[Quality evaluation of Desmodium styracifolium by fingerprint, pattern recognition combined with quantitative analysis of multi-components by single marker].

HPLC analysis was performed on a Phenomenex PS C18（4.6 mm×250 mm, 5 µm）column using methanol -0.2% formic acid (30:70) at a flow rate of 0.8 mL·min⁻¹. The column temperature was 30 °C and the detection wavelength was set at 335 nm. The injection volume was 10 µL. The HPLC fingerprint of Desmodium styracifolium was established with 10 common peaks, and 5 of them were identified as vicenin-1, schaftoside, isoorientin, isoschaftoside and isovitexin, respecivetly. The fingerprints of 21 batches of D. styracifolium samples were analyzed with similarity evaluation, cluster analysis, principal component analysis and partial least squares discriminant analysis. There was no significant difference among the quantitative results of these five ingredients verified by external standard method (ESM) and quantitative analysis of multi-components by single marker (QAMS) method. The application of fingerprint, pattern recognition combined with QAMS can provide more comprehensive references for the quality control and evaluation of D. styracifolium.

[Research on distribution and quality suitability division of Desmodium styracifolium].

The research on distribution and quality suitability division of Desmodium styracifolium were formulated by Maxent and ArcGIS model based on the content of schaftoside and polysaccharide of D. styracifolium and its field research in the south and southwest areas of China (Guangdong, Guangxi, Hainan and Yunnan), and the most suitable habitats of distribution suitability and quality suitability were screened. The distribution suitability results indicated that average air temperature in April,mean temperature of coldest quarter, soil type, coldness index were found as the four dominant factors contributing to the plant distribution. The quality suitability results indicated that: ①Polysaccharide content and precipitation in April show significant positive correlation;Schaftoside content and mean temperature of April, mean temperature of coldest quarter show significant negative correlation. Schaftoside content shows significant negative correlation with the precipitation in October and November and the sunshine duration in April and May, while there is a significant positive correlation between schaftoside content and precipitation in April and temperature seasonality standard deviation, and a highly significant positive correlation was found between schaftoside content and precipitation in February and March. ②The quality zoning map was drawn depend on general content of polysaccharide and schaftoside as the index of quality. And this research provides scientific location basis for the production regionalization, cultivation bases selection and directive breeding of D. styracifolium.

[Secondary metabolites from a deep-sea-derived actinomycete Micrococcus sp. R21].

To investigate cytotoxic secondary metabolites of Micrococcus sp. R21, an actinomycete isolated from a deep-sea sediment (-6 310 m; 142 degrees 19. 9' E, 10 degrees 54. 6' N) of the Western Pacific Ocean, column chromatography was introduced over silica gel, ODS, and Sephadex LH-20. As a result, eight compounds were obtained. By mainly detailed analysis of the NMR data, their structures were elucidated as cyclo(4-hydroxy-L-Pro-L-leu) (1), cyclo(L-Pro-L-Gly) (2), cyclo( L-Pro-L-Ala) (3), cyclo( D-Pro-L-Leu) (4), N-ß-acetyltryptamine (5), 2-hydroxybenzoic acid (6), and phenylacetic acid (7). Compound 1 exhibited weak cytotoxic activity against RAW264. 7 cells with IC50 value of 9.1 µmol x L(-1).

[Effects of storage time on quality of Desmodium styracifolium seeds].

The dynamic changes of germination percentage, germination potential, thousand-seed weight, antioxidase activity in Desmodium styracifolium seeds with different storage time were tested, and electrical conductivity, contents of soluble sugar, soluble protein, starch in seed leach liquor were also determined in order to reveal the mechanism of seed deterioration. The results as the following. (1) The germination percentage, germination potential and thousand-seed weight of D. styracifolium seeds declined, while the seed coat color darkened with the extension of storage time. (2) The activities of superoxide dismutase (SOD) and peroxidase (POD) decreased with the prolongation of storage period. The SOD activity declined fastest in 1,095-1,185 d of storage, while the POD activity declined significantly in 365-395 d of storage. (3) The electrical conductivity and the contents of soluble sugar, starch in seed leach liquor increased, while the content of soluble protein declined with the extension of storage time. (4) Correlation analysis indicated that the germination percentage, germination potential and thousand-seed weight of D. styracifolium seeds have a significantly positive correlation with SOD and POD activity, while have a significantly negative correlation with the electrical conductivity, contents of soluble sugar and starch. It can be concluded that during the storage of D. styracifolium seeds, physiological and biochemical changes including decrease in antioxidase activity, rise in electrical conductivity, degradation effluent of soluble sugar and starch, degradation of soluble protein were the main factors leading to the seed deterioration.

[De novo sequencing and analysis of root transcriptome to reveal regulation of gene expression by moderate drought stress in Glycyrrhiza uralensis].

Moderate drought stress has been found to promote the accumulation of active ingredients in Glycyrrhiza uralensis root and hence improve the medicinal quality. In this study, the transcriptomes of 6-month-old moderate drought stressed and control G. uralensis root (the relative water content in soil was 40%-45% and 70%-75%, respectively) were sequenced using Illumina HiSeq 2000. A total of 80,490 490 and 82 588 278 clean reads, 94,828 and 305,100 unigenes with N50 sequence of 1,007 and 1,125 nt were obtained in drought treated and control transcriptome, respectively. Differentially expressed genes analysis revealed that the genes of some cell wall enzymes such as ß-xylosidase, legumain and GDP-L-fucose synthase were down-regulated indicating that moderate drought stress might inhibit the primary cell wall degradation and programmed cell death in root cells. The genes of some key enzymes involved in terpenoid and flavonoid biosynthesis were up-regulated by moderate drought stress might be the reason for the enhancement for the active ingredients accumulation in G. uralensis root. The promotion of the biosynthesis and signal transduction of auxin, ethylene and cytokinins by moderate drought stress might enhance the root formation and cell proliferation. The promotion of the biosynthesis and signal transduction of abscisic acid and jasmonic acid by moderate drought stress might enhance the drought stress tolerance in G. uralensis. The inhibition of the biosynthesis and signal transduction of gibberellin and brassinolide by moderate drought stress might retard the shoot growth in G. uralensis.

[Analysis of quality variation and genetic diversity of Desmodium styracifolium from different provenances].

OBJECTIVE: To explore the quality variation and genetic diversity of Desmodium styracifolium from different provenances, and lay a foundation for rational exploitation on germplasm resources and fine variety breeding of D. styracifolium. METHOD: Amplified fragment length polymorphism (AFLP) markers were developed to analyze genetic diversity in D. styracifolium from 18 resources. NTSYSpc-2. 11F software was used to analyze the similarity among the D. styracifolium germplasms and construct the genetic phylogenetic tree. The schaftoside content in D. styracifolium from different provenances was determined by HPLC. RESULT: A total of 844 fragments were amplified with 8 primers, in which 717 were polymorphic bands, accounting for 84. 27% of the total detected variation. All the specimens from 18 resources could be grouped into 3 clusters by cluster analysis. The schaftoside contents of D. styracifolium germplasms differed significantly, with the highest content in the germplasm from Sanya, Hainan. CONCLUSION: Significant quality variation and genetic diversity can be observed among D. styracifolium germplasms. The diverse germplasm resources should be explored and the fine variety should be selected to breed.

[Cloning and expression regulation of 1-deoxy-D-xylulose-5-phosphate reductoisomerase cDNA from Alpinia officinarum].

The rhizome of Alpinia officinarum is a widely used Chinese herbal medicine. The essential oil in A. officinarum rhizome is mainly composed of 1, 8-cineole and other monoterpenes, as the major bioactive ingredients. In plants, monoterpenes are synthesized through the methylerythritol phosphate (MEP) pathway in the plastids, and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is an enzyme catalyzing a committed step of the MEP pathway. In the present study, the full-length cDNA encoding DXR was cloned from the rhizome of A. officinarum, using homology-based RT-PCR and rapid amplification of cDNA ends (RACE) techniques. The new cDNA was designated as AoDXR and submitted to GenBank to be assigned with an accession number HQ874658. The full-length cDNA of AoDXR was 1 670 bp containing a 1 419 bp open reading frame encoding a polypeptide of 472 amino acids with a calculated molecular mass of 51.48 kDa and an isoelectric point of 6.15. Bioinformatic analyses revealed that AoDXR showed extensive homology with DXRs from other plant species and contained a conserved plastids transit peptide, a Pro-rich region and two highly conserved NADPH-binding motifs in its N-terminal region characterized by all plant DXRs. The phylogenetic analysis revealed that AoDXR belonged to angiosperm DXRs. The structural modeling of AoDXR showed that AoDXR had the typical V-shaped structure of DXR proteins. The tissue expression pattern analysis indicated that AoDXR expressed strongly in leaves, weak in rhizomes of A. officinarum. Exogenous methyl jasmonate (MeJA) could enhance the expression of AoDXR and the production of 1, 8-cineole in A. officinarum rhizomes. The cloning and characterization of AoDXR will be helpful to reveal the molecular regulation mechanism of monoterpene biosynthesis in A. officinarum and provides a candidate gene for metabolic engineering in improving the medicinal quality of A. officinarum rhizome.

[Isolation of alkaloids from Corydalis saxicola by high-speed counter-current chromatography].

OBJECTIVE: To develop a method for the rapid isolation of alkaloids from the crude extract of Corydalis saxicola. METHODS: High-speed counter-current chromatography (HSCCC) was applied for the separation of alkaloids from Corydalis saxicola, with the biphase solvent systems comprised n-butanol-ethyl acetate-water-formic acid (5: 1: 5: 0.01) and n-butanol-ethyl acetate-methanol-water-formic acid (5: 5: 1:9:0.05). RESULTS: About 300 mg of crude extract was isolated by HSCCC, yielding 3.6 mg of scoulerine, 9.2 mg of isocorydine, 5.5 mg of dehydrocheilanthifoline, 7.5 mg of dehydrocavidine, 20.4 mg of palmatine and 20.9 mg of berberine, with purities of 71%, 92%, 85%, 76%, 90%, 97%, respectively. CONCLUSION: It is time-saving and simple to isolate alkaloids from Corydalis saxicola by HSCCC with high yields and purities.

[Study on the influence on the content of dehydrocavidine and total alkaloids in Corydalis saxicola by different drying methods].

OBJECTIVE: To evaluate the influence of five different drying methods on the contents of dehydrocavidine, a main active constituent, and total alkaloids in Corydalis saxicola. METHODS: The whole plant samples of C. saxicola were harvested at its florescence stage, and then immediately divided into four parts of root, stem, leaf, and inflorescence. Each part of sample was dried by the following five methods, drying in a sunshine, drying in a shade, oven-drying at 60 degrees C, vacuum-drying at room temperature and frozen vacuum-drying, respectively. Then the contents of dehydrocavidine and total alkaloids were determined by RP-HPLC and ultraviolet spectrophotometry. RESULTS: The contents of dehydrocavidine and total alkaloids in all the four parts of the sample processed by drying in a shade were significantly higher than those in the samples processed by the other four drying mehods. CONCLUSION: Different drying methods could significantly influence the contents of dehydrocavidine and total alkaloids in C. saxicola. The process of drying in a shade for a long time would be the best.