Study On The Anticancer Activity Of Oligopeptide From The Ruditapes Philippinarum Hydrolysate

Ruditapes philippinarums tast delicious, be full of nutrinents, high-protein, possesshigh protein, the types of amino acid and ration are reasonable, it has lots of medicinevalue, such as enhance immunity, antitumor, anti-atherosclerosis, antibacterial andanti-senility and so on. Traditional Chinese medical science thinks that clam have effects ofziyinmingmu, strengthening and nourishing, diuresis and relieving stranguria, softeningand resolving hard masses, resolving phlegm and so on. Prostate cancer is a disease ofcomplicated and a major cause of high morbidity and mortality, interacting environmentalwith genetic factors and sex hormones, the effect of traditional treatment is not good,toxicity and the recurrence rate is very high, which seriously influence global male’s healthand perplex medical personnel and patients. So whether we can find an alternate ofefficient, nontoxic and novel antitumor drug is especially important.Ruditapes philippinarums are used in the research, studies on preparation of antitumoroligopeptide by use of enzymatic hydrolysis of Ruditapes Philippinarums protein,ultrafiltration, Sephadex G-25gel chromatography and reversed-phase HPLC methodwere carried out, to investigate the antitumor effect of Ruditapes Philippinarumoligopeptides,PC-3and DU-145cell lines were employed as an in vitro model in this study,We determined the effect of Ruditapes Philippinarum oligopeptides on cell proliferation inPC-3and DU-145cells and examined their underlying mechanism with a combination ofcell and molecular biological methods,preliminary discussed process of solid beveragewhich include the Ruditapes Philippinarum oligopeptides and fruit flavour,it can providecertain basis for the development of antitumor drugs.Research contentsTrypsin、alcalase、papaw and dispase was used to hydrolysis,screening the bestenzyme by determining content of free ANN in the enzymatic hydrolysate and the value offishy smell and optimizing hydrolysis conditions. and below3KDa molecular weightfractions was obtained. The Ruditapes Philippinarum hydrolysates was isolated by ultrafiltration,purified on Sephadex G-25gel chromatography and demonstrated the purityof oligopeptides by reversed-phase HPLC and its peptide sequence analysis was detected.The hormone-independent PC-3, DU-145prostate cancer cells were treated withRuditapes Philippinarum oligopeptides at three different concentrations.Concentration-dependent responses of the effect of Ruditapes Philippinarum oligopeptideson cell proliferation was examined using MTT assay, AO/EB staining and FCM wereemployed for observation of apoptosis on PC-3、DU-145after treating with RuditapesPhilippinarum oligopeptides.Research on the eliminating Ruditapes Philippinarum hydrolysates smell, theRuditapes Philippinarum hydrolysates was treated with red tea、light salt brine、saltsolution、β-cyclodextrin、activated carbon、aether yeast、aether yeast combined withactivated carbon,fishy smell and loss ratio of ANN were investigated mainly in theresearch,screening the conditions of deodorization and optimized it.The solid beverageprocess is combining fresh fruit juice with deodorization of hydrolysates,then carrying onthe blending of sour agent, sweetener, stabilizer and so on,concentrating、drying andautoclaving,the physicochemical index was determinated at last.Research result1. Trypsin was the best hydrolysis enzyme, enzymolysis conditions were pH8.0,temperature50℃，enzyme concentration1200u/g and8h, the amino acid sequence wasidentified as Asp-Trp-Pro-His, and its molecular weight was607.6Da.2. MTT assay showed Ruditapes Philippinarum oligopeptides that inhibitedproliferation of PC-3and DU-145cells in both concentration-dependent andtime-dependent manners (P＜0.05).3. Treatment with Ruditapes Philippinarum oligopeptides for24h significantlyinhibited the growth of PC-3、DU-145cells. Cells form irregular, died cells were found andfloated in the culture media.4. AO/EB double staining disclosed that there were more viable apoptotic cells inRuditapes Philippinarum oligopeptides treated groups than control group. Early apoptosis cells were found in10mg/mL Ruditapes Philippinarum oligopeptides treated groups,lateapoptosis cells and dead cells appeared in20mg/mL and30mg/mL RuditapesPhilippinarum oligopeptides treated groups, Thus, certain concentration of RuditapesPhilippinarum oligopeptides resulted in a decrease in proliferation and an increase inapoptosis in PC-3、DU-145cells.5. FCM showed that after being exposed to10,20,30mg/mL RuditapesPhilippinarum oligopeptides for24h, the early stage of apoptosis of PC-3, DU-145cellswas observed and apoptosis rate significantly higher than those in the control group;PIstaning by FCM, SubG1appeared before G0/G1, cells rearly changed during G0/G1,decreased during S and increased during G2/M.6. The best deodorization was Angela highly active dry yeast joint active carbon, theconditions were firstly fermentation reaction1h at temperature30℃Angela highly activedry yeast, then centrifugal filtration and added active carbon, the conditions were pH4.0,temperature30℃,volume of addition1.0%and30min, the contents of ionization ANNwas2.878mg/g and loss ratio was7.9%, basically had no fishy smell and had yeastpeculiar odour, clearly.The best fruit juice was orangeade,observed orange and bright,smelt fresh orangeade flavor, tasted sweet and sour,no astringent. The solid beveragesolubility was good, sanitary index were up to the standard.