RESUMO
Eighteen compounds including eleven previously undescribed diterpenes were isolated from the leaves of Croton mangelong. The structures were determined by HRESIMS, IR, NMR, X-ray diffraction and ECD spectroscopic analysis. All isolates were assayed for their anti-hyperglycemic activities in insulin resistance (IR) 3T3-L1 adipocytes, and compound 4 was tested for its anti-diabetic activity in vivo. Results suggested compound 4 could effectively reduce blood glucose level in diabetic SD rats in a dose of 30 mg/kg.
Assuntos
Células 3T3-L1 , Croton , Diterpenos , Hipoglicemiantes , Folhas de Planta , Ratos Sprague-Dawley , Folhas de Planta/química , Diterpenos/farmacologia , Diterpenos/química , Diterpenos/isolamento & purificação , Croton/química , Animais , Camundongos , Hipoglicemiantes/farmacologia , Hipoglicemiantes/química , Hipoglicemiantes/isolamento & purificação , Ratos , Estrutura Molecular , Diabetes Mellitus Experimental/tratamento farmacológico , Masculino , Resistência à Insulina , Glicemia/efeitos dos fármacos , Adipócitos/efeitos dos fármacosRESUMO
Eight compounds were isolated from ethyl acetate fraction of 80% ethanol extract of the hulls of Garcinia mangostana by silica gel, Sephadex LH-20 column chromatography, as well as prep-HPLC methods. By HR-ESI-MS, MS, 1D and 2D NMR spectral analyses, the structures of the eight compounds were identified as 16-en mangostenone E(1), α-mangostin(2), 1,7-dihydroxy-2-(3-methy-lbut-2-enyl)-3-methoxyxanthone(3), cratoxyxanthone(4), 2,6-dimethoxy-para-benzoquinone(5), methyl orselinate(6), ficusol(7), and 4-(4-carboxy-2-methoxyphenoxy)-3,5-dimethoxybenzoic acid(8). Compound 1 was a new xanthone, and compound 4 was a xanthone dimer, compound 5 was a naphthoquinone. All compounds were isolated from this plant for the first time except compounds 2 and 3. Cytotoxic bioassay suggested that compounds 1, 2 and 4 possessed moderate cytotoxicity, suppressing HeLa cell line with IC_(50) va-lues of 24.3, 35.5 and 17.1 µmol·L~(-1), respectively. Compound 4 also could suppress K562 cells with an IC_(50) value of 39.8 µmol·L~(-1).
Assuntos
Antineoplásicos , Garcinia mangostana , Garcinia , Xantonas , Humanos , Garcinia mangostana/química , Células HeLa , Espectroscopia de Ressonância Magnética , Xantonas/farmacologia , Garcinia/química , Extratos Vegetais/química , Estrutura MolecularRESUMO
Nine compounds were isolated from the 90% ethanol extract of Salacia polysperma by silica gel, Sephadex LH-20 column chromatography, together with preparative HPLC methods. Based on HR-ESI-MS, MS, 1D and 2D NMR spectral analyses, the structures of the nine compounds were identified as 28-hydroxy wilforlide B(1), wilforlide A(2), 1ß,3ß-dihydroxyurs-9(11),12-diene(3),(-)-epicatechin(4),(+)-catechin(5),(-)-4'-O-methyl-ent-galloepicatechin(6), 3-hydroxy-1-(4-hydroxy-3-methoxy-phenyl)propan-1-one(7),(-)-(7S,8R)-4-hydroxy-3,3',5'-trimethoxy-8',9'-dinor-8,4'-oxyneoligna-7,9-diol-7'-aldehyde(8), and vanillic acid(9). Compound 1 is a new oleanane-type triterpene lactone. Compounds 1, 3, 4, 7-9 were isolated from the Salacia genus for the first time. All compounds were assayed for their α-glucosidase inhibitory activity. The results suggested that compound 8 exhibited moderate α-glucosidase inhibitory activity, with an IC_(50) value of 37.2 µmol·L~(-1), and the other compounds showed no α-glucosidase inhibitory activity.
Assuntos
Salacia , Triterpenos , Salacia/química , alfa-Glucosidases , Triterpenos/farmacologia , Espectroscopia de Ressonância Magnética , Etanol , Estrutura MolecularRESUMO
Over-expression of the pathway specific positive regulator gene is an effective way to activate silent gene cluster. In the curret study, the SARP family regulatory gene, vasR2, was over-expressed in strain Verrucosispora sp. NS0172 and the cryptic gene cluster responsible for the biosynthesis of pentaketide ansamycin was partially activated. Two tetraketides (1 and 2) and a triketide (3) ansamycins, together with five known compounds (4-8), were isolated and elucidated from strain NS0172OEvasR2. Their NMR data were completely assigned by analysis of their HR-ESI-MS and 1H, 13C NMR, HMQC, HMBC and 1H-1H COSY spectra.
Assuntos
Micromonosporaceae , Policetídeos , Rifabutina/metabolismo , Micromonosporaceae/genética , Micromonosporaceae/metabolismo , Família Multigênica , Policetídeos/metabolismoRESUMO
Constitutively expression of the pathway-specific activators is an effective method to activate silent gene clusters and improve natural product production. In this study, nine shunt products of aminoansamycins (1-9) were identified from a recombinant mutant strain S35-LAL by overexpressed the large-ATP-binding regulator of the LuxR family (LAL) gene aas1 in Streptomyces sp. S35. All the compounds showed no anti-microbial, anti-T3SS and cytotoxic activities.
Assuntos
Produtos Biológicos/metabolismo , Lactamas Macrocíclicas/metabolismo , Família Multigênica , Streptomyces/genética , Organismos Geneticamente Modificados , Streptomyces/metabolismoRESUMO
Polycyclic tetramate macrolactams (PoTeMs) are a group of hybrid PK-NRP natural products having a variable set of carbocyclic rings, a conserved assembly pathway, and diverse bioactivities. We report here the identification of seven new PoTeMs, clifednamides D-J (3-9), along with the known clifednamides A (1) and B (2) through rational pathway refactoring and heterologous expression. Remarkably, clifednamides D (3), G (6), and H (7) feature an unprecedented 27,28-seco skeleton. The cytotoxic activities of compounds 1-9 indicated that the hydroxy group of C-25, the methyl group of C-30, the inner five-membered ring, and the intact macrocycle are all critical for the activities. Meanwhile, the cytochrome P450 enzyme CftS023A and the hydroxylase CftS023E involved in oxidative tailoring of clifednamides were found to decorate the fused 5-6 bicyclic intermediates. Accordingly, the biosynthetic pathway for clifednamides was proposed.
Assuntos
Antibacterianos/biossíntese , Antibacterianos/química , Streptomyces/química , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Vias Biossintéticas , Linhagem Celular Tumoral , Sistema Enzimático do Citocromo P-450/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Espectroscopia de Ressonância Magnética , Testes de Sensibilidade Microbiana , Oxigenases de Função Mista/metabolismo , Estrutura Molecular , Oxirredução , Microbiologia do Solo , Streptomyces/metabolismoRESUMO
Ansamycins is a group of type I polyketides characterized by the unique starter unit 3-amino-5-hydroxybenzoic acid. This family of secondary metabolites shows diverse biological activities, well-known members of which include rifamycin, geldanamycin, and maytansine. Previously, we isolated an AHBA synthase gene-positive strain Streptomyces sp. XZQH13 containing a "silent" ansamycin biosynthetic gene cluster ast. The constitutive expression of the Large-ATP-binding regulators of the LuxR family regulator gene astG1 located within the cluster triggered the expression of the biosynthetic genes. Reverse transcription-PCR experiments showed that the expression of the key biosynthetic genes, astB4, astD1, and astF1, was induced in the astG1 overexpression mutant compared to the wild type. This led to the isolation of two known ansatrienins, hydroxymycotrienin A (1) and thiazinotrienomycin G (2), which were identified by analysis of the mass spectral and NMR spectral data, from the mutant. These observations suggest that astG1 is probably a pathway-specific positive regulator for the biosynthesis of ansatrienin.
Assuntos
Alanina/análogos & derivados , Regulação Bacteriana da Expressão Gênica , Rifabutina/análogos & derivados , Streptomyces/genética , Streptomyces/metabolismo , Tiazinas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Alanina/metabolismo , Vias Biossintéticas/genética , Perfilação da Expressão Gênica , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Família Multigênica , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Rifabutina/metabolismoRESUMO
Echosides, isolated from Streptomyces sp. LZ35, represent a class of para-terphenyl natural products that display DNA topoisomerase I and IIα inhibitory activities. By analyzing the genome draft of strain LZ35, the ech gene cluster was identified to be responsible for the biosynthesis of echosides, which was further confirmed by gene disruption and HPLC analysis. Meanwhile, the biosynthetic pathway for echosides was proposed. Furthermore, the echA-gene, encoding a tri-domain nonribosomal peptide synthetase (NRPS)-like enzyme, was identified as a polyporic acid synthetase and biochemically characterized in vitro. This is the first study to our knowledge on the biochemical characterization of an Actinobacteria quinone synthetase, which accepts phenylpyruvic acid as a native substrate. Therefore, our results may help investigate the function of other NRPS-like enzymes in Actinobacteria.
Assuntos
Proteínas de Bactérias , Peptídeo Sintases , Streptomyces , Compostos de Terfenil , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Genoma Bacteriano , Dados de Sequência Molecular , Peptídeo Sintases/genética , Peptídeo Sintases/metabolismo , Streptomyces/enzimologia , Streptomyces/genética , Compostos de Terfenil/química , Compostos de Terfenil/metabolismoRESUMO
Divergolides are a group of structurally unprecedented ansamacrolactam antibiotics with antibacterial and antitumor activities. A biosynthetic gene cluster predicted to encode the biosynthesis of divergolides was cloned and sequenced from endophytic Streptomyces sp. W112. The gene cluster of divergolides (div) spans a DNA region of 61-kb and consists of 20 open reading frames (ORFs) that encode polyketide synthases (PKSs), enzymes for the synthesis of AHBA and PKS extender units, and post-PKS modifications, proposed regulators, and putative transporters. Disruption of the AHBA synthase gene (divK) completely abolished the production of divergolides proved its involvement in the biosynthesis of divergolides. Bioinformatics analysis suggested that the regulatory gene div8 in div gene cluster might encode a positive regulator for the biosynthesis of divergolides. Constitutive overexpression of div8 improved the production of divergolides E, implying that div gene cluster maybe responsible for the biosynthesis of divergolides. These findings set the stage for fully investigating the biosynthesis of divergolides and rational engineering of new divergolide analogs by genetic modifications, and pave the way to further improve the production of divergolides.
Assuntos
Proteínas de Bactérias/genética , Macrolídeos/metabolismo , Família Multigênica , Streptomyces/genética , Aminobenzoatos/metabolismo , Antibacterianos/biossíntese , Antibacterianos/química , Proteínas de Bactérias/metabolismo , Vias Biossintéticas/genética , Clonagem Molecular , Regulação Bacteriana da Expressão Gênica , Biblioteca Genômica , Hidroliases/genética , Hidroliases/metabolismo , Hidroxibenzoatos/metabolismo , Macrolídeos/química , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Fases de Leitura Aberta/genética , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Espectrometria de Massas por Ionização por Electrospray , Streptomyces/metabolismoRESUMO
Plant microbiota (the microorganisms that live in any associations with plant tissues) represents a rather unexplored area of metagenomic research compared with soils and oceans. Constructing a metagenomic library for plant microbiota is technically challenging. Using all the biomass without pre-enrichment could lead to vast proportions of the host plant DNA in the metagenomic library, doubtless obliterating the microbial contribution. Therefore, the first and essential step is to enrich for the constituent microorganisms from plant tissues. Here, a strong enrichment for plant microbiota was achieved by coupling SDS (sodium dodecyl sulfate) with NaCl, creating a predominantly microbial metagenomic library that contains 88% bacterial inserts. 16S rDNA sequence analysis revealed that the metagenomic DNA of enrichments originates from very diverse microorganisms. At least 74 distinct ribotypes (at a 97% threshold) from seven different bacterial phyla were identified and mainly distributed among Actinobacteria and Proteobacteria. Additionally, a simplified version of Amplified Ribosomal DNA Restriction Analysis (ARDRA) was developed for a quick and efficient assessment of the enriching procedures. This work opens further insight into the great biotechnical potential of plant microbiota, holding more potential for drug discovery through a metagenomic strategy, and paving the way for recovery and biochemical characterization of functional gene repertoire from plant microbiota.
Assuntos
Bactérias/crescimento & desenvolvimento , Bactérias/isolamento & purificação , DNA Bacteriano/genética , Biblioteca Gênica , Plantas/microbiologia , Biodiversidade , DNA Bacteriano/química , DNA Ribossômico/química , DNA Ribossômico/genética , Genes de RNAr , Genótipo , Dados de Sequência Molecular , Filogenia , RNA Bacteriano/genética , RNA Ribossômico 16S/genética , Ribotipagem , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
From the branch tissue of Maytenus hookeri, the endophytic strain Lz531 was isolated, and determined to belong to Streptomyces, according to its 16S rRNA sequence. From the extracts of the fermentation broth of Streptomyces sp. Lz531, two new and four known compounds were isolated. The two new compounds were identified as cyclo(L-Pro-L-Val-L-Val) (1) and 13-methyl-N-(2-phenylethyl)tetradecanamide (2).
Assuntos
Maytenus/microbiologia , Miristatos/isolamento & purificação , Peptídeos Cíclicos/isolamento & purificação , Streptomyces/química , Miristatos/química , Peptídeos Cíclicos/química , Streptomyces/metabolismo , SimbioseRESUMO
Gastrodia elata Bl. is an achlorophyllous orchid plant feeding on the fungus Armillaria mellea. The plant lives underground during its life cycle except for florescence. Gastrodianins, members of the superfamily of monocot mannose-binding protein (MBP), have been identified from Gastrodia elata, yet their physiological functions in the plant are rarely understood. Aspects of expression of gastrodianins in growth and development of the plant will be helpful to dissect their functional roles. Two types of cDNA clones with complete cDNA sequences matching the known gastrodianins were obtained from G. elata Bl.f.glauca S.chow (Orchidaceae) and designated gastrodianin-4A (ga4A) and gastrodianin-4B (ga4B), respectively. But only one isoform was found to be expressed in all different parts of a single plant. Based on the RNA gel blot analysis, gastrodianins were much more abundantly expressed in the fully opened flowers than the underground corms where an enhanced expression was found in the out layers of secondary corms. By RNA in situ hybridization gastrodianin transcripts were distinctly detected in the cortical cells and vascular cells of corms. Strong transcript accumulations were observed in two to eight layers of cortical cells in secondary corms. From its peripheral tissue expression pattern and level in corms and flowers, the gastrodianin may account for a possible defense against phytopathogens or insects.
Assuntos
Gastrodia/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Lectinas de Ligação a Manose/genética , Proteínas de Plantas/genética , Sequência de Aminoácidos , Clonagem Molecular , DNA de Plantas/análise , DNA de Plantas/metabolismo , Lectinas de Ligação a Manose/química , Dados de Sequência Molecular , Proteínas de Plantas/química , RNA de Plantas/análise , RNA de Plantas/metabolismoRESUMO
Geraniol may accumulate up to 86-98% of the leaf essential oils in geraniol chemotypes of the evergreen camphor tree Cinnamomum tenuipilum. A similarity-based cloning strategy yielded a cDNA clone that appeared to encode a terpene synthase and which could be phylogenetically grouped within the angiosperm monoterpene synthase/subfamily. After its expression in Escherichia coli and enzyme assay with prenyl diphosphates as substrates, the enzyme encoded by the putative C. tenuipilum monoterpene synthase gene was shown to specifically convert geranyl diphosphate to geraniol as a single product by GC-MS analysis. Biochemical characterization of the partially purified recombinant protein revealed a strong dependency for Mg2+ and Mn2+, and an apparent Michaelis constant of 55.8 microM for geranyl diphosphate. Thus, a new member of the monoterpene synthase family was identified and designated as CtGES. The genome contains a single copy of CtGES gene. Expression of CtGES was exclusively observed in the geraniol chemotype of C. tenuipilum. Furthermore, in situ hybridization analysis demonstrated that CtGES mRNA was localized in the oil cells of the leaves.