Purification of a novel angiogenesis inhibitory factor from Dasyatis akajei cartilage

The isolation and purification procedure for Dasyatis akajei cartilage angiogenesis inhibitory factor-I (DCAIF-I) was developed.DCAIF-I yield was improved by investigating the extractconditions, and DCAIF-I purification was achieved by combining several types of column chromatographies.The angiogenesis inhibitory bioactivity of DCAIF-I was identified by two models, such as the collagenase model and the blood vessel formation model of the chorioallantoic membrane of chicken embryo(CAM).The orthogonal experiment method was used to optimize the extract conditions, such as temperature, pH, stirring rate, and extract time.Under the optimized conditions, it was found that a high concentration of protein with the yield of 0.84% was obtained at 30℃, pH 6.2, stiring rate of 100 r/min, and extract for 72 h.The combination and parameter selection of ion exchange chromatography, gel filtration chromatography and reverse-phase high performance liquid chromatography achieved DCAIF-I purification to obtain the SDS-PAGE-purity preparation, The purifiedDCAIF-I was homogeneous as a single band of 62kDa in 12% SDS-PAGE stained with coomasie brilliant blue.The bioactivity of DCAIF-I was identified by the model of inhibitory activity of collagenase and the blood vessel formation of the CAM.The identification of collagenase model showed that the absorption value of DCAIF-I treated group was between the values of two control groups, which indicated that DCAIF-I could inhibit the dissolution of collagen protein with the inhibition rate of 36.3%.The identification of the CAM experiment indicates that the large area of blood vessels in DCAIF-I treated group exhibited heavy loss of color, accom panied with the decreased density of vessels.Quantitative analysisof CAM indicated that the blood vessels quantity of DCAIF-I treated group was decreased obviously with the increased dosage of DCAIF-I.Therefore, these findings have special importance in showing that DCAIF-I had the inhibitory activity on collagenase and the inhibitoryeffect of angiogenesis in the CAM.Moreover, the do sage of DCAIF-I correlated with the inhibitory effect of angiogenesis in the CAM.