The Preparation Of Collagen Peptides From Squid Skin And Its Effects On Rheumatoid Arthritis

Malignant tumor is a serious threat to peoples’ health and life with highmorbidity and high mortality.The present treatments for cancer have been found,suchas Antineoplastic chemotherapy, radiotherapy and sergery.Though chemotherapeuticshave some certain curative effects for cancer,it is hard to achieve the desiredtherapeutic effect caused by weak selectivity, big side effect and drugtolerance.Searching for new antineoplastic drugs of which become efficient, lowtoxicity and good specificity is a very promising development route.Because of small molecular weight, no immunogenicity, high activity and smallside effects, especially for good inhibitory activity on tumor cell lines which havebecome multidrug resistance ones,studies on peptides has been a hot area which isconcerned by domestic and foreign researchers. In the text, research of thepreparation, separation and purification and antitumor properties of enzymatichydrolysate from Oratosquilla Oratoria meat by using of enzymatis hydrolysis,ultrafiltration,sephadex G-25gel chromatography and RP-HPLC comprehensivly.Thepresent study provides theoretical basis for the application of antitumor peptideswhich obtained through enzymolysis from Oratosquilla Oratoria meat.1. enzymolysis technology of Oratosquilla Oratoria: The polypeptide was extractedfrom Oratosquilla Oratoria by a method of enzymatic hydrolysis techniques.Objective to estabish a perfect condition for enzymatic hydrolysis,a series ofsingle-factor experiments,such as time,temperature, pH,enzyme dosages, were madeto investigate the effects of the extraction ratio for optimal levels,using responsesurface analysis to optimize the main process parameters and getting a regressionequation: Y=－43.73356+7.45713X₁+0.75917X₂+0.33395X₃+1.9687X₄+0.16X₁X₂－0.0076X₁X₃+0.03X₁X₄－0.0225X₂X₃+0.4625X₂X₄－0.0051X₃X₄－0.43707X₁²－1.46604X₂²－0.00207567X₃²－0.29907X₄². The optimum conditions for extractionwere found from the equation: temperature55℃,pH8.36,time3.95h,enzyme dosage0.92%,under these conditions, The extraction ratio of polypeptide was0.896mg·g^-1,the predictive value was0.88mg·g^-1and the degree of hydrolysis was26.52%. 2.the separation and purification of peptides: The hydrolysate was isolated andpurified by the method of ultrafiltration and Sephadex G-25gel and RP-HPLC.Thenthe antitumor activities of every fractions obtained were detected by MTT method. Byusing ultrafiltration,three fractions were obtained from the hydrolysate,which wereO1,O2,and O3. The fraction O3got the highest activity during three fractions thatwere purified, and the PC-3inhibition rate was60.03%at the concentration of15mg·mL^-1.O3was further fractionated into three portions after using Sephadex G-25column,which were OS1,OS2and OS3. The fraction OS2got the highest activityduring three fractions,and the IC50of OS2on PC-3、DU^-145and A549cells are6.702mg·mL^-1,5.665mg·mL^-1,6.723mg·mL^-1.MTT assay showed great concentration-dependent in proliferation of tumor cells.Then,three components were got throughRP-HPLC after the OS2was collected,which were HOS1,HOS2and HOS3,andduring which, HOS2showed the highest antitumor activity was collected and purifiedwith RP-HPLC,a peptide （FOHS） with high purity was finally obtained.The aminoacid sequence of FOHS was identified as Phe Tyr Met Asn His.3. Primary research on the physico-chemical and functinonal properties of peptides:（1） Antioxidant Activity of FHOS: After determined,the scavenging rate ofsuperoxide anion radical is31.78%and we can also find the reducing power of A700is0.259.the IC₅₀of DPPH scavenging activity rate and The scavenging rate ofhydroxyl radical are5.504mg mL^-1�'�3.268mg mL^-1respectivly.Which comparedwith the antioxidant activity of Vc acted at0.1mg·mL^-1,the scavenging of hydroxylradical and DPPH scavenging activity are both showed powerful.（2） Antitumor Activity of FHOS: the antitumor activities of FHOS with acted onPC-3and A549cells were detected by MTT method.and then the IR of two cellsrespectively calculated after72h were70.1%and90.0%.（3） The morphocytology observation of tumor cells inhibited by FHOS:HEstaining showed that two kind of tumor cells are both found typical apoptosischaracteristics,such as nucleus pycnosis, condensed chromatin and apoptoticbody,etal.