A random amplified polymorphic DNA (RAPD) primer to assist the identification of a selected strain, aizu K-111 of Panax ginseng and the sequence amplified

Panax ginseng is widely used as a Chinese medicine, but it takes a long time to reach harvest and to establish its qualified strains. In the course of searching high quality Panax ginseng, we found a useful random amplified polymorphic DNA (RAPD) primer, which showed a 725 base pair band for a selected elite strain Aizu K-111 (now called Kaishusan) including its cultured tissues, while the other strains did not necessarily show this band. We sequenced the DNA fragment amplified and designed primers to improve electrophoretic profiles, based on the sequence.     

Development of species specific AFLP-derived SCAR marker for authentication of Panax japonicus C. A. MEYER

Panax japonicus is an important medicinal plant. The aim of this study was to develop species-specific molecular markers for P. japonicus. Amplified fragment length polymorphism (AFLP) was compared among P. japonicus, P. ginseng and P. quinquefolius. A clear species-specific AFLP marker for P. japonicus was generated. After isolation and sequencing of the AFLP fragment, a DNA sequence (293 bp) was obtained and named JG14. Oligonucleotide primer (23 mer) was designed for amplifying 191 bp of the sequence of JG14. PCR analysis revealed a clear amplified band for P. japonicus but not in 3 other Panax species (P. ginseng, P. quinquefolius and P. notoginseng). This sequence characterized amplified regions (SCAR) marker will be used for rapid authentication of P. japonicus among other related Panax species. This is the first report of species-specific SCAR marker development in P. japonicus.     

Characters of nrDNA ITS region sequences of fruits of Alpinia galanga and their adulterants.

The fruits of Alpinia galanga (L.) Sw. are used as a traditional Chinese medicine; but the dry fruits of A. conchigera, A. suishaensis, A. maclurei and A. polyantha are also used as the medicine in local areas. Because dry fruits of these related plants are similar to those of Alpinia galanga (L.) Sw. in odor, morphological characters and chemical components, and even anatomical characters, it is difficult to identify the medicine. Nuclear ribosomal DNA internal transcribed spacer (ITS) regions of the five taxa were directly sequenced using an automated sequencer. Sequence analysis showed that the ITS 1 ranges from 177 to 178 base pairs (bp), and the ITS 2 from 225 to 234 bp. The size of the 5.8S coding region is 164 bp for all species. Also, the pairwise sequence divergence is higher and some molecular markers were determined. According to these molecular markers, Alpinia galanga (L.) Sw. and the related species can easily be distinguished from each other. Therefore, evidence from nrDNA ITS sequence variation can identify the medicine at the DNA level.     

Authentication of Panax notoginseng by 5S-rRNA spacer domain and random amplified polymorphic DNA (RAPD) analysis

The great majority of Panax species are well-known herbal medicines in the Orient, and many of them share a close resemblance in appearance and chemical composition. Among these Panax species, the root of P. notoginseng (Sanqi) is a unique herb that has distinct clinical usage. Here, the 5S-rRNA spacer domains were isolated from P. notoginseng, P. japonicus var. major, P. stipuleanatus, P. quinquefolius, P. ginseng, P. zingiberensis, and P. wangianus, and four common adulterants of P. notoginseng including Curcuma wenyujin, Curcuma longa, Bletilla striata and Gynura segetum. The spacer domains were sequenced and compared, which showed over 75 % DNA identity among all Panax species, but not for the adulterants. In addition, random amplification of polymorphic DNA (RAPD) analysis was used to distinguish different members of Panax genus as well as the morphological variants of P. notoginseng. These molecular methods could be used in the authentic identification of P. notoginseng from other Panax species.     

Genetic polymorphism of medicinally-used Codonopsis species in an internal transcribed spacer sequence of nuclear ribosomal DNA and its application to authenticate Codonopsis Radix

Codonopsis Radix has been prescribed as the roots of Codonopsis pilosula, C. pilosula var. modesta and C. tangshen in Chinese Pharmacopoeia. In order to find out genetic markers for identifying the 3 taxa and to authenticate Codonopsis Radix, the molecular analysis of the internal transcribed spacer sequence of nuclear ribosomal DNA was conducted on Codonopsis plants collected widely from Gansu Prov. and Chongqing city of China, the main producing areas of Codonopsis Radix. Significant genetic polymorphism was observed, represented by 11 types of ITS sequences in C. pilosula, 5 types in C. pilosula var. modesta and 5 types in C. tangshen. Among the determined sequences, 1, 1 and 2 types were thought to be of pure lines of each taxon, respectively, designated as types P0, PM0, T1 and T3, and the rest might be derived from hybridization. Hybrid lines were inferred to be resulting from the combination of these pure lines. The informative sites for discriminating the 3 taxa were detected at the nucleotide positions 122nd, 226th, 441st and 489th from upstream of the ITS sequence. For discrimination of the types of C. tangshen, including one type T0 registered in GenBank, the nucleotides at positions 135th, 489th and 500th were informative. Botanical sources of the crude drugs produced in a wide range of the southeast Gansu Prov. were C. pilosula, just those from Wenxian of Gansu Prov. were C. pilosula var. modesta. The crude drugs produced in Chongqing were derived from C. tangshen.     

西洋参cDNA文库构建及表达序列标签(EST)分析

为研究西洋参根的基因表达谱和挖掘其功能基因， 采用表达序列标签(EST)技术首次建立了四年生西洋参根的EST文库。通过BLAST与Gene Ontology分析获得与人参皂苷生物合成相关、 编码P450和糖基转移酶等的基因序列11个， 与生长素调节生长发育相关、 编码生长素响应因子4和生长素调节蛋白等的基因6个， 与水分胁迫相关、 编码蛋白dehydrin和DC2.15 like蛋白等的基因7个， 与编码抗氧化酶如peroxidase和catalase相关的基因6个。另外， 从西洋参根的EST文库获得抗病基因12个， 分别编码转录因子WRKY家族蛋白和ribonuclease蛋白家族， 62个EST是其他物种尚未报道的新基因。可见， EST技术作为功能基因组研究的重要手段可在西洋参功能基因的开发与研究中发挥重要作用， 这些基因的发现为进一步克隆基因全长、 研究基因功能、 改良西洋参品质、 阐明西洋参生长缓慢的分子机制等方面奠定了基础。     

Genetic profiling of Hypericum (St. John's Wort) species by nuclear ribosomal ITS sequence analysis

A PCR-based DNA amplification method was applied to genetically distinguish the popular dietary supplement Hypericum perforatum L. (common St. John's Wort) from other related Hypericum species. Nuclear ribosomal gene sequences of the internal transcribed spacer (ITS) region were analyzed for 50 Hypericum taxa native to the Old and New Worlds, representing 11 of the 36 currently accepted taxonomic sections. This study provides a genetic method for authentication of commercial H. perforatum preparations. In addition, these data allow a preliminary assessment of phylogenetic relationships within the genus, revealing three strongly supported monophyletic clades, plus several secondary monophyletic groupings. Using ITS gene sequences, we were able to distinguish H. perforatum from all other species of Hypericum included in this study.     

Molecular authentication of Panax ginseng species by RAPD analysis and PCR-RFLP

In order to develop convenient and reproducible methods for the identification of ginseng drugs at a DNA level, randomly amplified polymorphic DNA (RAPD) and PCR-restriction fragment length polymorphism (PCR-RFLP) analyses were applied within Panax species. To authenticate Panax ginseng among ginseng populations, RAPD analysis was carried out using a 20 mer-random primer. The similarity coefficients among the DNA of ginseng plants analyzed were low, ranging from 0.197 to 0.491. In addition, by using PCR-RFLP analysis, very different fingerprints were obtained within Korean ginseng plants. These results suggest that these methods are able to authenticate the concerned Panax species. Broader application of this approach to authenticate other morphologically similar medicinal materials is rationalized.     

SEN—V非编码区基因的克隆与序列分析

目的：调查TTV阴性的非甲-庚型肝炎患者中SEN-V感染情况。对SEN-V5非编码区部分基因进行克隆与序列分析。方法：采用巢式PCR技术检测40例TTV阴性的非甲-庚型肝炎患者血清SEN-VDNA，对PCR产物进行克隆，测序和分析。结果：40例病例中SEN-VDNA阳性38例（92%），对其中6株SEN-V基因克隆测序，并与基因库中的SEN-V（GenbankNo.AX025677)序列比较，其核苷酸序列同源性在81%-94%。结论：在TTV阴性的非甲-庚型肝炎患者中存在SEN-V感染。SEN-V的致病性及其与非甲-庚型肝炎的关系有待进一步研究。     

Authentication of Stephania tetrandra S. Moore (Fang Ji) and differentiation of its common adulterants using microscopy and HPLC analysis

Stephania tetrandra S. Moore (Hang Fang Ji) is used in traditional Chinese medicine as a diuretic, an antiphlogistic, and an antirheumatic. The name “fang ji” is applied to at least four different genera of plants, including Aristolochia fangchi Y. C. Wu ex L. D. Chow and S. M. Hwang, Cocculus orbiculatus (L.) DC., Stephania tetrandra S. Moore, and Sinomenium acutum Rehder and E. H. Wilson. Due to similarity in the use of their common names, Stephania tetrandra S. Moore is often confused with Aristolochia fangchi Y. C. Wu ex L. D. Chow and S. M. Hwang, which has potentially dangerous consequences. To aid rapid and easy differentiation between the roots of these four species, so as to avoid possible contamination, detailed macroscopic and microscopic observations were made using stereo-and light-microscopy. The powdered samples were further analyzed using HPLC.     

Application of random amplified polymorphic DNA (RAPD) to detect genotoxic effect of trifluralin on maize (Zea mays)

Trifluralin is a widely used dinitroaniline herbicide throughout the world. However, limited efforts have been made to study its genotoxic effects on different plants. The present study aimed to evaluate the herbicide’s genotoxic potential on maize (Zea mays) by using the random amplified polymorphic DNA (RAPD) technique. For this purpose, maize seedlings were treated with aqueous solutions of trifluralin at concentrations ranging from 0.5 to 3 ppm for 7 days. In the RAPD analyses, 15 primers were used and 91 bands were obtained, with an average of 6.06 bands per primer in the control seedlings. After trifluralin treatment, significant changes were observed in RAPD profiles. These changes included loss of normal bands and appearance of new bands, in comparison to the control group, and they were dose dependent. In addition, root growth and total soluble protein level in trifluralin-treated seedlings were analyzed and compared for genomic template stability (GTS), which was performed for the qualitative measurement of changes in randomly amplified polymorphic DNA (RAPD) profiles. The results showed that GTS, root growth, and total soluble protein content of the seedlings gradually decreased with an increase in trifluralin concentration. These findings suggest that the RAPD technique is a useful biomarker assay to evaluate the genotoxic effects of herbicides on plants.     

人参和西洋参抗衰老药理作用的对比研究

目的：研究人参和西洋参粉剂在抗脂质过氧化、抗缺氧、抗疲劳、抗低温应激反应及调节免疫方面的异同。方法：采用２０ｇ·Ｌ－１乙醇提取制成的人参和西洋参粉剂。结果：西洋参在抗脂质过氧化、抗缺氧、抗低温应激反应方面强于或优于人参；人参在抗疲劳及对正常免疫功能的影响方面强于西洋参；而对糖皮质激素造成的低下的免疫功能的调节作用，二药的作用差异不显著。结论：西洋参抗衰老药理作用基本强于人参，但二药作用特点有所不同。     

三七甲羟戊酸激酶PnMVK1基因的克隆和生物信息学分析

药用植物三七通过甲羟戊酸途径(mevalonic acid pathway)合成三萜皂苷生物合成的前体。甲羟戊酸激酶(mevalonate kinase,MVK)则是甲羟戊酸合成途径中ATP依赖的限速酶之一。本研究根据课题组已获得的三七[Panax notoginseng(Burk.)F.H.Chen]转录组数据中的MVK1转录本序列,利用RT-PCR方法获得三七MVK1(PnMVK1)基因的全长cDNA序列;对PnMVK1蛋白进行理化性质、蛋白二级结构及三维结构预测分析,并预测了该蛋白的结构与功能;利用实时荧光定量PCR方法检测了PnMVK1基因在三七的根、茎、叶、花中的表达情况。序列分析表明,克隆获得的三七PnMVK1基因长为1 164 bp,编码387个氨基酸,GenBank登录号JQ957844。生物信息学预测PnMVK1蛋白不含跨膜区,不含信号肽,具有GHMP激酶等保守结构域。PnMVK1基因在三七的根中表达丰度最高。本研究成功克隆并分析了三七MVK1的全长序列,为进一步阐明三七三萜代谢途径奠定基础。     

Identification of fungi based on the nucleotide sequence homology of their internal transcribed spacer 1 (ITS1) region

In this study, we examined the identification of fungi based on the sequence homology of the internal transcribed spacer 1 (ITS1) region. A newly designed primer pair could amplify the target region of all 42 strains tested. The PCR products were sequenced and the sequence homologies were searched by BLAST. It was demonstrated that this method is a reliable identification method at the genus or species level. At present, available databases are still insufficient to identify some fungi, but with the accumulation of further data in the ITS1 database, this method will be available for the identification of fungi.     

Authentic identification of stigma Croci (stigma of Crocus sativus) from its adulterants by molecular genetic analysis

Stigma Croci, stigma of Crocus sativus L., is a precious traditional Chinese medicine, which is commonly used to activate blood circulation and to dissipate blood stasis. Three plant species, Carthamus tinctorius L., Hemerocallis fulva (L.) L. and Hemerocallis citrina Baroni, could carry the name Stigma Croci in the commercial markets of South East Asia. However, C. sativus is the only one that has proven its effectiveness, while the others could act as adulterants. The authentic identification of C. sativus on the market is difficult. By using molecular genetic method, the spacer domains of 5S-rRNA were cloned from the genomic DNAs that were isolated from C. sativus, C. tinctorius, H. fulva and H. citrina. The cDNAs encoding the spacer domains, about 300 to 500 bp, were sequenced. The nucleotide sequences of these four species showed great diversity, which could serve as markers for authentic identification of Stigma Croci to distinguish from its substitution and counterfeit.     

Gynostemma pentaphyllum: identification of major sapogenins and differentiation from Panax species.

Four main dammarane-type aglycones of gypenosides, extracted from the aerial parts of Gynostemma pentaphyllum were identified by gas chromatography-mass spectrometry. By detecting these aglycones as well as the aglycones of ginsenosides, a difference in sapogenin composition between Gynostemma pentaphyllum and Panax species was observed, which can be used in the differentiation of these plant drugs.     

人参、西洋参及其主要活性成分的抗糖尿病作用研究进展

本文参考国内外文献,综述了人参、西洋参及其主要活性成分控制血糖、改善糖尿病相关的代谢紊乱及其相关机制的研究进展。     

Novel biological activity of the region (106-126) on human prion sequence

We report that the synthetic peptide Prp106-126 (KTNMKHMAGAAAAGAVVGGLG-COOH) and the reversed peptide Prp126-106 (GLGGVVAGAAAAGAMHKMNTK-COOH) of human prion (hPrp) can express the decarboxylase activity for oxaloacetate in the presence of trifluoroethanol, similar to that of Oxaldie 1 (LAKLLKALAKLLKK-CONH2) reported previously. The degree of the relative activity of Prp106-126 and Prp126-106 to Oxaldie 1 is 0.47 and 0.21, respectively. Based on this experimental result, we applied the informational system method (ISM) developed by Veljkovic et al. to the amino acid sequence of Prp106-126 and Prp126-106 to extract a common factor. The same spectra were obtained, indicating that the same periodicity may be conserved on their sequences, as a necessary factor for expressing the same biological activity, irrespective of the orientation of the primary sequence.     

Application of automated flow injection analysis (FIA) to dissolution studies

The application of FIA to dissolution studies is described. Propantheline bromide, salicylamide and sulfamethizole were chosen as model drugs to investigate the utility of FIA method for dissolution studies. In each case the FIA system with the appropriate chemistry manifold was coupled with the rotating basket apparatus, A fully automated monitoring of dissolution rates was achieved. A complete dissolution profile in tabulated form is provided by the computer of the system at the end of the experiment.

Automation of any type of solid dosage forms agitation technique can be easily acquired by adapting a FIA system.