An Investigation On The Anticoagulating Activity Fraction (AAF) Of Bombyx Batryticatus(BB) And Quantitative Analysis The Components Associated With This Effect

[Abstract]Objective To explain the chemical nature of anticoagulating activity of BB by extracting and separating BB and to quantifily analysis the main components associated with anticoagulating activity.Methods The prepared technology was chosen by TT assay. Proteins and peptides, amino acids, oxalate ammonium(OA) were identified and determined by chemical assay such as TLC, UV spectrometry, titrimetric analysis and amino acid auto-analysis, et al. Also study on the effect of oxalate ammonium on AA of BB with adding OA to BB extract solution. Then separated the AAF by HPLC/MS and ion exchange chromatography.Results The AAF was prepared as the following procedures. Sephadex G-25 column was equilibrated with 0.02 mol/L Tris-HCl (pH7.4) buffer solution containing 0.05mol/L NaCl at 4℃. Then added 30mL crude extract sample to the column at the speed of 2.5mL/10min and eluted the column with the same buffer solution at the same speed, collected the elution solution with a tube of 2.5mL and determined the elution solution with TT and UV. Combined those elution whose TT was higher than 100s as the AAF. Six spots could be developed from the sample of AAF in this procedure, being at the same bands with similar color as the controlling spots of standard amino acid. The content of dry AAF was 4.31%. Its main components were protein and peptides, amount 68.4% of dry extract. The content of total amino acids was 2. 10g/mL. The amount of Glu, Ala and Val were much higher, amount to 22.76%, 14.97% and 7.96% of total amino acids, respectively. In additions, the content of ammonium oxalate was 0.42%, and amounted for 9.74% of dry extract. The total ion concentration by univalent ion was 0.165mol/mL. The reducing substances that can be oxidized by KMnO4 equal to the 0.174mol/mL by oxalate, and positive ion, negative ion and neutral molecules in these were 8.27%,57.50% and over 34%, respectively. Several activity peaks could be detected from the sample chromatography by HPLC. The NO.2 peak was composed of a series components with m/z at 1073,1223,1284,1346, et al. OA could lengthen significantly the blood clotting time of BB extract and gel filtrate solution.Conclusion The prepared technology was practical. The AAF had effect on anticoagulation significantly. The identified and determined methods were accurate and could be controlled. TLC could be used to identify amino acid. The classic chemical analysis could be used to determinate main components including protein and peptides, OA and ion concentration. OA had effect on BB by lengthen TT.