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Doi:10.1016/j.phytochem.2006.06.033

An antibacterial hydroxy fusidic acid analogue from Liam Evans a, John N. Hedger b, David Brayford b, Michael Stavri c, Eileen Smith c, Gemma O’Donnell c, Alexander I. Gray d, Gareth W. Griffith e, Simon Gibbons c,* a Hypha Discovery Ltd., School of Biosciences, University of Westminster, 115 New Cavendish Street, London W1W 6UW, UK b School of Biosciences, University of Westminster, 115 New Cavendish Street, London W1W 6UW, UK c Centre for Pharmacognosy and Phytotherapy, The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N 1AX, UK d Department of Pharmaceutical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 0NR, UK e Institute of Biological Sciences, University of Wales, Aberystwyth, Penglais, Aberystwyth SY23 3DD, UK Received 3 May 2006; received in revised form 19 June 2006 A fusidane triterpene, 16-deacetoxy-7-b-hydroxy-fusidic acid (1), was isolated from a fermentation of the mitosporic fungus Acremo- nium crotocinigenum. Full unambiguous assignment of all 1H and 13C data of 1 was carried out by extensive one- and two-dimensionalNMR studies employing HMQC and HMBC spectra.
Compound 1 was tested against a panel of multidrug-resistant (MDR) and methicillin-resistant Staphylococcus aureus (MRSA) strains and showed minimum inhibitory concentration values of 16 lg/ml.
Ó 2006 Elsevier Ltd. All rights reserved.
Keywords: Acremonium crotocinigenum; Fusidane triterpene; Fusidic acid; Antibacterial; MRSA; MDR; Staphylococcus aureus Reserve, Pichincha Province, Ecuador in 1986, and cur-rently held in the University of Westminster culture collec- Our studies on the production of metabolites by taxa of tion. Acremonium is a polyphyletic genus, often confused tropical rainforest fungi in fermentation, have led to the with Cephalosporium and is related to a number of ascomy- isolation and characterisation of a new metabolite, desig- cete teleomorphs (It contains some 105 spe- nated 16-deacetoxy-7b-hydroxy-fusidic acid (1), which is cies, including a number which have been shown to structurally related to the commercial antibiotic, fusidic produce biologically active metabolites ( acid, a widely used therapeutic for methicillin-resistant Previous studies on A. crotocinigenum found sesquit- Staphylococcus aureus (MRSA) infections which is still of erpenoid compounds of the isocrotonic acid type ( interest as a template for antibiotic activity improvement The detection of 1 was part of a programme for screen- component of fermentation liquors from shake cultures of ing tropical fungi for new antibiotics with activity against an isolate of the mitosporic fungus Acremonium crotocini- MRSA. There is currently an acute need for new effective genum, cultured from rotting wood in Rio Palenque Forest antibiotics for MRSA treatment, especially since theappearance of vancomycin resistant (VRSA) strains ( Corresponding author. Tel.: +44 207 7535913; fax: +44 207 7535909.
E-mail address: (S. Gibbons).
Liquid fermentation was used in conjunction 0031-9422/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2006.06.033 L. Evans et al. / Phytochemistry 67 (2006) 2110–2114 with bioautography, to qualitatively indicate the presence methyl singlets, one methyl doublet, four olefinic carbons of antibacterial compounds, facilitating the isolation of and a carbonyl of a carboxylic acid (dC 173.8), were indic- compound 1 by vacuum liquid chromatography.
ative of a fusidane class triterpene of the fusidic acid type).
By careful analysis of the HMBC, HMQC and COSY spectra it was possible to show that 1 was a new fusidic acid analogue. Assuming that the methyl doublet was C-28 ofthe fusidane skeleton, the protons of this group coupled H-4 formed part of a spin system with a deshielded methine H 3.73, H-3) and two methylene groups (at C-2 and C-1).
In the HMBC spectrum, C-1 was coupled to by the protons of methyl-C19 (dH 0.95) which showed further couplings to C-10 (2J), C-9 (3J) and C-5 (3J). In the COSY spectrum, H- 5 (dH 2.31 m) coupled to both protons of a methylene moi-ety (C-6, dH 1.45, 1.67), which further coupled to adeshielded oxymethine proton (C-7, dH 3.00, t). Inspectionof the HMBC spectrum showed that the carbon associatedwith this deshielded proton was coupled to by the protons of a further angular methyl singlet (C-30), which showedadditional couplings to a methine carbon (C-9) and two Bioautography of the Diaion HP20 resin extract of the quaternary carbons (C-8, dC 45.6 and C-14, dC 49.6). This fermentation filtrate led to the isolation of compound 1 completed the resonances for the A and B rings of com- as a white solid. High-resolution ESI-TOFMS in the posi- pound 1. Inspection of the COSY spectrum showed that tive mode suggested a molecular formula of C the proton associated with C-9 (H-9) formed part of a nals in the 1H and 13C NMR spectra () for five CH–CH–CH2–CH spin system which allowed identifica-tion of positions C-9, C-11, C-12 and C-13, respectively.
C-11 was deshielded (dC 68.7, dH 4.37) indicating that anoxygen should be placed here. Furthermore, H-13 (delin- Table 11H (400 MHz) and 13C NMR (100 MHz) spectral data and 1H–13C long- eated by inspection of the HMQC spectrum) was also range correlations of 1 recorded in CDCl3 deshielded (dH 3.05) suggesting that it was allylic and that an olefinic carbon (C-17) should be placed at the neigh-bouring carbon, which is typical for fusidic acid metabo- of a methyl group (C-18) coupled to C-13 (3J), C-14 (2J) and to a methylene carbon (C-15, 3J). CH2-15 coupled to a deshielded allylic methylene group (dH 2.68, 2.86 (CH2- 16)) which again was supportive of being alpha to an ole- finic carbon (C-17, dC 160.4). This completed rings C and D of 1. H-13 and H2-16 both gave a 2J coupling to C-17 and a 3J coupling to C-20, suggesting a C-17,20 double bond. In the HMBC spectrum C-17 was also coupled to by the protons of an allylic methylene (C-22, dH 2.44) which also coupled to a carbonyl carbon of a carboxylic acid group (C-21) and an olefinic methine carbon (C-24, dC 124.0). A further methylene (C-23) could be placed between C-22 and C-24 by couplings observed in the COSY spectrum. Finally, two deshielded geminal methyl groups could be placed on an olefinic carbon (C-25) via their HMBC correlations to this carbon and to the olefinic partner C-24 finalising the C-24–C-25 double bond. These resonances completed the eight carbon chain of the fusi- dane triterpene skeleton. HRESI-MS of 1 suggested a molecular formula of C29H46O5 [M]+ (475.3422). From the chemical shift values of H-3, H-7 and H-11, hydroxyl groups must be placed at these positions. From the molec- ular formula and chemical shift of the C-21 carbon, a car- L. Evans et al. / Phytochemistry 67 (2006) 2110–2114 IR spectra were recorded on a Nicolet 360 FT-IR spec- trophotometer and UV spectra on a Thermo Electron Cor- Cultures were maintained on malt extract agar (Oxoid) and for long term storage on malt extract agar plugs sub- merged in sterile distilled water at room temperature, aspart of the University of Westminster culture collection Fig. 1. Key COSY (double headed arrow) and HMBC (single headed (Culture No. cc56). The isolate was identified as A. crotoci- nigenum by David Brayford, initially through DNAsequencing of a PCR product amplified from the variableITS (internal transcribed spacer) region of the ribosomal boxylic acid must be placed at C-21 and this is identical to RNA locus using the conserved primers ITS1F and ITS4 The final consideration was to assign stereochemistry of DNA sequence databases with 508 bp of DNA sequence hydroxyl groups at C-3, C-7 and C-11. The hydrogens at from this PCR product using the FASTA algorithm C-3 and C-11 were assigned as equatorial (rel b) on the basis of no large discernable couplings for these signals, revealed the most closely related sequence accession to be which would make the hydroxyl groups at these positions AJ621773 (Acremonium crotocinigenum), which showed both a and axial. The coupling constant for H-7 (d 3.99, 98.2% identity over a 513 bp overlap. This A. crotocinige- t) was 8.0 Hz indicating an axial-axial interaction with num strain was isolated from the basidiome of Trametes the axial partner of CH2-6. This would make H-7 axial (a) and the OH at this position therefore equatorial (b).
This was further supported by an NOE between H-7 and the conidia, chlamydospores and colony appearance with CH3-30 indicating that they are both on the alpha face of were used to confirm that cc56 was indeed morpho- Fusidic acid and all known analogues to date have no logically the same as A. crotocinigenum. Further confirma- substitution on carbon 7. Compound 1 possesses an hydro- tion was obtained by direct comparison of cc56 with strain xyl at this position. A. crotocinigenum has also been found CABI 112775 (syn. CBS 129.64) kindly supplied by the to produce analogues with an hydroxyl on carbon 16 and 1 International Mycological Institute, Egham, UK. The is the first member of this class to be completely unsubsti- ITS sequence for isolate cc56 has been deposited in the GenBank database (accession number DQ882846).
Compound 1 was tested against a battery of drug-resis- tant bacteria and where active, possessed a minimum inhib- occasionally more active than erythromycin and norfloxa- Inoculum for the fermentation was prepared by vigor- cin, was significantly less potent than the fusidic acid ously shaking twenty 8 mm diameter plugs, excised from an actively growing culture of A. crotocinigenum on 2%malt extract agar (Oxoid), in 10 ml of sterile distilled watercontaining 2–3 ml of glass beads (VWR). The resulting mycelial suspension (2 ml) was added to each of twenty1000 ml conical flasks, containing 200 ml of sterilised 2% potato dextrose broth (Difco). The flasks were incubatedon a rotary shaker (200 rpm) for two weeks at 26 °C.
NMR spectra were recorded on a Bruker AVANCE Biomass was removed from the culture broth by filtering 500 MHz spectrometer. Chemical shift values (d) were through muslin prior to filtration through a Whatman No.
reported in parts per million (ppm) relative to appropriate 1 filter paper. The filtrate was then extracted with Diaion internal solvent standard and coupling constants (J values) HP20 resin (400 ml; Mitsubishi) which had previously been are given in Hertz. Accurate mass measurements were washed with HPLC grade methanol (Merck) and thor- determined on a Micromass Q-TOF Ultima Global Tan- oughly conditioned with distilled water. The resin was dem Mass Spectrometer. The sample was run under elec- removed, washed with distilled water (2 · 1000 ml) and trospray ionisation mode using 50% acetonitrile in water eluted with HPLC grade methanol (2 · 1000 ml). The and 0.1% formic acid as solvent. [Glu]-fibrinopeptide B methanolic eluent was evaporated to dryness.
L. Evans et al. / Phytochemistry 67 (2006) 2110–2114 Bioautographic analysis was performed using Staphylo- coccus aureus (NCTC 6571) as the test organism.
The crude HP20 resin extract of the culture filtrate was S. aureus inoculum was prepared by seeding a 100 ml dissolved in methanol (5 ml) and combined with an equiv- conical flask containing sterile nutrient broth (10 ml), the alent mass of silica gel (flash chromatography grade; BDH; flask was shaken overnight at 200 rpm at 37 °C.
1.6 g) and evaporated. The slurry was packed in a pre-col- The inoculum was applied to the run TLC plates by umn cartridge assembled in a Biotageä chromatography gently dabbing with sterilised foam. The seeded plates were apparatus along with a 40 mm diameter silica gel column.
subsequently incubated overnight at 37 °C in a humidified The column was eluted with the following mobile phase chamber. The incubated plates were then sprayed with fractions: 100% dichloromethane (100 ml), 2% methanol/ nitro-blue tetrazolium (Sigma Ltd.) in order to stain the dichloromethane (200 ml), 4% methanol/dichloromethane live S. aureus and then re-incubated for 1 h to develop, (200 ml), 6% methanol/dichloromethane (200 ml), 8% the undeveloped areas of the plates indicating the presence methanol/dichloromethane (200 ml) and finally 10% meth- anol/dichloromethane (200 ml). None of the fractions wereobserved to contain the desired metabolite which had cor- related to a zone of inhibition in the bioautographical anal-ysis. The column was therefore further eluted with 20% S. aureus strain ATCC 25923 was the generous gift of E.
methanol/dichloromethane and a series of 30 ml volume Udo (Kuwait University, Kuwait). S. aureus RN4220 con- fractions were collected. TLC analysis showed the target taining plasmid pUL5054, which carries the gene encoding compound to be present in fractions 7–18. These fractions the MsrA macrolide efflux protein, was provided by J.
were combined, evaporated to dryness and re-dissolved in 9:1 ethyl acetate/n-hexane (2 ml) for further fractionation the TetK tetracycline efflux protein, was provided by E.
on a 10 mm diameter Biotage column, using isocratic 9:1 ethyl acetate/n-hexane as the mobile phase, fractionated overexpresses the norA gene encoding the NorA MDR into 7 ml fractions. The target compound was contained efflux protein was provided by G. Kaatz ( in fractions 5–15, these were combined, dried and reconsti- ). All Staphylococcus aureus strains were cultured on tuted in 8% methanol/dichloromethane for further iso- nutrient agar and incubated for 24 h at 37 °C prior to cratic fractionation using the same solvent and a 10 mm MIC determination. Bacterial inocula equivalent to the Biotage column. Fractions of 3 ml volume were collected, 0.5 McFarland turbidity standard were prepared in normal TLC analysis showed the compound to be solely present saline and diluted to give a final inoculum density of in fractions 12–32. These fractions were combined and 5 · 105 cfu/ml. The inoculum (125 ll) was added to all the dry weight of pure compound determined to be wells and the microtitre plate was incubated at 37 °C for 18 h. The MIC was recorded as the lowest concentrationat which no bacterial growth was observed as previously 3.5. Thin layer chromatography and bioautography analysis Thin layer chromatography (TLC) separation was 3.7. 16-Deacetoxy-7b-hydroxyfusidic acid (1) achieved using silica gel plates and three solvent systems(9:1 dichloromethane/methanol; 6:4 ethyl acetate/n-hexane White powder; ½aŠ21 À 113:64 (c 0.08, CHCl and 9:1 ethyl acetate/n-hexane). All solvents used were (ACN) kmax (log e): 233 (3.96) nm; IR mmax (thin film) Metabolites were visualised on the TLC plates by spray- 1436.56, 1375.42, 1255.07 1053.01, 934.01, 653.86; 1H ing with a 4% vanillin/concentrated sulphuric acid solution (m/z): 475.3422 [M+H]+ (calc. for C29H47O5, 475.3418).
Table 2MICs of 1 and standard antibiotics in lg/ml All MICs were determined in duplicate.
L. Evans et al. / Phytochemistry 67 (2006) 2110–2114 Gibbons, S., Udo, E.E., 2000. The effect of reserpine, a modulator of multidrug efflux pumps, on the in vitro activity of tetracyclineagainst clinical isolates of methicillin resistant Staphylococcus aureus We acknowledge the contribution of the late David (MRSA) possessing the Tet(K) determinant. Phytother. Res. 14, Brayford, who sadly passed away before the publication of this paper. We thank the Engineering and Physical Sci- Gyimesi, J., Melera, A., 1967. Structure of crotocin, an antifungal ences Research Council (Grant No. GR/R47646/01). We antibiotic. Tetahedron Lett. 17, 1665–1673.
also thank Ing. Raul Camacho and staff of the Facultad Kaatz, G.W., Seo, S.M., Ruble, C.A., 1993. Efflux-mediated fluoroqui- nolone resistance in Staphylococcus aureus. Antimicrob. Agents de Ciencias, Escuela Superior Politecnica de Chimborazo, Riobamba, Ecuador for assistance with field work in Ecua- Kirk, P.M., Cannon, P.F., David, J.C., Stalpers, J.A., 2001. Ainsworth dor, and Cal & Piedad Dodson for permission to collect in and Bisby’s Dictionary of the Fungi, ninth ed. CAB International, Pearson, W.R., Lipman, D.J., 1988. Improved tools for biological sequence comparison. PNAS 85, 2444–2448.
Rastup-Andersen, N., Duvold, T., 2002. Reassignment of the 1H NMR spectrum of fusidic acid and total assignment of 1H and 13C NMRspectra of some selected fusidane derivatives. Mag. Res. Chem. 40, Centers for Disease Control (CDC), 2002. Public health dispatch: vancomycin-resistant Staphylococcus aureus – Pennsylvania. Morbid.
Ross, J.I., Farrell, A.M., Eady, E.A., Cove, J.H., Cunliffe, W.J., 1989.
Characterisation and molecular cloning of the novel macrolide- Chang, S., Sievert, D.M., Hageman, J.C., Boulton, M.L., Tenover, F.C., streptogramin B resistance determinant from Staphylococcus epide- Downes, F.P., Shah, S., Rudrik, J.T., Pupp, G.R., Brown, W.J., rmidis. J. Antimicrob. Chemother. 24, 851–862.
Cardo, D., Fridkin, S.K.and the Vancomycin-Resistant Staphylococ- Schol-Schwarz, M.B., 1965. Cephalosporium crotocinigenum sp. nov.
cus aureus Investigative Team, 2003. Infection with vancomycin- Trans. Brit. Mycol. Soc. 48, 51–53.
resistant Staphylococcus aureus containing the vanA resistance gene.
Søtofte, I., Duvold, T., 2001. A new potent fusidic acid analogue. Acta Gams, W., 1971. Cephalosporium-artige Schimmelpilze (Hyphomycetes).
White, T.J., Bruns, T., Lee, S., Taylor, J.W., 1990. Amplification and Gustav Fischer Verlag, Jena, 262 pp.
direct sequencing of fungal ribosomal RNA genes for phylogenetics.
Gardes, M., Bruns, T.D., 1993. ITS primers with enhanced specificity for In: Innis, M.A., Gelfand, D.H., Sninsky, J.J., White, T.J. (Eds.), PCR basidiomycetes – application to the identification of mycorrhizae and Protocols: A Guide to Methods and Applications. Academic Press,

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