RESUMO
Three new compounds (1, 6, 9), with six known compounds (2-5, 7-8) were obtained from water-soluble extract of Cimicifuga dahurica (Turcz.) Maxim. by bioactivity-guided isolation. Their structures were elucidated by chemical and spectral analysis, including 1D, 2D NMR data and HRESIMS. H2O2-induced neurotoxicity on PC12 cells model were conducted to evaluate the neuro-protective capability of these compounds. The piscidic acid derivatives compounds 4-7 showed marked neuro-protective effect at certain concentration.
Assuntos
Cimicifuga/química , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Triterpenos/química , Triterpenos/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Peróxido de Hidrogênio/metabolismo , Hidroxibenzoatos/química , Hidroxibenzoatos/isolamento & purificação , Hidroxibenzoatos/farmacologia , Neurônios/citologia , Neurônios/metabolismo , Fármacos Neuroprotetores/isolamento & purificação , Células PC12 , Ratos , Triterpenos/isolamento & purificaçãoRESUMO
Three new phenolic compounds (1-3), along with five known compounds (4-8) were isolated from the rhizome of Cimicifuga dahurica (Turcz.) Maxim. Their structures were elucidated by spectroscopic methods including 1D-NMR, 2D-NMR and HR-MS techniques. DPPH method and protective effect on PC12 cells against H2O2-induced oxidative damage model were carried to evaluate the antioxidant capability of these compounds. Compound 5 showed significant antioxidant activity with IC50 values 9.33µM in DPPH assay and compound 2 displayed marked neuro-protective effect with 87.65% cell viability at the concentration of 10µM. Additionally, the possible structure-activity relationships of these phenolic compounds were tentatively discussed.
Assuntos
Antioxidantes/química , Cimicifuga/química , Fármacos Neuroprotetores/química , Polifenóis/química , Animais , Antioxidantes/isolamento & purificação , Biologia Computacional , Fármacos Neuroprotetores/isolamento & purificação , Células PC12 , Polifenóis/isolamento & purificação , Ratos , Rizoma/química , Relação Estrutura-AtividadeRESUMO
A variant strain of rabbit hemorrhagic disease virus, designated "whn-1", was isolated and identified in China. The virus lacked haemagglutinating activity at 25, 37 and 4 degrees C, respectively, and gave negative results in the HAT after two passages in experimentally infected rabbits, but gave positive results in Agar Diffusion Reaction (ADR) and Counter Immunoelectrophoresis (CIE). Using electron microscopy, negatively stained particles of the RHDV isolate showed that the virions was approximately 35 nm in diameter. The capsid protein VP60 gene of whn-1 strain was cloned into pMD18-T vector by RT-PCR assays and sequenced. The obtained VP60 gene sequence has been submitted to GenBank with the accession number: DQ069280. The whole VP60 gene of whn-1 was 1740 nt in size and encodes 579 aa. Alignment with other 16 strains of RHDV in the world, including such "RHDVa" strains as France 99-05, France-Reu-00, Germany-Triptis and ChinaTP, in addition to RCV and EBHSV, showed that the homology of RHDV strains were 90.0-98.0% for nucleotide sequence, 94.3-99.0% for amino acid sequence, respectively. The results indicated that the sequences of VP60 gene of different RHDV isolates, including non-haemagglutinating whn-1 strain and low-haemagglutinating Rainham strain, were relatively highly homologous, and the major variant amino acid were located within region C (301-328 aa) and region E(344-434 aa), which were specific to "RHDVa" strains. Moreover, the molecular characterisation of VP60 protein of RHDV whn-1 strain, such as Hydrophilicity plot, Flexible regions, Antigenic index, etc., were compared with reference RHDV strains of Spanish-AST/89, France-99-5 and UK-Rainham in this article. From the experiment, it's concluded that, the "whn-1" strain is probably an antigenic variant of "RHDVa", and the 3 amino acids of Phe (304), Ala (305), Ser (309), and 5 amino acids of Gly (359), Asn (365), Ala (369), Ala (370), Asn (386), located in P2 region in the VP60 protein, probably played an important role in the haemagglutination activity.
Assuntos
Vírus da Doença Hemorrágica de Coelhos/genética , Vírus da Doença Hemorrágica de Coelhos/isolamento & purificação , Proteínas Estruturais Virais/genética , Animais , Infecções por Caliciviridae/veterinária , Infecções por Caliciviridae/virologia , China , Clonagem Molecular , Contraimunoeletroforese , Hemaglutinação por Vírus/genética , Hemaglutinação por Vírus/fisiologia , Vírus da Doença Hemorrágica de Coelhos/imunologia , Fígado/virologia , Microscopia Eletrônica de Transmissão , Dados de Sequência Molecular , Filogenia , RNA Viral/genética , Coelhos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Análise de Sequência , Homologia de Sequência de Aminoácidos , Homologia de Sequência do Ácido Nucleico , Proteínas Estruturais Virais/química , Proteínas Estruturais Virais/imunologia , Vírion/ultraestruturaRESUMO
The 1074-bp phyCs gene (optimized phyC gene) encoding neutral phytase was designed and synthesized according to the methylotrophic yeast Pichia pastoris codon usage bias without altering the protein sequence. The expression vector, pP9K-phyCs, was linearized and transformed in P. pastoris. The yield of total extracellular phytase activity was 17.6 U/ml induced in Buffered Methanol-complex Medium (BMMY) and 18.5 U/ml in Wheat Bran Extract Induction (WBEI) medium at the flask scale, respectively, improving over 90 folds compared with the wild-type isolate. Purified enzyme showed temperature optimum of 70 degrees and pH optimum of 7.5. The enzyme activity retained 97% of the relative activity after incubation at 80 degrees for 5 min. Because of the heavy glycosylation the expressed phytase had a molecular size of approximately 51 kDa. After deglycosylation by endoglycosylase H (EndoH(f)), the enzyme had an apparent molecular size of 42 kDa. Its property and thermostability was affected by the glycosylation.
