RESUMO
In this paper, high-speed counter-current chromatography (HSCCC), assisted with ESI-MS, was first successfully applied to the preparative separation of three macrolide antibiotics, brefeldin A (12.6 mg, 99.0%), 7'-O-formylbrefeldin A (6.5 mg, 95.0%) and 7'-O-acetylbrefeldin A (5.0 mg, 92.3%) from the crude extract of the microbe Penicillium SHZK-15. Considering the chemical nature and partition coefficient (K) values of the three target compounds, a two-step HSCCC isolation protocol was developed in order to obtain products with high purity. In the two-step method, the crude ethyl acetate extract was first fractionated and resulted in two peak fractions by HSCCC using solvent system n-hexane/ethyl acetate/methanol/water (HEMWat) (3:7:5:5 v/v/v/v), then purified using solvent systems HEMWat (3:5:3:5 v/v/v/v) and HEMWat (7:3:5:5 v/v/v/v) for each fraction. The purities and structures of the isolated compounds were determined by HPLC, X-ray crystallography, ESI-MS and NMR. The results demonstrated that HSCCC is a fast and efficient technique for systematic isolation of bioactive compounds from the microbes.
Assuntos
Distribuição Contracorrente/métodos , Fermentação , Compostos Macrocíclicos/isolamento & purificação , Penicillium/metabolismo , Acetatos/química , Brefeldina A/análogos & derivados , Brefeldina A/química , Brefeldina A/isolamento & purificação , Brefeldina A/metabolismo , Cromatografia Líquida de Alta Pressão , Cristalografia por Raios X , Meios de Cultivo Condicionados/química , Compostos Macrocíclicos/química , Compostos Macrocíclicos/metabolismo , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Solventes/química , Espectrometria de Massas por Ionização por Electrospray , Espectrofotometria UltravioletaRESUMO
A novel compound named as brefeldin A formylate (1), together with two known compounds, brefeldin A (2) and ergosterol (3), was isolated from the Penicillium sp. strain HLKG-44, which was isolated from polluted environment in Fujian Province. Their structures were identified based on the spectral and X-ray crystallographic analyses. The compound 1, brefeldin A formylate, exhibited moderate cytotoxic activity against the human lung cancer cell line A549 with IC(50) value of 18.9 microg/ml by the MTT assay protocol.
Assuntos
Antineoplásicos/isolamento & purificação , Penicillium/química , Antineoplásicos/química , Antineoplásicos/farmacologia , Brefeldina A/análogos & derivados , Brefeldina A/química , Brefeldina A/isolamento & purificação , Cristalografia por Raios X , Ensaios de Seleção de Medicamentos Antitumorais , Ergosterol/química , Ergosterol/isolamento & purificação , Humanos , Conformação Molecular , Estrutura MolecularRESUMO
Brefeldin A (BFA) is a macrolide lactone antibiotic, possessing antitumor, antiviral, antifungal activities. In this work, a separation strategy involving one-step macroporous resin adsorption chromatography combined with crystallization was established for BFA purification from Eupenicillium brefeldianum CCTCC M 208113 fermentation broth. Among six macroporous resin adsorbents tested, the non-polar resin HZ830 had the best adsorption and desorption performance. The static equilibrium adsorption data fitted well with the Freundlich equation, and the adsorption kinetic followed the pseudo-second order model. Through experimental optimization of column adsorption and desorption, BFA in purity of 90.4% (w/w), 92.1% (w/w) yield was obtained by a one-step macroporous resin adsorption chromatography, using a stepwise elution protocol. Furthermore, high purity (>99%, w/w) of BFA crystals were prepared from E. brefeldianum CCTCC M 208113 fermentation broth in an overall recovery of 67.0% (w/w), using a combination of adsorption chromatography packed with non-polar macroporous adsorbent HZ830 and crystallization in acetone.
Assuntos
Brefeldina A/isolamento & purificação , Cromatografia Líquida/métodos , Eupenicillium/metabolismo , Adsorção , Cromatografia Líquida/instrumentação , Meios de Cultivo Condicionados , Difusão , Etanol , Eupenicillium/química , Fermentação , Cinética , Reagentes de Laboratório/química , Modelos Lineares , ÁguaRESUMO
In this work, a robust brefeldin A-synthesizing fungus, Eupenicillium brefeldianum ZJB082702, was bred from a Murraya paniculata endophytic fungus E. brefeldianum A1163. Using one-factor-at-a-time experimental design, optimization of media composition for E. brefeldianum ZJB082702 fermenting brefeldin A was conducted. Outcomes indicated that mixed carbon source and mixed nitrogen source were of c ritical importance to brefeldin A fermentation. After 6d culture in the optimized fermentation media, composed of (gl(-1)) 13.33 starch, 26.67 glucose, 1.0 yeast extract powder, 1.0 corn steep liquor, 0.5 soybean meal, 0.75 NaNO(3), 2.5 malt extract, 6.0 CaCO(3), 3.0 MgSO(4), 4.0 KH(2)PO(4), 1.0 × 10(-2) CuSO(4), brefeldin A yield peaked at 1304.7 mgl(-1), 648.2 mgl(-1) in 500 ml baffled flask and 15 l stirred fermentor respectively, formed as a growth associated type of secondary metabolite based on fermentation profile analysis.