Enzymatic Preparation And Antitumor Activity Of Polypeptides From Ruditapes Philippinarum

Ruditapes philippinarum(commonly known as variegated clams,clams)is highly productive and rich in antitumor,antioxidation,antihypertensive and enhance immu-nity and other bioactive substances in china.It can be seen that the ruditapes philippinarumhas a broad development and application prospects.In this study,the protein of ruditapes philippinarum was prepared by enzymatic method,ultrafiltration,ion-exchange chromatography andgel chromatography.And the antitumor activity of high purity polypeptide was investigated,to provide a theoretical basis for the develop-pment of functional food of ruditapes philippinarum.The main results of the paper are as follows:(1)Extraction of antitumor polypeptides from ruditapes philippinarum: Enzym-olysis conditions were optimized using single factor and orthogonal experiment,and theamino nitrogen(ANN)content and tumor cell A549 inhibition ratewere determined.The optimum enzymatic extraction conditions were confirmed as: enzyme in solid matter,2000 U/g,solid-liquid ratio(wet sample),1:1(w/w),pH value,2.0,enzymolysis temperature,37?,andreaction time,1.0 h.Under this condition,the content of ANN in the solution of ruditapes philippinarum was 0.1470 g/100 mL.The A549 inhibition rate of enzymatic hydrolysate of crude productsobtained from philippines clam was 99.83% when the concentration was 20 mg/mL.(2)Isolation and purification of polypeptides from philippines clam: The crude extracts of ruditapes philippinarum were prepared with the best enzymatic extraction conditions.Three peptide components with different molecular weights(MWCO I>10 KDa,MWCO?5-10 KDa,and MWCO?<5 KDa)which were separated by ultrafiltration from crude extracts were investigated.The MWCO ? show highestanti-tumor activity,IC50 was 6.88 mg/mL.MWCO? was further isolated by SP Sephadex C-25 ion exchange column.The MWCO ?-? indicated highest antitumor activity,IC50 was 6.65 mg/mL.MWCOIII-IV was further purified by Sephadex G-25 gel filtration column,three components were isolated,and the antitumor activity of MWCOIII-IV-A was the highest,IC50 was 4.82 mg/mL.Amino acid composition analysis showed that,MWCO?-?-A contained 15 kinds of amino acids,among them,glutamic acid,leucine and aspartic acid and other acidic amino acid content was higher and accounting for 25.43%,9.16% and 8.51% of the total amino acids respectively.(3)The antitumor activity of peptide MWCO? from philippines clam: The high activity MWCO ? obtained by ultrafiltration was evaluated in vivo antitumor efficacy.The results showed thathigh dose of MWCOIII had significant inhibitory effect on the growth of A549 tumor bearing mice,and the inhibition rate was 49.01%.Compared with the model group of A549 tumor bearing mice,the growth and development of A549 tumor bearing mice were significantly improved.Low,medium and high doses of MWCOIII could promote the growth of spleen and thymus,and the growth promoting effect of spleen and thymus had a positive correlation with the dosage of MWCOIII.MWCO?could inhibit the expression of VEGF,CD31 and BCL-2 protein and promote the expression of BAX protein in A549 tumor bearing mice.MWCO ? could promote the secretion of IL-2 and IFN-? in A549 tumor-bearing mice.In conclusion,MWCO? could inhibit the tumor of A549 bearing mice body growth,and enhance the immune function of A549 micein vivo.In this paper,the extraction,isolation and purification,in vitroand in vivo antitumor activity of antitumor peptide from ruditapes philippinarum were studied,which laid a foundation for the development of functional food of ruditapes philippinarum and provided scientific basis for the further development and utilization of ruditapes philippinarum the of the philippines clam polypeptide.