Sepration Of Anticancer Pepetide From Spirulina And Preparation Of Chitosan Nanoparticles

Spirulina is an excellent natural food. It’s rich of protein, balanced nutrients and varietyof biological active substasces. It’s recommended as Global Ideal Food by FAO. Recentyears, many researchers have found that spirulina has a variety of functions, just asanti-fatigue、anti-radiation、anti-virus、anti-tumor and strengthening the immune system.Spirulina has become a hotspot in research.The study used spirulina powder as raw material, extracted spirulina protein by usingultrasonication and repeated freezing and thawing. Then hydrolyzated the protein withdifferent enzymes to gain different molecular weight hydrolyzate by ultrafitration.MTT assay was used to measure the inhibition of tumor cells in vitro. Separated andpurificated the hydrolyzates with Sephadex G-50. Again MTT assay was used to filter a betteranti-tumor peptide. It was interacted with the chitosan to form a complex of chitosannanoparticles and did a series of characterization. The results are as follows：（1） Using the degree of hydrolysis as an indicator, Studied the hydrolysis conditions oftrypsin, alcalase, papain and pepsin by L（934）orthogonal experiment. The optimal conditionsof trypsin were PH=8, Temperature is42℃, the ratio of enzyme and substace （E/S） is3%,and the degree of hydrolysis was38.49%. The optimal conditions of alcalase were pH=8.5,Temperature is50℃, E/S was5%and the degree of hydrolysis was31.16%. The optimalconditions of pepsin were pH=2, Temperature is37℃, E/S was6%and the degree ofhydrolysis was7.07%. The optimal conditions of papain were pH=6.5, Temperature is55℃,E/S was4%and the degree of hydrolysis was27.08%.（2） MTT assay was used to study the inhibition of different molecular weight enzymatichydrolysis on MCF-7and HepG-2cells in vitro. When the concentration of the drug was1mg/mL, the inhibiton trend of the hydrolysis was better than spirulina protein. The trypsinhydrolysis had an obvious inhibition on the two cells. The inhibition rates of5-10KDhydrolysis on two cells in vitro was45.19%and23.5%.（3） The detection wavelength, eluent and the volume of the sample was studied throughSephadex G-50. Finally the elution condition was determined. The speed was0.5mL/mL, theeluent was ultra-pure water，the volum was2mL and the detection wavelength was215nm.Three peaks were collected. The concentration of the peptide was57%,41%and50%respectively. （4） MTT assay was used to study the inhibition of different compoents purified fromhydrolysis on MCF-7and HepG-2cells in vitro. When the concentration of the drug was500μg/mL, The inhibition rates of the three compoents on MCF-7cell in vitro was86%、98%and6%. The inhibition rates of the three compoents on HepG-2cell in vitro was76%、95%and28%. The Y₂had the strongest inhibition. The IC50of MCF-7and HepG-2cell was61.36and60.96μg/mL.（5） The chitosan nanoparticles （CS NPs） was prepared by Ion-crosslinked method. Theresults showed that when the concentration of TPP was from0.5mg/mL to1mg/mL, the CSNPs can form and have an appropriate size. Then prepared Y₂-CS NPs under this situation.Wecan see that the size of the complex increased with the concentration of TPP. When theconcentration was0.5mg/mL, the system was most stable. The Zeta potential was41.5mv,the average size was152.7nm. Meanwhile we characterized the complex with many methodsto verify it was successfully prepared.（6） The effect of different concentration of Y₂on entrapment efficiency and drug loadingof Y₂-CS NPs were studied. The drug loading decresed with the increased concentration of Y₂.When the concentration of Y₂was0.2-1.0mg/mL, the drug loading was42%-19%. But theentrapment efficiency increased slowly, from5%-15%.（7） MTT assay was used to study the CS NPs and Y₂-CS NPs on MCF-7and HepG-2cells in vitro. When the concentration of the CS NPs was50-100μg/mL, the inhibition rateson MCF-7cell in vitro was16.95%. The inhibition rates of Y₂-CS NPs on MCF-7cell in vitrowere26.05%. CS NPs and Y₂-CS NPs had no inhibition on HepG-2cell in vitro.