| Curo trifasciata antitumor peptides was prepared by hydrolysis in this article. Using the degree of anti-tumor activity as an indicator to optimize hydrolysis conditions.The polypeptide composition was isolated and purified by ultrafiltration and Sephadex column. Mass spectrometry was used to analysis the components of peptides whose anti-tumor activity is good, notation software solution combines PEAKS to get polypeptide sequence,then synthesis the polypeptide.finally hybrid Peptide and synthetic peptide with good anti-tumor activity were interacted with the chitosan to form a complex of chitosan nanoparticles.The results were as follows:(1) Using crude protein extraction rate as an indicator, salt mention, Tris-HCl extraction and enzymatic extraction were compared, the final choice of extraction method was enzymatic extraction whose extraction rate reached 84.15%.Another advantage of this method was that the protein extraction process along with the enzymatic so polypeptides could be obtained directly, Also this process without a lot of salt mixed in this process, subsequent separation and purification were facilitated.(2) Curo trifasciata meat was hydrolyzed by trypsin, alcalase, papain and protamex under certain conditions to obtain peptides.After ultrafiltration of enzyme solution,12 kinds of peptides were obtained. MTT assay showed that : Trypsin hydrolysates E has better anti-tumor activity, when the concentration of hydrolysate was 1mg/m L,the inhibition rate of Hep G-2 was 92.95%,the inhibition rate of MCF-7 is 67.08%,so E were selected to isolation and purification.(3) Using the degree of anti-tumor activity as an indicator, Studied the hydrolysis conditions of alcalase, pepsin and protamex whose hydrolysates’ s anti-tumor activity was low by L9(34) orthogonal experiment. Among the inhibition rate of breast cancer cells(MCF-7),the optimal enzymatic species and hydrolysis conditions were papain and p H was 8, T was 55 ℃, E / S was 1.5%.when the concentration of hydrolysate was 1mg/m L,the inhibition rate of MCF-7 was 92.37%. Among the inhibition rate of liver cancer cells(Hep G-2),the optimal enzymatic species and hydrolysis conditions were protamex and p H was 8, T was 40℃, E/S was 2.0%.when the concentration of hydrolysate was 1mg/m L,the inhibition rate of Hep G-2 was 94.16%.After hydrolysates were ultrafiltrate, component F and M whose anti-tumor activity were better were selected to isolation and purification.(4) After Sephadex G-15, E was separate into four components E1, E, E3 and E4; M was separate into three components M1, M2 and M3; F was separate into five components F1, F2, F3, F4 and F5.MTT assay showed that when the concentration of these components was 500 μg / m L, E1, E2, M2 and F4 had better anti-tumor activity. The inhibition rate of liver cancer cells(Hep G-2) were 81.91%,70.65%,74.70% and 78.60% respectively. The inhibition rate of breast cancer cells(MCF-7) were 34.70%,73.70%,62.93% and 70.59% respectively.E1 had the most prominent anti-tumor activity.(5)By the means of a mass spectrometry,two stage mass spectrometry mass spectrometry and PEAKS,six sequence of peptides were got.Their charge-mass ratio were 769.490(E1-1), 852.549(E1-2), 777.399(E2-1), 827.464(M2-1), 771.574(F4-1) and 861.159(F4-2),respectively.Their sequences were : RGVKGPR;KLGPKGPR;SSPGPPVH; EMLQPPL;PGKPLFL;SCCSCDED,respectively.The MTT antitumor experiment of synthetic peptide showed that E2-1 had the most prominent anti-tumor activity whose half inhibitory concentration(IC50) of MCF-7 was 112.03 ?g/m L.(6)The chitosan nanoparticles of E1 and E2-1 were prepared by Ion-crosslinked method. The optimal experimental conditions were determined by single factor experiment: C(TPP) was 0.8 mg / m L, p H(CS) was 4.5,T was 50 ℃.The CS / TPP nanoparticles’ s particle size and Zeta potential were 191.2 nm and 31.2 mv,respectively.With optimum conditions the E1-CS / TPP nanoparticles’ s particle size and Zeta potential were 224.8 nm and 38.4mv. E2-1-CS / TPP nanoparticles’ s particle size and Zeta potential were 210.6 nm and 36.5mv. The dosage were 15.22% and 21.39%, embedding rate were 28.93% and 43.15%. MTT assay showed that the peptide who was packaged by chitosan coated had a slight decrease in anti-tumor activity when the quality was same. Take dosage into consideration, when peptide content was same its antitumor activity was similar. The vitro test certified: the release rate of The nanoparticles E1-CS/TPP was reduced in the gastric fluid when compare with the control test, but continued to release in the intestinal juice,cecum and colon for the purpose of delayed release. |
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