铁皮石斛化学成分的分离与鉴定

目的研究铁皮石斛(Dendrobium officinale Kimura et Migo.)中的化学成分。方法采用大孔吸附树脂、正相硅胶、ODS、Sephadex LH-20等柱色谱法和制备型高效液相色谱法等分离技术,对铁皮石斛乙醇提取物化学成分进行分离、纯化,并通过化合物的理化性质与波谱特征鉴定其化合物结构。结果从乙醇提取物中分离并鉴定了9个化合物,分别为反式-3,4,5-三甲氧基肉桂醇(trans-3,4,5-trimethoxyl-cinnamyl alcohol,1)、广玉兰赖宁苷(magnolenin C,2)、officinalioside(3)、moellenoside A (4)、松脂素-4-O-β-D-吡喃葡萄糖(pinoresinol-4-O-β-D-glucopyranoside,5)、erianthridin(6)、ephemeranthol A(7)、2,7-dihydroxy-3,4-dimethoxyphenanthrene(8)和毛兰菲(confusarin,9)。结论化合物1～5为首次从石斛属植物中分离得到。     

铁皮石斛乙醇提取物的分离与鉴定

目的研究铁皮石斛(Dendrobium officinale Kimura et Migo.)的化学成分。方法采用正相硅胶、ODS、Sephadex LH-20柱色谱法和制备型高效液相色谱法等分离技术,对铁皮石斛乙醇提取物的化学成分进行分离、纯化,并通过化合物的理化性质与波谱特征对所分离化合物进行结构鉴定。结果与结论从铁皮石斛的乙醇提取物中分离出9个化合物,分别鉴定为densiflorol B(1)、4-methoxy-2,5-dihydroxy-phenathrene (2)、 3′,4-dihydroxy-3,5′-dimethoxystilbene (3)、peroxiatractylenolideⅢ(4)、4,4′,7,7′-tetrahydroxy-2,2′,8,8′-tetramethoxy-1,1′-di-phenanthrene (5)、nobilin A (6)、2,7-dihydroxy-3,4-dimethoxyphenanthrene (7)、thymidine (8)、glaberide I 4-O-β-D-glucopyranoside (9)。其中,化合物3、4、5、8、9为首次从石斛属植物中分离得到。     

黔产铁皮石斛对阿司匹林诱导胃黏膜上皮细胞GES-1损伤的保护作用研究

目的 探讨铁皮石斛全提物对阿司匹林诱导人胃黏膜上皮细胞GES-1细胞损伤的药效作用.方法 体外培养人胃黏膜上皮细胞株GES-1细胞,经阿司匹林处理后,建立阿司匹林诱导GES-1细胞损伤模型;以阿司匹林联合铁皮石斛全提物各浓度预处理GES-1细胞为铁皮石斛组,以阿司匹林联合奥美拉唑预处理细胞为阳性组,并设立阴性对照组,采用MTS法检测细胞存活率,并通过比色法测定细胞上清液中LDH含量.结果 当阿司匹林的浓度为18.5 mmol/L处理GES-1细胞24 h时,细胞的抑制率为40％;铁皮石斛浓度范围0～500 μg/mL内,受损细胞存活率随铁皮石斛剂量增加而升高,LDH释放量逐渐降低(P＜0.05).结论 铁皮石斛对于阿司匹林诱导GES-1细胞损伤具有保护作用.     

铁皮石斛野生居群基于RAMP标记的遗传多样性评价

本文运用RAMP标记16对引物组合,对铁皮石斛9个野生居群的112个样本进行了检测,共得到123条扩增带,其中86条(占69.92%)具有多态性,每对引物组合可扩增出3～8条多态性带,平均5条,表明铁皮石斛不同居群间存在较丰富的遗传多样性;铁皮石斛居群间遗传相似系数的变化范围为0.250～0.813,平均值为0.550。利用RAMP扩增条带数据进行聚类分析,可将9个铁皮石斛野生居群划分为3个类群,聚类结果表现出较好的地域相关性,显示RAMP标记可以有效地评价铁皮石斛野生居群的遗传多样性及遗传结构。     

DNA fingerprinting of Cannabis sativa using inter-simple sequence repeat (ISSR) amplification

Chemical analysis of cannabinoid, and Inter-Simple Sequence Repeat (ISSR) fingerprinting of DNA were used to identify different samples of Cannabis sativa L. for forensic purposes. Three samples were classified into two types, tetrahydrocannabinol (THC) and cannabidiol (CBD) chemo-types, by high performance liquid chromatography (HPLC). The two samples of the CBD type were not distinguished by their HPLC patterns. ISSR fingerprinting identified polymorphic DNA patterns between these samples. ISSR fingerprinting clearly differentiated between cannabis samples that could not be achieved by HPLC analysis.     

SNP, ARMS and SSH authentication of medicinal Dendrobium officinale KIMURA et MIGO and application for identification of Fengdou drugs

Dried stems of Dendrobium officinale have been used as crude drugs in traditional Chinese medicine (TCM) with good tonic efficacy. Sequences of chloroplast, nuclear and mitochondria genes and the method of genomic DNA (gDNA) suppression subtraction hybridization (SSH) were used to authenticate different populations during the process of good agriculture practice (GAP) and crude drug quality control. Six populations could be authenticated successfully by nine single sucleotide polymorphism (SNP) sites and six pairs of diagnostic primers for amplification refractory mutation system (ARMS) were also designed to identify six populations on the basis of single nucleotide polymorphism (SNPs). The remainder two populations (JSR, GGL) with the same sequences could be authenticated by SSH. One population-specific fragment was obtained by SSH and a pair of specific primers (SSH-JB01, SSH-JB02) on the specific sequence was designed to authenticate GGL population from the other populations tested. As the resultants were population-specific, the botanic origins of fifty "Fengdou" drug samples from markets could be classified. It is evident that the combined methods provide a high throughput and reliable approach for identification of D. officinale plants and "Fengdou" drugs.     

Genetic diversity of Centella asiatica in China analyzed by inter-simple sequence repeat (ISSR) markers: combination analysis with chemical diversity

Centella asiatica is an important plant species used in traditional Chinese medicine. To help the efficient use and conservation of this species, the genetic diversity of C. asiatica populations in China was investigated using inter-simple sequence repeat (ISSR) markers. Fourteen natural populations comprising 162 individuals were included to estimate genetic diversity. At the species level, genetic diversity was relatively high (P = 66.33%, H = 0.2183, I = 0.3305). At the population level, the genetic diversity of JH (Jinhua, Zhejiang Province, China) and JJ (Jiujiang, Jiangxi Province, China) populations was relatively high (P = 43.88%, 38.78%, H = 0.1610, 0.1301, I = 0.2376, 0.1957, respectively), whereas the genetic diversity of GA (Guang’an, Sichuan Province, China) and EM (E’mei, Sichuan Province, China) was relatively low (P = 10.2%, 5.1%, H = 0.0383, 0.0211, I = 0.0570, 0.0309, respectively). On the basis of Nei’s G _st value, more genetic differentiation among populations was determined (G _st = 0.6573). In addition, the 14 populations were clustered into four groups in view of abundant ISSR data, which further defined the genetic relationship among populations. Interestingly, the genetic clustering result was similar to previous chemical clustering results based on high-performance liquid chromatography (HPLC) data, which would also classify the 14 populations into four groups. Thus, we combined the clustering results and compared their difference. The combined analysis and genetic diversity data provide a scientific basis for conserving populations of relatively high genetic diversity such as JH and JJ populations and establishing good agricultural practices (GAP) for C. asiatica.     

The large single-copy (LSC) region functions as a highly effective and efficient molecular marker for accurate authentication of medicinal Dendrobium species

Having great medicinal values, Dendrobium species of “Fengdou” (DSFs) are a taxonomically complex group in Dendrobium genus including many closely related and recently diverged species. Traditionally used DNA markers have been proved to be insufficient in authenticating many species of this group. Here, we investigated 101 complete plastomes from 23 DSFs, comprising 72 newly sequenced and 29 documented, which all exhibited well-conserved genomic organization and gene order. Plastome-wide comparison showed the co-occurrence of single nucleotide polymorphisms (SNPs) and insertions/deletions (indels), which can be explained by both the repeat-associated and indel-associated mutation hypotheses. Moreover, guanine-cytosine (GC) content was found to be negatively correlated with the three divergence variables (SNPs, indels and repeats), indicating that GC content may reflect the level of the local sequence divergence. Our species authentication analyses revealed that the relaxed filtering strategies of sequence alignment had no negative impact on species identification. By assessing the maximum likelihood (ML) trees inferred from different datasets, we found that the complete plastome and large single-copy (LSC) datasets both successfully identified all 23 DSFs with the maximum bootstrap values. However, owing to the high efficiency of LSC in species identification, we recommend using LSC for accurate authentication of DSFs.     

Utilization of RAPD markers to assess genetic diversity of wild populations of North American ginseng (Panax quinquefolium)

The Catskill Mountains of New York State are an important source of wild-collected American ginseng (Panax quinquefolium) and, increasingly, of woods-cultivated ginseng. The objective of this study was to assess genetic diversity among 9 different wild ginseng populations in and adjacent to the Catskill Mountain region of New York State and to compare these to wild populations from other states including Kentucky, Tennessee, North Carolina, Pennsylvania, and Virginia, and one cultivated population from Wisconsin. Randomly amplified polymorphic DNA (RAPD) markers were used to estimate the genetic distance among samples from the 15 populations. Pooled DNA from 10 plants of each of 8 New York populations was initially screened with 64 random primers; subsequently, the 15 primers that exhibited the greatest number of reproducible polymorphic markers were selected for further experimentation. Gel electrophoresis with the selected 15 primers produced 124 highly reproducible polymorphic bands. The ratio of discordant bands to total bands scored was used to estimate the genetic distance within and among populations. Multidimensional scaling (MDS) of the relation matrix showed distinctly separate clusters between New York and non-New York populations, indicating separation between these two groupings. The MDS analysis was confirmed using pooled chi-square tests for fragment homogeneity. This study shows that RAPD markers can be used as population-specific markers for Panax quinquefolium, and may eventually be utilized as markers for ginsenoside assessment.     

一种适于ISSR分析的太子参DNA提取方法

目的获得能用于中药太子参简单序列重复区间DNA标记技术等分析研究的高质量DNA。方法以太子参为材料,采用改良CTAB法提取太子参DNA,经紫外分光光度计和琼脂糖凝胶电泳检测。结果改良的CTAB法能获得高质量的太子参DNA,OD260/OD280>1.8,蛋白质、多糖、RNA等去除较彻底,适于简单序列重复区间分析。结论改良的CTAB法所提取太子参DNA适于简单序列重复区间分析。     

Development and evaluation of a gastro-retentive delivery system for improved antiulcer activity of ginger extract (Zingiber officinale)

Aim was to develop and optimize multiunit gastro-retentive floating beads (FBs) intended for localized and prolonged release of ginger for treating gastric ulcers. Protective effect of ginger extract (GE) against ulcer is well documented, but therapeutic use is compromised due to poor bioavailability and physicochemical properties. GE was only slightly soluble (3.19?±?0.38?mg/ml) in simulated gastric fluid (SGF; pH 1.2). The solubility decreased in water to 0.69?±?0.03?mg/ml and further by 26% in the presence of calcium carbonate (0.5% w/v). We prepared FBs of GE using calcium carbonate and sodium alginate in different proportions. Beads were evaluated for diameter, buoyancy, entrapment, and porosity. In vitro dissolution showed a Fickian release with a cumulative release of >80% at 24?h. Preclinical evaluation was done in cold-restraint stress induced gastric ulcers, in albino rats, in terms of (i) ulcer index, hemorrhagic streaks (l), mucus content, (ii) oxido-nitrosative stress, and (iii) histopathology. GE loaded FBs (200?mg/kg) were significantly better than free GE and better/equivalent to cimetidine (10?mg/kg). The system was evaluated for therapeutic effect (curative), i.e. after the induction of ulcers. Most of the natural phytochemical or antioxidants show pretreatment effectiveness. We, however, developed and established GE FBs for sustained curative effect.     

Identification of the crude drug atractylodes rhizome (Byaku-jutsu) and atractylodes lancea rhizome (So-jutsu) using chloroplast TrnK sequence as a molecular marker

Novel methods for molecular authentication of Atractylodes-derived crude drugs (Jutsu) were established based on PCR-restriction fragment length polymorphism (RFLP) and direct sequencing of chloroplast trnK. Two regions inside the chloroplast trnK were selected as molecular markers for identification and discrimination of Atractylodes Rhizome (Byaku-jutsu) and Atractylodes Lancea Rhizome (So-jutsu). The Region 1 fragment (260 bp) amplified from So-jutsu and Wa-byaku-jutsu (Atractylodes Rhizome derived from A. japonica) gave 2 bands of 180 bp and 80 bp on agarose gel electrophoresis after digestion with a restriction endonuclease HinfI, whereas the fragment amplified from Kara-byaku-jutsu (Atractylodes Rhizome derived from A. ovata) remained undigested, which allowed unambiguous identification of Kara-byaku-jutsu. By direct sequencing of Region 2 (436 bp) and comparison of the nucleotide sequence data sets we could not only discriminate Byaku-jutsu and So-jutsu but also identify the original plant species of each crude drug specimen. A simple and reliable protocol for rapid preparation of DNA suitable for PCR from as little as 1 mg of Atractylodes-derived crude drugs was also described.     

Preliminary discovery of novel markers for human cell line activation test (h-CLAT)

The human cell line activation test (h-CLAT) is an OECD approved (Test No. 442E) assay to identify novel skin sensitizers. h-CLAT simulates dendritic cell activation in the skin sensitization pathway and is based on the measurement of CD54 and CD86 overexpression on monocytic, leukemic THP-1 cells. However, the current h-CLAT markers show inconsistent results with moderate and weak sensitizers. Moreover, these markers have accessory roles in cell adhesion and signaling rather than a direct role in cellular inflammation. Therefore, we have explored other inflammation-related markers in this study. PBMCs comprises a mixture of cells that resemble the complex immunological milieu in adults and were primarily used to identify markers. PBMCs (n = 10) and THP-1 cells were treated with 1-chloro-2,4-dinitrobenzene (DNCB, strong) and NiCl₂ (Ni, moderate) sensitizers or DMSO (control) and incubated for 24 h. The samples were subjected to RNA sequencing to obtain log₂fold change in gene expression. DNCB and NiCl₂ significantly upregulated 80 genes in both cell types. Of these, CD109, CD181, CD183, CLEC5A, CLEC8A & CD354 were experimentally validated. DNCB and Ni but not isopropyl alcohol (non-sensitizer) significantly induced the expression of all novel markers except CLEC8A. Moreover, the percentage induction of all novel markers except CLEC8A satisfied the OECD acceptance criteria. In summary, we identified five novel markers that may supplement the current repertoire of h-CLAT markers.     

Tityus perijanensis González-Sponga (Scorpiones, Buthidae): molecular assessment of its geographical distribution and venom lethality of Venezuelan populations.

An extensive field survey allowed us to expand the geographical distribution of the scorpion Tityus perijanensis in the Perijá range, western Zulia State, Venezuela, including areas where adult cases of severe scorpionism have been reported. 16S ribosomal RNA (rRNA) gene sequencing, DL(50) determination, and native PAGE suggest low genetic and venom proteomic divergence across the distribution range. The results also indicate phylogenetic divergence between T. perijanensis and T. discrepans, the species prevalent in northcentral Venezuela. T. perijanensis venom lethality (0.91-0.94 mg/kg) is comparable to that of the Brazilian T. serrulatus and ranks highest among toxic Venezuelan Tityus studied so far. The data indicate that the Perijá range should be included amongst the endemic areas of scorpionism of Venezuela and Colombia.     

Assessment of technical protocols for novel embryonic stem cell tests with molecular markers (Hand1- and Cmya1-ESTs): a preliminary cross-laboratory performance analysis

The Hand1- and Cmya1-ESTs are novel short-term tests for embryotoxic chemicals using genetically engineering mouse ES cells for luciferase reporter gene assays. These ESTs allow convenient determination of differentiation toxicity and cell viability in a short duration with high throughput 96-well microplates for prediction of embryotoxicity of chemicals. To assess the Hand1-EST technical protocol, we firstly compared reporter gene assay and cytotoxicity test data for a representative compound (hydroxyurea) from four different laboratories with tests carried out under the same experimental conditions. Extensive investigations of the Hand1- and Cmya1-ESTs were then performed to explore reproducibility by comparing a set of 6 well-known test chemicals, including hydroxyurea, across the laboratories. The results gave good correspondence in all four laboratories, indicating that transferability, intra-laboratory variability and inter-laboratory variability of the present technical protocols of the ESTs were sufficient to conduct further validation studies.     

Relevance of the C-terminal Arg-Phe sequence in gamma(2)-melanocyte-stimulating hormone (gamma(2)-MSH) for inducing cardiovascular effects in conscious rats

1. The cardiovascular effects by gamma(2)-melanocyte-stimulating hormone (gamma(2)-MSH) are probably not due to any of the well-known melanocortin subtype receptors. We hypothesize that the receptor for Phe-Met-Arg-Phe-amide (FMRFa) or Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-amide (neuropeptide FF; NPFFa), other Arg-Phe containing peptides, is the candidate receptor. Therefore, we studied various Arg-Phe containing peptides to compare their haemodynamic profile with that of gamma(2)-MSH(6 - 12), the most potent fragment of gamma(2)-MSH. 2. Mean arterial pressure (MAP) and heart rate (HR) changes were measured in conscious rats after intravenous administration of gamma(2)-MSH related peptides. 3. Phe-Arg-Trp-Asp-Arg-Phe-Gly (gamma(2)-MSH(6 - 12)), FMRFa, NPFFa, Met-enkephalin-Arg-Phe-amide (MERFa), Arg-Phe-amide (RFa), acetyl-Phe-norLeu-Arg-Phe-amide (acFnLRFa) and desamino-Tyr-Phe-norLeu-Arg-Phe-amide (daYFnLRFa) caused a dose-dependent increase in MAP and HR. gamma(2)-MSH(6 - 12) showed the most potent cardiovascular effects (ED(50)=12 nmol kg(-1) for delta MAP; 7 nmol kg(-1) for delta HR), as compared to the other Arg-Phe containing peptides (ED(50)=177 - 292 nmol kg(-1) for delta MAP; 130 - 260 nmol kg(-1) for delta HR). 4. Peptides, which lack the C-terminal Arg-Phe sequence (Lys-Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp-Arg-Pro-Gly (gamma(2)-pro(11)-MSH), desamino-Tyr-Phe-norLeu-Arg-[L-1,2,3,4 tetrahydroisoquinoline-3-carboxylic acid]-amide (daYFnLR[TIC]a) and Met-enkephalin (ME)), were devoid of cardiovascular actions. 5. The results indicate that the baroreceptor reflex-mediated reduction of tonic sympathetic activity due to pressor effects is inhibited by gamma(2)-MSH(6 - 12) and that its cardiovascular effects are dependent on the presence of a C-terminal Arg-Phe sequence. 6. It is suggested that the FMRFa/NPFFa receptor is the likely candidate receptor, involved in these cardiovascular effects.     

Classification of genetic variation for cadmium tolerance in Bermudagrass [Cynodon dactylon (L.) Pers.] using physiological traits and molecular markers

Cadmium (Cd) is one of the most toxic pollutants that caused severe threats to animal and human health. Bermudagrass is a dominant species in Cd contaminated soils, which can prevent Cd flow and spread. The objectives of this study were to determine the genetic variations in major physiological traits related to Cd tolerance in six populations of Bermudagrass collected from China, and to examine the genetic diversity and relationships among these accessions that vary in Cd tolerance using molecular markers. Plants of 120 accessions (116 natural accessions and 4 commercial cultivars) were exposed to 0 (i.e. control) or 1.5 mM CdSO₄·8/3H₂O for 3 weeks in hydroponic culture. Turf quality, transpiration rate, chlorophyll content, leaf water content and growth rate showed wide phenotypic variation. The membership function method was used to comprehensively evaluate Cd-tolerance. According to the average subordinate function value, four accessions were classified as the most tolerant genotypes and four accessions as Cd-sensitive genotypes. The trend of Cd tolerance among the six studied populations was as follows: Hunan > South China > North China > Central China > West South China and Xinjiang population. Phylogenetic analysis revealed that the majority of accessions from the same or adjacent regions were clustered into the same groups or subgroups, and the accessions with similar cadmium tolerance displayed a close phylogenetic relationship. Screening genetically diverse germplasm by combining the physiological traits and molecular markers could prove useful in developing Cd-tolerant Bermudagrass for the remediation of mill tailings and heavy metal polluted soils.