Preparation Of Bioactive Peptides From Tortoise (Chinemys Reevesii) Protein And Its Antitumor Effects In Vitro And In Vivo

Cancer is a kind of highly lethal disease worldwide that takes away millions of people’s lives every year. However, there’s not yet effective medical method or intervention to cure it so far. It’s still a major topic to seek for novel, efficient, and harmfulless anticancer drugs in the 21st century. As is well known to us all, bioactive peptide, which is composed of amino acids through peptide bond, is a category of compound with many functions such as antitumor, immunity enhancement, antivirus, and antihypertension. And the abundant structure types make it huge potential in medical application. Ever since 1995, about one third drugs newly come into the market were related to protein or peptide. Up to now, near one hundred polypeptide drugs are approved registration, and there are also over one hundred in clinical trial and hundreds in preclinical development. The worldwide rapid development of peptide drugs is refered to their much unique advertanges:They almost have no side effects unlike the micromolecular chemical drugs with various metabolic toxicity; they have no cumulative toxicity; they have remarkable pharmacodynamics and good molecular cognition; they have explicit structure-activity relationship which contribute a lot to their goal-oriented R&D; They are so small molecules that are easy to transform and synthesize. In a word, all of the above make peptide great capacity to be explored.In this dissertation, we mainly focus on the preparation of antitumor peptide with precious traditional Chinese medicine tortoise protein as raw material by enzymolysis technology. Firstly, for the preparation of peptide, we made a comparision between fermentation by bacillus subtilis and enzymolysis with protease of protamex, trypsin, and papain. The experiment parameters include pH value, temperature, time, amount of protease, and amount of culture. On account of few indexes, enzymolysis was finally choosed for its larger scale, easier operation, and especially their higher activity products. Papain was eventually adopted in our experimental scheme after optimization. Orthogonal experiment confirmed the best hydrolysis plan in which the parameters are:t=8h, pH=7.5, T=60℃, protease-substrate ratio0.5%, liquid-substrate ratio 3, respectively. MTT method was used to investigate the activity of peptides with human breast cancer cell MCF-7 as experiment subject.Ultrafiltration (UF) together with column chromatograph were used in the the separation and purification of peptides mixture. Molecule weight cut-off (MWCO) of UF membrane is 10 KDa. According to the molecule difference and polarity variety, sephadex G-75, sephadex G-50, and sephadex LH-60 were choosed as chromatograph filler. A peptide constituent (named TP-1) with antitumor activity was finally acquired which showed concentration-denpendent and time-dependent toxicity. RP-HPLC indicated that the single principal component in TP-1 is of high purity. IC50 is 2.5 mg/mL according to the activity test. In addition, TP-1 has little growth inhibition to normal fibroblast cell L929. Subsequently, FT-IR, UV, and CD spectrum manifested the existence of secondary structure of β-turn and random coil in TP-1, which may well be significant for the bioactivity of the new peptide. In the end of this part, the molecular weight of the principal component in TP-1 is determined as 8 KDa by LC-MS/MS and its amino acid sequence is QYSTKEDKYEEEIKLLTDKLKEAETRAEFAERSVAKLEKTIDDLEENLASA KEENVGIHQVLDQTLLELNNL with the accession number K7FRA9 in NCBI, accordingly. In the last part of this study, we explored the likely mechanism of antitumor action of TP-1 both in vitro and in vivo. Cell wound scratch assay and DAPI/PI fluorescent staning results demonstrated the ability of inhibition of cell migration. Flowcytometry test proved that cell growth inhibition happened in both time-and dose-dependent ways as the cell cycle was blocked at phase G2. Animal homograft tumor model was built to verify the antic ancer activity of TP-1 in vivo. An exciting pharmacological effect was visually depicted after abdominal cavity injection of TP-1 with 100 mg/kg-d for 10 consecutive days, in which a suppression rate of 34% tumor weight was observed.