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.     

Peyote identification on the basis of differences in morphology, mescaline content, and trnL/trnF sequence between Lophophora williamsii and L. diffusa

Genus Lophophora (Cactaceae) has two species: Lophophora williamsii Coulter, which is called peyote, and L. diffusa Bravo. Although it was reported that L. williamsii contained mescaline and L. diffusa did not, we found L. williamsii specimens that did not contain mescaline. This finding indicated that the two species could not be differentiated in terms of mescaline content. Moreover, the relationship between mescaline content and morphology of the two species is also unknown. In this study, we attempted to clarify the difference in morphology, mescaline content, and DNA alignment of the chloroplast trnL/trnF region between L. williamsii and L. diffusa. As a result, L. williamsii specimens were classified into two groups. Group 1 had small protuberances on the epidermis, contained mescaline, and the analyzed region on the trnL/trnF sequence was 881 base pairs (bp) long in all except one (877?bp). Group 2 had large protuberances on the epidermis, did not contain mescaline, and the analyzed region was 893?bp long. On the other hand, L. diffusa had medium-sized protuberances on the epidermis, did not contain mescaline, and the analyzed region was 903?bp long. Also investigated was the potential application of the PCR-restriction fragment length polymorphism (RFLP) method as a means of identification based on the trnL/trnF sequence. By applying the PCR-RFLP method, the two species could be distinguished and L. williamsii specimens could be differentiated into group 1 and group 2.     

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.     

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.     

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.     

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.     

Intraspecific variation in Cannabis sativa L. based on intergenic spacer region of chloroplast DNA

We analyzed the nucleotide sequences of the non-coding region of chloroplast DNA: the intergenic spacer between trnL (UAA) 3'exon and trnF (GAA). Two kinds of sequence, "type-1" and "type-2," were detected in 33 populations of Cannabis sativa. The length of the "type-1" fragment was 354 bp. In contrast, the "type-2" fragment from 3 populations was 353 bp long, with only one base deletion compared to "type-1." The fragment length from Humulus lupulus was 353 bp with a 1-bp deletion, and ten 1-bp substitutions compared to the sequences from C. sativa "type-1." Furthermore, we could clearly identify differences between C. sativa and H. lupulus using single-strand conformation polymorphism of PCR products (PCR-SSCP) analysis.     

A molecular marker that is specific to medicinal rhubarb based on chloroplast trnL/trnF sequences.

"Da-Huang" (Radix et Rhizoma Rhei, medicinal rhubarb), a famous and important Traditional Chinese Medicine, has often been confused with the adulterant species in the same genus, Rheum. Through sequencing the trnL (UAA)/trnF (GAA) regions of chloroplast DNA of thirteen species of Rheum (three medicinal rhubarb species and ten adulterant ones), a molecular marker of the medicinal species was found. A pair of PCR primers based on the sequences, was thus designed, which amplified a highly specific DNA fragment in medicinal rhubarb exclusively, and absent in the adulterants at all under an optimized PCR condition.     

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.     

不同产地蒺藜核糖体DNA内转录间隔区序列分析

目的通过测定核糖体DNA内转录间隔区(Ribosomal DNA internal transcribed spacer,rDNA-ITS)基因序列,确定不同产地的蒺藜在种质遗传上是否存在差异。方法利用PCR产物直接测序,测定不同产地蒺藜的rDNA-ITS基因序列。结果测得ITS碱基序列742 bp,其中ITS1全部序列267 bp,5.8 S全部序列167bp,ITS2全部序列209 bp。6个产地的蒺藜样品的ITS的碱基序列完全相同。结论不同地区的蒺藜在种质上没有发生变异。     

Molecular authentication of the traditional Chinese medicinal plant Euphorbia pekinensis

The ITS regions of Euphorbia pekinensis and six other Euphorbia species used as adulterants of E. pekinensis were sequenced to differentiate them. The sequences are identical among the individuals in the seven species studies. Diversity in DNA sequences among various species was found ranging from 8.3% to 43.8% in ITS1 and 7.6% to 36.6% in ITS2 region. Furthermore, based on the divergent ITS regions, species-specific primers, JDJp 1 and JDJp 2, were designed in the polymorphic regions of E. pekinensis to distinguish it from adulterants. These ITS-derived primers amplified a 281-bp-specific DNA fragment from E. pekinensis. No amplified product was observed using DNA of six adulterants.     

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.     

Phylogeny of medicinal Phyllanthus species in China based on nuclear ITS and chloroplast atpB-rbcL sequences and multiplex PCR detection assay analysis

Plants of the genus Phyllanthus are commonly used in India, China and Korea for medicinal purposes. Although they are widely cultivated and marketed, there has been uncertainty about the efficacy of different species. A prerequisite of Good Agricultural Practice (GAP) is the authentication of relevant species, and this is now made unequivocal by applying DNA sequence tools. In this study the phylogenetic relationships among 18 Phyllanthus species found in China were investigated by DNA sequence analyses of the nuclear internal transcribed spacers (ITS1 and ITS2) along with the combined chloroplast ATPB and RBCL sequences. Cladistic analysis indicated that this genus is paraphyletic and strongly supports the notion that two problematic and confusing species, P. niruri and P. amarus, are two individual, albeit closely related, species. We have also developed an ITS rDNA-based multiplex PCR assay to differentiate three medicinally important species, P. amarus, P. niruri and P. urinaria.     

DNA microarray for identification of the herb of dendrobium species from Chinese medicinal formulations

A DNA microarray for detecting processed medicinal Dendrobium species (Herba Dendrobii) was constructed by incorporating the ITS1-5.8S-ITS2 sequences of 16 Dendrobium species on a glass slide. Using fluorescence-labeled ITS2 sequences as probes, distinctive signals were obtained for the five medicinal Dendrobium species listed in the Chinese Pharmacopoeia. The established microarray was able to detect the presence of D. nobile in a Chinese medicinal formulation containing nine herbal components.     

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。     

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.     

Novel polymorphism of internal transcribed spacers (ITS) and their utilization in phylogenetic analysis of Neanthes glandicincta (Annelida: Polychaeta: Nereididae)

Sequences of internal transcribed spacers (ITS1 and ITS2) are increasingly being used to infer phylogenetic relationships at or below species levels. Here we report a novel case of ITS polymorphism within Neanthes glandicincta (Annelida: Polychaeta: Nereididae). Two types of ITS sequence (Type I and Type II) were cloned and sequenced, which showed significant differences both in nucleotide composition and length. Variations of these two types sequences also differed from each other with Type I was highly divergent while Type II was highly conserved. Phylogenetic trees inferred from ITS1 and ITS2 sequences showed striking discrepancy in N. glandicincta. Non-concerted evolution of multi-gene is suggested to be responsible for the high degree of polymorphism in ITS regions. Due to the two divergent types of ITS presented within a single N. glandicincta individual, the utilization of ITS regions for delineation of population or closely related species cannot be substantiated. The finding of different types of ITS in a single individual also stresses the need for analyzing a large number of clones whenever ITS sequences obtained by PCR amplification and cloning are being used in phylogenetic reconstruction.     

Oxidative DNA damage and total antioxidant status in rats during experimental gram-negative sepsis

Sepsis and septic shock remains as leading cause of death in adult intensive care units. It is widely accepted that gram-negative bacteria and their endotoxins cause sepsis and septic shock, predominantly. Enhanced generation of reactive oxygen species may be responsible for tissue injury in septic shock and endotoxemia. The aim of this study was to assess oxidative DNA damage and the total antioxidant status (TAS) in peripheral lymphocytes of rats during different intraperitoneal gram-negative sepsis stages. Adult male Sprague-Dawley rats were divided randomly into four groups. Control group was intraperitoneally inoculated with 2 mL of pyrogene-free saline (Group I, n = 6), and the other rats received an intraperitoneal inoculum with 2 mL of saline containing 2 x 10(8) CFU of Escherichia coli. The animals were killed at time zero (Group I, n = 6), at 6th (Group II, n = 7), 12th (Group III, n = 7), and 24th (Group IV, n = 7) hour after the E. coli inoculation. Oxidative DNA damage in peripheral lymphocytes of rats was evaluated by modified comet assay (single-cell gel electrophoresis). Formamidopyrimidine DNA glycosylase (Fpg) and Endonuclease III (Endo III) were used to detect oxidized purines and pyrimidines, respectively. Total antioxidant quantification was carried out using ABTS+ (2,2'-Azino-di-[3 ethylbenzthiazoline sulphonate]) radical formation kinetics (Randox kit) in serum samples. Significant elevations of basal levels of strand breaks (SB) in Group IV were observed as compared with Group I, II, and III. There was a significant increase in Fpg sites in Group III as compared with Group I and II. However, there was no significant difference in terms of Endo III sites in any of the groups. Although the TAS was decreased with the stages of sepsis, this moderate decrease was significant in only Group IV as compared with Group I. There was no statistically significant correlation between DNA damage and TAS for any of the groups.     

The use of polymerase chain reaction (PCR) for the identification of ephedra DNA in dietary supplements

As part of a continuing research effort to develop chemical and genetic authentication profiles of botanicals, an investigation was performed with the goal to detect, identify and verify Ephedra sinica Stapf DNA in dietary supplements such as plant mixtures and tablets/capsules. We amplified and sequenced the chloroplast psbA-trnH spacer from 21 Ephedra spp. and from two of their closest relatives, Gnetum gnemon L. and Welwitschia mirabilis Hook. Based on sequence comparisons, we identified regions unique to all of the Ephedra spp. samples analyzed. We concluded that the psbA-trnH spacer sequence could be used as a molecular marker. Based on this spacer sequence, we designed Ephedra spp.-specific primers that can help to identify Ephedra spp. DNA in plant mixtures containing as little as 1/1,000 part of Ephedra spp. tissue. We used a DNA extraction method that allows for quick DNA isolation from plant mixtures for PCR analysis.     

Authentication of medicinal Dendrobium species by the internal transcribed spacer of ribosomal DNA

Herba Dendrobii (Shihu) is a commonly used Chinese medicine derived from the stem of several orchid species belonging to the genus Dendrobium. It is rather expensive and adulteration is frequent. Proper authentication of the medicinal species is necessary to protect consumers and support conservation measures. DNA sequences of the internal transcribed spacer 2 (ITS 2) of 16 Dendrobium species were shown to be significantly different from one another by an average of 12.4% and from non-orchids and Pholidota (an adulterant of Shihu) by 29.8% and 18.8%, respectively. The intra-specific variation among the Dendrobium species studied was only about 1%. Therefore, ITS 2 regions could be adopted as a molecular marker for differentiating medicinal Dendrobium species from one another and also from non-orchids and adulterants.