Study On The Collagen Activity Peptide Of Holothuria Nobilis Selenka

Sea cumcumbers belong to invertebrate echinodermata and are listed as one of the "eight treasures" fortheirs high nutritional and pharmaceutical value. The total number of presently existing sea cucumberspecies is more than1000around the world and140species are in China. About40sea cumcumber speciesare edible, of which10species are higher value of goods. The distribution of Holothuria nobilis Selenka isin the south China Paracel Islands and Hainan Island and Western Australia waters. Holothuria nobilisSelenka is thick body wall, a high quality and large edible sea cucumber.The mainly active ingredients of sea cucumber are concentrated in glycosaminoglycan, polysaccharide,saponins, lipids and trace elements et al. Sea cucumber collagen and its active peptide research is less. Afte-rwards Cui Feng Xia et al study the active collagen peptide of sea cucumber in some ways, but theresearch is focus on small molecules peptide which the relative molecular mass is less than1000. So far,the collagen polypeptides of sea cucumber have not been reported.Holothuria nobilis Selenka is the research object in the paper. The active collagen polypeptides arefrom treatment of holothuria nobilis which is hydrolyzed by a flavourzyme. Collagen protein polypeptidewas separated and purified by various separation and purification techniques, of which the amino acidcomposition, amino acidsequence and a variety of physiological activity in vivo and in vitro were studied.The main research results of this paper are as follows:â’ˆThesea cucumber collagen hydrolyze to obtain target collagen polypeptide HNCP-F-A（Mw:1-10kDa）.Three components: HNCP-F-Aa,HNCP-F-Ab,HNCP-F-Ac were obtained by combination with ultra-filtration, gel column chromatography and HPLC.The polypeptide mass spectrum and main relative mole-cular mass of three components:2806Da、7201Da、1532Da were measured by the MALDI-TOF-MS.â’‰Objective collagen polypeptide amino acid composition analysis results show that Sea cucumbercollagen polypeptide mainly contains glycine（Gly）, alanine（Ala）, glutamate（Glu）, proline（Pro） andhydroxyproline（Hyp）. The total content of delicious amino acids, pharmacodynamic amino acids, essentialamino acids accounts for65.1%,58.08%, and24.96%of the mole percentage on the total amino acids fromcollagen polypeptide. So the collagen polypeptide has high medicinal value, tastes delicious and iscomplete in amino acid composition.. â’ŠCombined with MALDI-TOF/TOF MS-MS, we gained the denovo sequence results aboutHNCP-F-Ac: IPGYIMSSSNHDGGAP; GSNHSDSYWLMPR; PDMGVNSKYVGHLR; TMLAMVRVH-LYLR; DGNLYMGHRMMCSGY; DWEVAYDVHECPMCK; GDPKDDLFWVVCCKCLK. The part ami-no acid sequence from strip1of HNCP-F-Ab: NH₂-Gly-Ala-Pro-Gly-Pro-Met-Gly-Met-Met-Gly.â’‹Scavenging effect of the diphenyl-generation bitterness hydrazino radical（DPPH）, hydroxylradical（0H）, superoxide anion radical（O·–2） show that collagen polypeptide can remove them. The IC₅₀value of them is5.78mg/ml,4.20mg/ml,0.27mg/ml, respectively.â’ŒTheresults of Holothuria nobilis Selenka collagen polypeptides on physiological activity of D-gelmodel mice are as follows:â‘ Body weight,immune organs and viscera index of mice: The differenceswere not statistically significant significance（P>0.05） about their body weight, immune organ, organ indexin different dose groups.â‘¡Anti-oxidation level of serum,the skin tissue and hydroxyproline content in theskin tissue:comparing with the model group,the25mg/kg and50mg/kg dose group, T-AOC（Totalantioxidative capacity） activity of serum was significantly increased（P<0.05）. The SOD（Superoxidedismutate） activity of serum in the25mg/kg dose group was extremely significant increased（P<0.01）.The GSH-Px（Glutathione-Peroxidase）, SOD（Superoxide dismutate）, T-AOC（Total antioxidativecapacity） activity of different doses in skin tissue about sea cucumber collagen polypeptide is dualfunction.Comparing with the model group, the50mg/kg dose group, GSH-Px, SOD, T-AOC activity in skintissue was significantly improved （P<0.05）.Especially the activity of GSH-Px and T-AOC has significantlyimproved（P<0.01）,while in the100mg/kg high-dose group, T-AOC activity was significantly decreased（P<0.05）; hydroxyproline content of the skin was not statistically significant（P>0.05）.⒍The vitro activated partial thromboplastin time（APTT）, plasma prothrombin time（PT）, thrombintime（TT） of the New Zealand rabbit left ventricular arterial blood experimental results about the differentconcentration Holothuria nobilis Selenka collagen polypeptides show that the vitro coagulation indicators（APTT, PT, TT）was dual regulation. The concentration is100.0g/ml,200.0g/ml,400.0g/ml, comparin-g with the control group, PT was significantly shortened （P<0.05）; The concentration is100.0g/ml,200.0g/ml comparing with the control group, TT was significantly shortened（P<0.05）. PT and TT in the high concentration group（800.0g/ml）, comparing the control group, were prolonged, while TT extendedsignificantly（P>0.05）; APTT was shortened comparing with control group when the concentration is50.0g/ml,100.0g/ml,200.0g/ml, and there are significant differences（P<0.05）. In low concentrationsof12.5g/ml,25.0g/ml, APTT was prolonged, and was significantly prolonged（P<0.05） when theconcentration is12.5g/ml.The chemical structure and biological activity about Holothuria nobilis Selenka collagen polypeptidewere studied and a variety of modern chromatographic separation technologies were uesd to prepare andpurify macromolecular collagen polypeptides. Edman degradation and mass spectrometry were used to det-Ermine the polypeptide amino acid sequence and a variety of biological activities were studied. The resea-rch has fairly high academic significance,which fills the domestic gap, provides research data to investigatethe relationship of the sea cucumber physiological activity and structure, provides a scientific basis tofurther research and develop edible （medical）effect of sea cucumber.