Extraction Of Composite Amino Acids Of Silkworm Chrysalis Protein And Its Antihepatocarcinoma Effect In Vitro

Objective To study the optimal condition of extracting silkworm chrysalis protein (SCP) and composite amino acids of silkworm chrysalis protein(CAASCP), and study the inhibition effect of CAASCP on the human hepatic cancer cells in vitro.Methods Useing petroleum ether to defat China oak silkworm chrysalis and SCP was extracted from China oak silkworm chrysalis by technology of alkaline solution diffusion. Then, monofactorial test was applied to optimize the extraction conditions including ratio of substrate and water,time and temperature. Then,we can got scentless yellow protein by decolorization and deodorization with H2O2. After that, adopt Kjeldah method to determine contents of SCP. The CAASCP is prepared from SCP by hydrolysis with pepsin frist and after that acid hydrolysis. The paper discusses the relationgs between the substrate concentration,pH,the concentration of enzyme,hydrolysis temperature and hydrolysis time and degree of hydrolysis by single factor test.And discusses the optimal condition of enzyme hydrolysis by orthogonal test L16(45). We can got clear yellow CAASCP by vacuum distillation,neutralization,decolorization and so on. SMMC-7721 cell strain was used to study inhibition effect of CAASCP on the human hepatic cancer cells in vitro. The safe concentration of drug was calculated after detecting the toxicity of the drug to QSG-7701 by MTT means. Then, different dose(10"3 mg/ml,10-2mg/ml,10-1 mg/ml,1 mg/ml and 10 mg/ml) of CAASCP were added into each cell respectively, and blank control,cell control without drug was set at the same time.And each group was set up 5 holes again.Each hole was add 100 ul cell suspension which cell density is 4×104/ml,which to raise 24,48 and 72h discem.We evaluate multiplication restrain effect of CAASCP to hepatic cancer cell by MTT means to determin OD; detect apoptosis ratio by using Hoechst33258(10 mg/L)dyeing method and inverted microscope to observe morph change;using Flow cytometry to determine cell cycle and Annexin V/PI staining method to determine apoptosis.Results The condition of defat silkworm chrysalis by petroleum ether is that:ratio of substrate and water,1:4;temperature,50℃;time,4h.Repeat 4 times,ratio of defat silkworm chrysalis can achieve upwards 96%. The content of protein is 83.5%,the ratio reclaim of protein is 32.59%.The optimal enzyme hydrolysis condition is determined as follow:the substrate concentration,1:5;pH,4.0;the concentration of enzyme,1.5%; hydrolysis temperature,60℃;hydrolysis time,5h.After enzyme hydrolysis,we use hydrochloric acid hydrolysis the solution,and the ratio of hydrolyse can reach about 70%.In liquor,total amino acids is 2.241 g/L,necessary amino acids content is 0.835 g/L.Necessary amino acids occupy 37.26% of total amino acids.Cytotoxicity experiment indicate that:the maximum non-toxic concentration of CAASCP was 10mg/ml.We use defferent concentration of CAASCP to affect SMMC-7721 cell strain,after 24,48 and 72h,the cell growth inhibiting ratio of each group is 9.0%-53.5%,12.4%～63.4% and 16.8%-70.4%.Compare to cell control,difference OD between each group has significance(P<0.05).And display that it dependence on dose and time. The Half inhibitory concentration (IC50) of CAASCP to SMMC-7721 cell strain was 4.691 mg/ml,1.469 mg/ml and 0.451 mg/ml after 24,48and 72h.The result of Hoechst33258(10 mg/L) dyeing method and Flow cytometry indicated that CAASCP can obviously promote apoptosis of SMMC-7721 cell strain.Conclusions The main protein of silkworm chrysalis protein is globulin which dissolve in water and acid partly and maximum dissolubility in alkaline solution.So we can raise yield of protein utility by using alkaline solution to extraction silkworm chrysalis protein. Hydrolysis protein by enzyme and acid has many graces such as gentle hydrolysis condition,less enzyme dosis,destroy less amino acid,hydrolysis thorough and so on. The composite obtained by the full range of amino acids,high content of essential amino acids.We can discern that CAASCP has highly inhibition effect on the SMMC-7721 cell strain in vitro. CAASCP possesses a fine applying prospect for its low toxicity.