列当的遗传距离为0.979% 至1.149%, 与草苁蓉的遗传距离为1.066% 至1.224%。研究结果表明psbA-trnH基因间区可以作为条形码来鉴定肉苁蓉及其混淆品。     

Genetic diversity of Ephedra plants in mongolia inferred from internal transcribed spacer sequence of nuclear ribosomal DNA

Ephedrae herba has been used for treating colds, relieving coughs and asthma from ancient times. We previously reported the distribution of Ephedra sinica, E. equisetina, E. przewalskii, E. regeliana, E. monosperma and Ephedra sp. in Mongolia, and among them E. sinica and E. equisetina were potential new resources of Ephedrae herba of Japanese pharmacopoeia grade, based on our field survey and subsequent molecular and chemical assessments. However, the Ephedra population in southwestern areas showed a high possibility of having hybrid origins. Further field surveys in southwestern areas, and sequence analysis of the partial nuclear internal transcribed spacer 1 (ITS1) region, besides trnK and 18S ribosomal RNA (rRNA) gene regions, were conducted in order to obtain detailed evidence of hybridization status. As a result, the distribution of E. glauca in western area and E. lomatolepis in western-most area was confirmed. The ITS sequences from all 8 Ephedra species collected in Mongolia were roughly divided into 5 types (types I-V). Type II sequence, having several additive nucleotides, was found in Ephedra sp., E. glauca, E. regeliana and E. sinica, which provided useful information for tracing hybrid origins. Morphological, genetic and distribution evidence suggested that the hybridization of Ephedra species occurred widely in southwestern Mongolia, and several Ephedra species including E. przewalkskii and E. intermedia were involved in these events. Integrated with our previous report, trnK-, 18S- and ITS-types from pure lines of each species are proposed. In addition, we propose a practicable method for detecting additive peaks on a direct sequencing electropherogram.     

Survey of <Emphasis Type="Italic">Ephedra </Emphasis>resources in the Northern Areas of Pakistan and their genetic diversity

More than 400 species of medicinal plants grow in the Northern Areas of Pakistan, including Ephedra plants. To investigate the wild Ephedra plant resources in the area, we surveyed the medicinal plants and collected 71 specimens from 18 collecting sites to analyze their genetic variation. The DNA sequences of the internal transcribed spacers 1 and 2 (ITS 1 and 2) of nuclear ribosomal DNA and a noncoding sequence of chloroplast DNA (trn L/F) were analyzed. This DNA data analysis and external morphological features were used to confirm the species of the specimens, and it was found that E. intermedia was the major species in the area and that E. gerardiana and E. przewalskii were present sporadically. Although it inhabits a relatively small area in comparison with the northwestern Chinese provinces, the DNA sequence of E. intermedia in the Northern Areas of Pakistan was significantly more heterogeneous than the same species grown in those neighboring regions. Most of the E. intermedia specimens contained more than 0.7% ephedrine alkaloids, fulfilling the requirement of the Japanese Pharmacopoeia; thus, the Ephedra plants in the area are a genetic and medicinal resource of great importance.     

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.     

Molecular authentication of the ethnomedicinal plant Sabia parviflora and its adulterants by DNA barcoding technique

Sabia parviflora Wall. ex Roxb. is a traditional herb widely used by Chinese people, especially by the Buyi ethnic group which resides in Guizhou and Yunnan provinces. According to the Chinese Ethnic Pharmacopeia, the species is commonly used for soothing the liver and for the treatment of icteric hepatitis, hemostasis, and inflammation. However, due to the similar morphological characters of Sabia species and higher market demands, there are many substitutes and adulterants of S. parviflora. In this study, the differential identification of 6 Sabia species and 7 adulterants were investigated through DNA sequence analysis of three candidate DNA barcodes (trnH-psbA, rbcL-α, matK). Based on sequence alignments, we concluded that not only the trnH-psbA spacer sequence can distinguish S. parviflora from other Sabia species, but the matK + rbcL-α sequences also can differentiate it from the substitutes and adulterants. The classification tree of all samples based on rbcL-α sequences indicated that the rbcL region can identify samples into a family/genus level. Our results suggest that the three candidate barcodes can be used for the identification of S. parviflora and to distinguish it from common substitutes or adulterants.     

Authentication of the traditional medicinal plant Eleutherococcus senticosus by DNA and chemical analyses

Shigoka (SGK), the rhizome of Eleutherococcus senticosus, is a traditional medicine used as a tonic in northeastern Asia and far eastern Russia. We analyzed the nuclear ribosomal DNA internal transcribed spacer (ITS) sequence of the medicine available on the Japanese and Chinese markets and found that at least 3 species were used as the source plant of the commercial SGKs and that only 70% of all samples was made from the correct species. Furthermore, we performed the quantitative determination of 3 marker compounds, eleutheroside B (EB), syringaresinol diglucoside (Syr), and isofraxidin (Iso) by ultraperformance liquid chromatography (UPLC)/mass spectrometry (MS). We found that EB and Iso are specific to the correct source plant of SGK. Of them, EB is thought to be the best marker compound for quality assurance of the SGK from the viewpoint of its pharmacological activity.     

DNA条形码鉴定洋金花及其伪品

为建立有毒中药洋金花及其伪品DNA条形码鉴定方法,采用国际通用的条形码序列ITS2、psbA-trnH、matK和rbcL对洋金花原植物白花曼陀罗及其伪品毛曼陀罗、曼陀罗和木本曼陀罗4个种共计20份材料进行了比较研究。PCR及测序成功率分别为ITS2(100%)、matK(100%)、psbA-trnH(90%)、rbcL(85%)。采用CodonCode Aligner进行序列拼接,采用MEGA 4.1计算白花曼陀罗及其伪品的种内、种间的K2P距离,并基于K2P模型构建NJ树。结果显示ITS2序列共有30个单核苷酸多态性(SNPs)位点、psbA-trnH序列有33个单碱基的插入和缺失I。TS2和psbA-trnH序列种间遗传距离大于种内,matK和rbcL种内和种间没有明显Barcoding Gap。4个条形码序列及其组合获得的分子系统树(ITS2、psbA-trnH、matK、rbcL、matK+rbcL)均分成了两大支,木本曼陀罗单聚为一支,该分子证据支持将Brugmansia提升为属水平。实验结果表明ITS2及psbA-trnH序列可以作为洋金花及其伪品鉴定用的条形码序列。     

DNA barcode and identification of the varieties and provenances of Taiwan's domestic and imported made teas using ribosomal internal transcribed spacer 2 sequences

The major aim of made tea identification is to identify the variety and provenance of the tea plant. The present experiment used 113 tea plants [Camellia sinensis (L.) O. Kuntze] housed at the Tea Research and Extension Substation, from which 113 internal transcribed spacer 2 (ITS2) fragments, 104 trnL intron, and 98 trnL-trnF intergenic sequence region DNA sequences were successfully sequenced. The similarity of the ITS2 nucleotide sequences between tea plants housed at the Tea Research and Extension Substation was 0.379–0.994. In this polymerase chain reaction-amplified noncoding region, no varieties possessed identical sequences. Compared with the trnL intron and trnL-trnF intergenic sequence fragments of chloroplast cpDNA, the proportion of ITS2 nucleotide sequence variation was large and is more suitable for establishing a DNA barcode database to identify tea plant varieties. After establishing the database, 30 imported teas and 35 domestic made teas were used in this model system to explore the feasibility of using ITS2 sequences to identify the varieties and provenances of made teas. A phylogenetic tree was constructed using ITS2 sequences with the unweighted pair group method with arithmetic mean, which indicated that the same variety of tea plant is likely to be successfully categorized into one cluster, but contamination from other tea plants was also detected. This result provides molecular evidence that the similarity between important tea varieties in Taiwan remains high. We suggest a direct, wide collection of made tea and original samples of tea plants to establish an ITS2 sequence molecular barcode identification database to identify the varieties and provenances of tea plants. The DNA barcode comparison method can satisfy the need for a rapid, low-cost, frontline differentiation of the large amount of made teas from Taiwan and abroad, and can provide molecular evidence of their varieties and provenances.     

DNA barcode and identification of the varieties and provenances of Taiwan’s domestic and imported made teas using ribosomal internal transcribed spacer 2 sequences

The major aim of made tea identification is to identify the variety and provenance of the tea plant. The present experiment used 113 tea plants [Camellia sinensis (L.) O. Kuntze] housed at the Tea Research and Extension Substation, from which 113 internal transcribed spacer 2 (ITS2) fragments, 104 trnL intron, and 98 trnL-trnF intergenic sequence region DNA sequences were successfully sequenced. The similarity of the ITS2 nucleotide sequences between tea plants housed at the Tea Research and Extension Substation was 0.379–0.994. In this polymerase chain reaction-amplified noncoding region, no varieties possessed identical sequences. Compared with the trnL intron and trnL-trnF intergenic sequence fragments of chloroplast cpDNA, the proportion of ITS2 nucleotide sequence variation was large and is more suitable for establishing a DNA barcode database to identify tea plant varieties. After establishing the database, 30 imported teas and 35 domestic made teas were used in this model system to explore the feasibility of using ITS2 sequences to identify the varieties and provenances of made teas. A phylogenetic tree was constructed using ITS2 sequences with the unweighted pair group method with arithmetic mean, which indicated that the same variety of tea plant is likely to be successfully categorized into one cluster, but contamination from other tea plants was also detected. This result provides molecular evidence that the similarity between important tea varieties in Taiwan remains high. We suggest a direct, wide collection of made tea and original samples of tea plants to establish an ITS2 sequence molecular barcode identification database to identify the varieties and provenances of tea plants. The DNA barcode comparison method can satisfy the need for a rapid, low-cost, frontline differentiation of the large amount of made teas from Taiwan and abroad, and can provide molecular evidence of their varieties and provenances.     

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 diversity of 5S-rRNA spacer domain in Fritillaria species revealed by PCR analysis.

Beimu (bulbs of Fritillaria) is an important traditional Chinese herbal medicine commonly used as an antitussive and expectorant. There are about 25 species and varieties of Fritillaria that carry the name Beimu on commercial markets. The price for each Beimu may differ by more than 100-fold. However, the identification of the origin of a particular species on the market is difficult. Here, a molecular method was used to identify various species of Fritillaria regardless of their geographical origin. The 5S-rRNA coding sequence is highly conserved in higher eukaryotes, but the spacer sequence of the 5S-rRNA gene is variable among different species. Total genomic DNA from fresh leaves and bulbs of Fritillaria cirrhosa, F. puqiensis, F. anhuiensis, and F. thunbergii was extracted. The 5S-rRNA spacer region of the extracted DNAs was amplified by PCR with a pair of primers located within the conserved coding region. The isolated cDNA clones (approximately 600 bp) covering the 5S-rRNA spacer domain were sequenced. By aligning the isolated nucleotide sequences of the four Fritillaria species, sequence diversity was found in the spacer region. Furthermore, a unique EcoR I site was used for the rapid identification of different species of Fritillaria. To our knowledge, this is the first report on the detection of 5S-rRNA spacer domain sequences of Fritillaria and their use to identify species.     

Structure of feruloylhistamine, a hypotensive principle of Ephedra roots

From the crude drug "maō-kon", the underground part of Ephedra plant, we have isolated a new hypotensive imidazole alkaloid feruloylhistamine the structure of which has been determined as shown in formula I based on chemical and physical evidence.     

5SrRNA基因间隔区DNA序列在穿心莲道地性中的可行性研究

目的探讨5SrRNA基因间隔区DNA序列在穿心莲道地性研究中的可行性。方法提取不同产地穿心莲总DNA后,PCR特异扩增5SrRNA基因间隔区,采用荧光标记末端终止子双脱氧末端终止法测序,对测序结果进行多序列联配分析。结果成功得到321bp的5SrRNA基因间隔区碱基序列,经过多序列联配分析发现,不同产地穿心莲的此段5SrRNA基因间隔区DNA序列之间没有差异。结论5SrRNA基因间隔区DNA序列不适合用于研究穿心莲药材的道地性。     

重楼属药用植物DNA条形码鉴定研究

为评价DNA条形码候选序列对重楼属药用植物的鉴定作用, 探讨重楼属药用植物鉴定新方法, 本研究对重楼属11个物种17份样品的psbA-trnH、rpoB、rpoC1、rbcL、matK和核ITS2序列进行PCR扩增和测序, 比较各序列扩增和测序效率、种内和种间变异, 进行barcoding gap分析, 采用BLAST1和Nearest Distance方法评价不同序列的鉴定能力。结果显示, ITS2序列在所研究的重楼属药用植物中的扩增和测序效率均为100%, 其种内种间变异、barcoding gap与其他DNA条形码候选序列相比具有明显的优势, ITS2序列在重楼属中的鉴定成功率达到100%, 而生物条形码协会 (CBOL) 植物工作组推荐的matK和rbcL序列的鉴定成功率分别为52.9% 和5.9%, 二者联合鉴定能力没有提高, 对于ITS2序列扩大至29个物种67份样品依然具有100%的鉴定成功率。实验结果表明, ITS2序列能够准确鉴定重楼属药用植物, 可以作为潜在的药用植物通用条形码序列。     

Phylogenetic analysis of the DNA sequence of the non-coding region of nuclear ribosomal DNA and chloroplast of Ephedra plants in China

Twenty-four Ephedra plants belonging to 8 species grown in the northern and western parts of China were phylogenetically analyzed for their non-coding DNA sequences, internal transcribed spacers (ITSs) of nuclear ribosomal DNA as well as trnL intron and intergenic spacers between trnL and trnF (trnL/ trnF) of the chloroplast. Based on the ITS sequences, the 8 species could be divided into 3 groups: Group 1 (Ephedra intermedia, E. sinica, E. przewalskii), Group 2 (E. equisetina, E. monosperma, E. gerardiana), and Group 3 (E. likiangensis, E. minuta). The species classified into Group 1 grow mainly in the north, Group 3 in the south and Group 2 in the center, suggesting their genetic and geographic relationships. A specific primer set was designed to classify the 3 groups by routine PCR. Combined analysis of ITS and trnL/ trnF differentiated the 8 Ephedra species.