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Isolation and purification of macrocyclic components from Penicillium fermentation broth by high-speed counter-current chromatography
- Gao, Xiang, Zhuang, Rongqiang, Guo, Jiannan, Bao, Jian, Fang, Meijuan, Liu, Yan, Xu, Pengxiang, Zhao, Yufen
- Journal of separation science 2010 v.33 no.2 pp. 277-284
- Penicillium, X-ray diffraction, acetates, brefeldin A, countercurrent chromatography, fermentation, high performance liquid chromatography, methanol, nuclear magnetic resonance spectroscopy, partition coefficients
- 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.