Preparation And Identification Of The Argopecten Irradias Hydrolysate And Its Anti-Tumor Effects In Vivo And In Vitro

Most of marine bioactive extracts exhibit antitumor activity. Scallop is one of the marine organisms, and it presents a very good application and development prospect in marine anticancer drugs research because it has rich nutritional value and biologically active component. In this paper, the argopecten irradias hydrolysate was extracted and purified, and its inhibitory effect and mechanism on hepatoma were also studied. The main contents and results are summarized as follows:1. The preparation condition of the argopecten irradias hydrolysate was put forward, and its high antioxidant activity in vitro was proved.With the non-visceral argopecten irradias as the material, the optimal hydrolytic conditions were determined as follows: the ratio of solid to liquid 1: 6.25, p H 3.53, enzyme quantity of the acid protease 1312.82 U/g, reaction temperature 50℃, hydrolysis time 4 h. Under the conditions, the DPPH free radical scavenging activity of the argopecten irradias hydrolysate was 91.59%. The further study showed that there was no significant difference between the hemolysis degree(93.59%) of the hydrolysate(10 mg/m L) and it(93.63%) of vitamin C(0.1 mg/m L); anti-linoleic acid peroxidation capacity of the hydrolysate(10 mg/m L) reached 96.26% which was higer than it(67.74%) of vitamin C(1.0 mg/m L) and much higher than it(40.65%) of vitamin C(0.1mg/m L). The results indicated that the hydrolysate had obvious antioxidation activity.2. The amino acid components and content of the argopecten irradias hydrolysate were determined, and the primary structure of the polypeptide from argopecten irradias in the hydrolysate was identified.The argopecten irradias hydrolysate contained 14 kinds of amino acids determined by reversed phase high performance liquid chromatography(RP-HPLC) with precolumn derivatization. And the polypeptide from argopecten irradias(PAI) from it contained 13 kinds of amino acids which were arginine(Arg), cysteine(Cys), histidine(His), threonine(Thr), glycine(Gly), proline(Pro), Valine(Val), aspartic acid(Asp), lysine(Lys), tyrosine(Tyr), methionine(Met), serine(Ser) and leucine(Leu) according to the content. The amino acid sequence of the PAI were detected by 4800 Plus MALDI TOF/TOF mass spectrometer which were as follows: PAI-1(m/z 706.3) Cys-Cys-Ser-His-Thr-Arg, PAI-2(m/z 718.3) Asn-Gly-Trp-Val-Thr-Arg, PAI-3(m/z 730.3) Gly-Asn-Pro-Met-Arg-Arg, PAI-4(m/z741.4) Asp-His-Trp-Lys-Arg, PAI-5(m/z908.5) Cys-Thr-Tyr-Gly-Pro-Val-Leu- Arg.3. The influnce of the argopecten irradias hydrolysate on the two human hepatoma cells cultured in vitro was studied, and its anti-tumor effects in vitro was proved.The CCK-8 assay showed that the argopecten irradias hydrolysate in the different concentrations could inhibit the growth of human hepatoma cells Hep G2 and SMMC-7721(P<0.01), and the inhibition rate increased with the increasing concentration of the hydrolysate. After treated with argopecten irradias hydrolysate of 12 mg/m L, the inhibition rates on the two hepatoma cells were respectively 60.4% and 64.2%, significantly higher than them of the other groups(P<0.01); compared with control group, the morphology of two hepatoma cells were obviously changed, characterized by cell shrinkage, transparency decrease and nuclear chromatin pyknosis, and the apoptotic bodies were found;and the flow cytometry analysis showed that early apoptosis rates and late apoptosis rates were significantly increased(P<0.01), and the positive rates of p53 were obviously enhanced(P<0.01), while the positive rates of Bcl-2 were decreased significantly(P<0.01). The results showed that the potential molecular mechanisms of its anti tumor effects were : ①it could up-regulate the expression of p53, arrest the cells, and induced apoptosis by cysteine aspartic acid protease way; ② it could down-regulate the expression of Bcl-2 to induce tumor cell apoptosis through a mitochondria-dependent pathway.4. The influnce of the argopecten irradias hydrolysate by intragastric administration on the H22 tumor-bearing mice was studied, and its anti-tumor effects in vivo was proved.After the argopecten irradias hydrolysate was given to H22 tumor-bearing mice by gavage at different dosage, the tumor inhibition rates on mice of low dose group(500 mg/kg·bw), medium dose group(1000 mg/kg·bw) and high dose group(1500 mg/kg·bw) were 28.16%, 41.93% and 84.00%, respectively, which presented a dose dependent relationship. Comparing to the model group, the serum levels of MDA and 8-iso-PGF2α in medium dose group were decreased significantly(P<0.05), while its serum levels of SOD, IL-2, IL-12, GSH-Px and TNF-α were increased significantly(P<0.05); the tumor tissue extracts levels of mutant p53 protein, survivin protein, MDA, 8-OHd G and 8-iso-PGF2α in medium dose group were decreased significantly(P<0.05), while the levels of SOD and GSH-Px were increased significantly(P<0.05). Meanwhile pathological observation also indicated that the hydrolysate exhibited obvious anti-hepatoma biological effects. On the bases of the above study, it indicated that the argopecten irradias hydrolysate had obvious inhibitory effects on mice with H22 hepatocarcinoma cells, especially the medium dose group had optimum effect. Its anti-tumor effect may be realized possiblely through the following ways: ① enhancing the immunity and the antioxidant capacity of the body; ② regulating cytokine levels and indirectly inducing tumor cell apoptosis; ③ decreasing the expression of tumor markers protein and inhibitor of apoptosis protein, and suppressing the genesis and development of tumour.