Preparative isolation and purification of macrolactin antibiotics from marine bacterium Bacillus amyloliquefaciens using high-speed counter-current chromatography in stepwise elution mode.

Preparative high-speed counter-current chromatography (HSCCC) was successfully applied to the isolation and purification of two macrolactin antibiotics from marine bacterium Bacillus amyloliquefaciens for the first time using stepwise elution with a pair of two-phase solvent systems composed of n-hexane-ethyl acetate-methanol-water at (1:4:1:4, v/v) and (3:4:3:4, v/v). The preparative HSCCC separation was performed on 300 mg of crude sample yielding macrolactin B (22.7 mg) and macrolactin A (40.4 mg) in a one-step separation, with purities over 95% as determined by HPLC. The structures of these compounds were identified by MS, (1)H NMR and (13)C NMR. Our results demonstrated that HSCCC was an efficient technique to separate marine antibiotics, which provide an approach to solve the problem of their sample availability for drug development.