Optimization of the Extraction Technology of Senecio scandens by Orthogonal Design Combined with Informa- tion Entropy Weighting Method / 中国药房

OBJECTIVE：To optimize the extraction technology of Senecio scandens ，and to provide reference for the further development and utilization of the medicinal material. METHODS ：The method of reflux extraction with 80 ℃ water bath was used to extract S. scandens . HPLC method was adopted to determine the contents of chlorogenic acid and hyperoside. The determination was performed on Diamonsil C 18 with mobile phase consisted of 0.2％ glacial acetic acid water solution-methanol （82∶18，V/V）at the flow rate of 1.0 mL/min；the column temperature was set at 35 ℃；the detection wavelength was 327 nm，and the sample size was 5 μL. UV-Vis spectrophotometry was used to determine the contents of total flavonoids（by rutin ）and total alkaloids （by matrine）and the detection wavelengths were set at 509 nm and 208 nm，respectively. Based on single factor tests ，using ethanol volume fraction （A，％），solvent folds （B，mL/g），extraction time （C，h），extraction times （D，times）as factors ，using the contents of chlorogenic acid ，hyperoside，total flavonoids and total alkaloids as indexes ，and t he weight coefficients of above indicator components were calculated based on information entropy weighting method so as to calculate their comprehensive scores ， then L 9（34）orthogonal design was used to further optimize the extraction technology. The validation tests were also performed. RESULTS：The optimal extraction technology of S. scandens included that 8-fold 50％ ethanol，extracting for 3 times，lasting for 1.5 h each time. Results of 3 times of validation tests showed that RSDs of the contents of chlorogenic acid ，hyperoside，total flavonoids and total alkaloids were all lower than 1.5％（n＝3）. CONCLUSIONS ：The optimized technology is reproducible ， stable and feasible ，and can be used for the extraction of S. scandens .

Cloning and functional characterization of farnesyl diphosphate synthase in Senecio scandens / 中国中药杂志

As a secondary metabolite, sesquiterpenes are not only have important functions in plant defense and signaling, but also play potential roles in basic materials for pharmaceuticals, cosmetic and flavor. As a traditional Chinese herbal medicine, Senecio scandens exhibits effects of anti-inflammatory and immunosuppressive, as well as invigorating the blood and removing extravasated blood. Over 600 sesquiterpenes with diverse structures were isolated from S. scandens and related species in the same genus. To characterize sesquiterpenes synthesis, two FPS genes(SsFPS1 and SsFPS2) were identified in S. scandens through transcriptomic analysis. Bioinformatic analysis showed that both SsFPSs have conserved motifs for FPS function. Both SsFPSs exhibited constitutive gene expression in S. scandens tissues and SsFPS2 accumulated higher transcript in leaves and roots than SsFPS1. Meanwhile consistent with constitutive sesquiterpene accumulation in S.scandens tissues, most of these sesquiterpenes were detected in leaves and roots more than stems and flowers. Recombinant expression through Escherichia coli metabolic engineering, SsFPS1 or SsFPS2 was co-transformed with ZmTPS11(maize β-macrocarpene synthase) into BL21 competent cells. The results showed that the content of β-macrocarpene was increased by co-transformation with SsFPSs. It is demonstrated that SsFPS1 and SsFPS2 catalyzed E,E-FPP formation and provided FPP precursor for downstream sesquiterpene synthases. Characterization of SsFPSs provided the foundation for the exploration of biosynthesis of sesquiterpenoid with diverse structures and potential pharmaceutical values in S.scandens, and provide an important theoretical basis for the development of S. scandens abundant resources.

Sequence analysis and SSR marker mining of a peroxidase gene in Senecio scandens / 中草药

Objective: To study the relationship between the sequence structure and function of a peroxidase gene (POD) and mine and confirm SSR locus of this gene in Senecio scandens. Methods: A POD gene was obtained from full-length cDNA library of S. scandens. Then a series of online bioinformatics tools were used to analyze nucleic acid sequence and amino acid structure of this gene. According to its nucleotide sequence, one SSR locus was searched and a pair of primers were designed. The PAGE electrophoresis confirmed that this primer pair had polymorphism among several landraces. Results: The target gene had the structural characteristics of typical PODs. It showed a high conservation in functional domains. PAGE electrophoresis showed that the primer could be stably amplified in different landraces with obvious polymorphisms. Conclusion: The results expanded the understanding of the function and sequence of a POD in S. scandens. The newly developed marker could be effectively applied to the detection of POD gene and breeding projects, and it also provided reference for POD researches of other species.

Identification of stable reference gene by qRT-PCR in Senecio scandens / 中国中药杂志

As a traditional Chinese medicine, Senecio scandens is rich in important compounds such as flavonoid and sesquiterpenoid. Based on the transcriptome data of S. scandens, 15 candidate reference genes were selected including ABCT, ACT1, ACT2, ACT3, ACBP, ARF, ATPS, EF-H, EF-1α, ETIF, GAPDH, GTPB, MPS, UCE and 60S. Firstly, 9 candidate genes with relatively stable expressions such as ACT1, ACBP, ARF, ATPS, EF-1α, GAPDH, MPS, UCE and 60S were screened from different tissues of S. scandens by RT-PCR. Then, qRT-PCR was used to quantitatively analyze gene expression of these nine candidates in S. scandens with or without stress treatments. Further analysis of these gene expression data by geNorm and NormFinder showed that ACT1 exhibited the stablest expression in all samples and could serve as a reference gene for future study of S. scandens, and provide an endogenous control for gene expression analysis.

Simultaneous Determination of Total Content of p-Hydroxybenzoic Acid plus o-Hydroxybenzoic Acid and p-Hydroxybenzeneacetic Acid in Senecio Scandens Buc / 中国药师

Objective: To set up an HPLC method for the simultaneous determination of p-hydroxybenzoic acid plus o-hydroxy-benzoic acid and p-hydroxybenzeneacetic acid in Senecio scandens Buch. Methods:The column was a Shiseido( Fine Chemicals) Cap-cell Pak C18 (250 mm × 4. 6 mm, 5 μm) column at the room temperature. The mobile phase was methanol-water-formic acid (13∶87∶0. 5) at a flow rate of 1. 0 ml·min-1 . The detection wavelength was 240nm. Results: p-Hydroxybenzoic acid plus o-hydroxybenzoic acid had a favorable linear relationship within the range of 0. 025-0. 400 mg·ml-1 , the regression equation was Y=5. 94 × 106 X+2.46×104(r=0.9998),theaveragerecoverywas97.59% andRSDwas1.22%. p-Hydroxybenzeneaceticacidhadafavorableline-ar relationship within the range 0.05-0.80 mg·ml-1, the regression equation was Y=4.09 ×106X+1.12 ×104(r=0.999 8), the average recovery was 98. 07% and RSD was 1. 90%. Conclusion:The method is simple, feasible and reproducible. It can be used in the quality control of p-hydroxybenzoic acid plus o-hydroxybenzoic acid and p-hydroxybenzeneacetic acid in Senecio scandens Buch.

Study Progress in Chemical Constituents and Pharmacological Effects of Senecio Scandens Buch.-Ham / 中国药师

Senecio scandens Buch.-Ham. , also called Jiuliming, is a plant of Senecio cruentus. It mainly contains alkaloids,phe-nolic acids,flavonoids,volatile oils,terpenoids and the other ingredients, and the main pharmacological effects include antibiosis,anti-inflammation,anti-virus, antitumor, antioxidation and free radical scavenger, toxicological effects and so on. The study progress in chemical constituents and pharmacological effects of Senecio scandens Buch.-Ham. was reviewed in the paper to provide available infor-mation for more research.

Sensitive genes screening of mice liver injury induced by hepatotoxicant and their biological function analysis / 中华中医药杂志

Objective:To scan the common differentially expressed genes in mice liver tissue following subacute exposure to hepatotoxicants,including alcohol,carbon tetrachloride,BCG & LPS,the alcohol extracts of Rhizoma Dioscoreae Bulbiferae and Senecio scandens Buch.-Ham.,and apply to the rapid toxic evaluation of TCM and other xenobiotics.Methods:The gene expression profiles of mice liver tissue,after respective administration of alcohol,CCl4,BCG associated with LPS,the alcohol extracts of Rhizoma Dioscoreae Bulbiferae and Senecio scandens Buch.-Ham.to healthy mice,were analyzed by use of mouse genome cDNA microarray.The correlations between the common differential expressed genes and the liver injury were analyzed based on the biological functions of those differentially expressed genes.Results:Among all of the five drug administration groups,there were 7 known function genes(BC05200,NM-019410,NM-173019,AB028272,AK088925,AK030862 and AK088816)and 1 unknown function gene(BC069871)differentially expressed.Beside AK088816,the other 7 genes were all down-regulate expressed.Conclusion:The common differentially expressed genes participate in the process of saccharometabolism,apoptosis,cell growth cycle,cytoskeleton and signal transmission,protein folding and protein ubiquitin.The abnormal expression of the common genes were closely correlated to the liver,and might be important for rapid hepatoxicity elucidation of traditional Chinese medicine and other xenobiotics.