The Separation, Purity And Determination Of Monascopyridine A And Monascopyridine B From Monascus PHDS26

In this work, monascopyridine A(MPA) and monascopyridine B(MPB) were separated and purified from Monascus aurantiacus pksCT gene disruption mutant PHDS26, and an accurate HPLC method for the simultaneous determination MPA and MPB was established and validated. More details are as follows:Firstly, MPA and MPB were isolated and purified from Monascus PHDS26 by chromatography on a silica gel column and semi-preparative HPLC. The conditions of chromatography on a silica gel column were methanol as extraction solvent,n-hexane-ethyl acetate(7:3,v/v) as eluent, silica gel of 200-300 mesh as stationary phase; the conditions of semi-preparative HPLC were C18 preparative column Zorbax SB-C18(9.4×150 mm, 5 μm) and a mobile phase of acetonitrile-H2O(60:40,v/v), the detection wavelength was 300 mm, flow rate was 2.5 mL/min. 95% purity of MPA and MPB were obtained under these conditions.Secondly, MPA and MPB were extracted by supersonic wave auxiliary way from cell of Monascus PHDS26 ferment lysate. Various effective factors were researched and the technological conditions were optimized by orthogonal experiment. It showed that these conditions are methanol concentration 90%, temperature 40 ℃,supersonic wave time 10 min,solid-liquid ratio 1:25,for 2 times.Thirdly, a specific HPLC method had been developed and validated for the simultaneous determination of MPA and MPB. Chromatographic separation was achieved at 25 ℃ using a 250 × 4.6 mm I.D., 5μm Hypersil ODS-2 column(Thermo), with isocratic elution of water/acetonitrile(40:60, v/v) at a flow rate of 0.8mL/min. The linear equations of MPA and MPB were Y=33.386X+17.021 and Y=54.691X+0.409, respectively, and the correlation coefficients were 0.9999 and0.9997, respectively. The linear ranges of MPA and MPB were from 0.5-200 μg/mL and 0.5-300 μg/mL, respectively; and the RSD were 2.0%(within-day),4.8%(between-day) and 2.1%(within-day), 4.6%(between-day), respectively. The average recoveries of the method for MPA and MPB were 99.9% and 94%, respectively. The mycelia and fermented liquid of Monascus aurantiacus AS3.4384 and its pksCT genedisruption mutant PHDS26 were analyzed directly by this method. The results showed that MPA and MPB only be detected in the mycelia of PHDS26, and their yield reached the maximum in 16 th day, 2073.7 μg/g and 1961.7 μg/g, respectively.