Isolation And Characterization Of Bioactive Substances From Bitter Melon Seeds

An antifungal protein(MAP13) was isolated from bitter melon seeds by anion exchange chromatograpy column (DEAE-650C) and cation exchange chromatograpy column(CM-650C). It was identified to be homogeneous by SDS-PAGE, and the relative molecular weight was estimated to be about 12.7kDa. It was composed of two subunits linked by a disulfide bond, whose molecular weight were 6 kDa and 7 kDa, respectively. It showed inhibitory activity in vitro against plant pathogenic fungi Physalospora piricola,Fusarium oxysporum ,Pythium aphanidermatum and Alternaria alternata(Fr.) Keissl. IC50 of MAP13 against Pythium aphanidermatum was 93.6μM. The possible antifungal mechanism was proposed to be the destruction of cell wall of fungi. Using the assay systems of DPPH , the result showed that MAP13 had good antioxidant activity , and that IC50 against DPPH was 20.9μmol/L.An active fraction(P2) was separated from bitter melon seeds by gel permeation chromatography on a Sephadex G-75 column and reversed phase HPLC on LUNA C18 column. The homogeneity of P2 was identified by cLC capillary liquid chromatography. The result of mass spectrometry analysis of P2 showed that P2 consisted of two components(1533.54,1555.57Da), which had similar properties.P2 was proved to have strong growth inhibitory activity in vitro against plant pathogenic fungi Physalospora piricola,Fusarium oxysporum, Pythium aphanidermatum, Fusarium solani, Botrytis cinerea, Fusarium oxysporum f.sp.nelonis, Alternaria alternata(Fr.) Keissl and Mycosphaerella arachidicola. IC50 against Fusarium solani was 18μg/mL. The possible antifungal mechanism was different from MAP13.P2 had apparent inhibitory effect on the growth of HepG2 and MDCK cells in vitro. IC50 against HepG2 and MDCK was 3.87μg/mL and 5.99μg/mL, respectively. The results showed that inhibition capability of these two peptides against HepG2 was higher than against MDCK in the same dose, and much in the low concentration.