Study On The Biochemical Characterization Of Collagen And Activity Of Collagen Peptide Extracted From The Body Wall Of Sea Cucumber Stichopus. Japonicus

Sea cucumbers belonging to the phylum Echinodermata of invertebrate are traditional tonic consumed by Chinese and Japanese because of it's high nutritional and medical value. There are more than 900 kind of sea cucumbers in the world and more than 140 in China. However, only 40 kind of sea cucumbers found to be edible in which S. japonicas is the best of edible sea cucumbers distributed in the seaside of North China.The major edible parts of sea cucumber are the body wall mainly consisting of collagen, mucopolysaccharides and glycosides. During the past 30 years, sea cucumbers were used to cure tumor or diabetes successfully by herbalist doctor. Furthermore, sea cucumbers also were found to have activity of antitumor, antifungi and anticoagulant by domestic and foreign research. There is little information about the activity of sea cucumber collagen peptide contrasted to a lot of research on the activity of mucopolysaccharides, glycosides, lipids and trace element.Contrasted to vertebrate, collagen of sea cucumber had more difficulty to dissolve. In this paper, pepsin-solubilized collagen (PSC) without telopeptides was prepared from the body wall of sea cucumber S. japonicus and the biochemical characterization and the molecular structure of PSC were studied. Collagen peptides without mucopolysaccharides and glycosides were extracted from the body wall of S. japonicus by a kind of commercial enzyme. Antioxidation in vitro and in vivo and regulation of tyrosinase mRNA in B16F10 murine melanoma cells by collagen peptide were explored. Furthermore, the relationship of chemical properties and antioxidant activities of collagen peptide further suggests the antioxidant activity mechanism of peptides. The paper was aimed to broaden the research field of collagen, produce high-valued functional food and medicine from sea cucumber and provide a valuable scientific basis for the industrialization development of sea cucumber. The main results are as follows:1. In this paper, the nutritive composition of sea cucumber body wall was determined. The composition features of amino acid showed that contents of total amino acids, total essential amino acids, total delicious amino acids and total drug-effective amino acids in dry samples of body wall were 69.9%, 19.0%, 35.2% and 37.6%, respectively. Contents of moisture in fresh body wall was about 91.44%. Contents of crude protein, mucopolysaccharides, fat, glycosides and ash in dry samples of body wall were 70%, 13%, 1%, 0.04% and 4.2%, respectively. Ts of crude protein in body wall of raw S. japonicus was 67.58℃determined by DSC. Using Van Gieson staining technology to observe the structure of the different part of raw sea cucumber. The results showed that collagen fibrils and myofibrils were distributed in different part of S. japonicus body wall among which the most parts were collagen fibrils.2. The intact collagens were isolated from the body wall of Stichopus japonicus by a new method and their biochemical characterization were studied. The results showed that pepsin-solubilized collagens(PSC) without telopeptide could be prepared at low temperature. Through the ultraviolet scanning spectra from 190 to 400 nm, the PSC presented maximum absorption at 220 nm which was consistent with that of typeⅠcollagen. The compositional features of the amino acid were with the high contents of glycine(329 residues/1000) and hydroxyproline(66 residues/1000), but with low contents of tyrosine, phenylalanine and histidine. No cysteine was detected. The ratio of hydroxyproline to proline was 0.69. It was concluded that PSC was typicalⅠcollagen. Combined with the results of SDS-PAGE and Sephacryl S-300 HR, PSC was (α1)3 trimers whileαchain(about 135 kDa) resembledα1 chain of type-Ⅰcollagen of vertebrate. The content of total polysaccharide determined with phenol-sulfuric acid method was 0.61%and the glycosaminoglycan measured with cationic dye of methylene blue was 0.48%. The collagen could be separated from glycosaminoglycan by DEAE-52. The thermal denaturation temperature was 57℃as measured by DSC, about 5℃lower than that of type-Ⅰcollagen of calf.3. The molecular structure of PSC was studied in this chapter. The peptide maps of PSC digested by V8 protease, in comparison with calf skin typeⅠcollagen, was performed on 12% gel. The results suggested that calf skin typeⅠcollagen was more tolerant to digestion by V8 protease than PSC. V8 protease exhibited a high degree of specificity for glutamic acid and aspartic acid residues and proteins. Thus, glutamic acid and aspartic acid residues of skin collagen of calf might be lower than that of PSC. Judging from the electrophoretic patterns of 2D-PAGE, the pI of PSC consisting ofα1 chain was about 4.5~6.0. Spots ofα1 chain were excised and identified using peptide mass fingerprint and marix-assisted laser desorption ionization time-of-flight and time-of-flight mass spectrometry(MALDI-TOF- TOF). The results showed that peptides AQVAANTK, YPGCSGSR, IIIDDIR, ELTDVINK, GHVVSQHR, YTGGNGVVR, GSGWNGSDAVHTNMTNTR, GTGGNSGNK were found to exist inαchain of PSC .4. Collagen peptides were extracted from body wall of S. japonicus, H. mexicana and P. graeffei with flavor enzyme after the sea cucumber materials were soaked in ethanol for 1 week to eliminate glycosides. Collagen peptides were purified from mucopolysaccharides by ethanol precipication. The ratio of ethanol to hydrolyte was 3:1 (V:V). The molecular weight distribution of the three kind of collagen peptides was separated into three major peptide fragments with average molecular weights of 1700, 750 and 75 Da, respectively. The compositional features of the amino acid showed that contents of total amino acids, total essential amino acids, total delicious amino acids and total drug-effective amino acids in dry samples of collagen peptides of S. japonicus, H. mexicana and P. graeffei were 99.21%, 98.72%, 50.27%; 25.01%, 15.41%, 12.57%; 53.82%, 56.1%, 26.32%; 54.35%, 51.65%, 26.12% respectively. However, collagen had high nutritional and medical value.5. The scavenging effects of collagen peptides on superoxide, hydroxyl radical were studied by the method of chemiluminescence system. The results showed that all of the three kind of collagen peptides had excellent scavenging effects on superoxide and hydroxyl radical. Furthermore, the scavenging effects increased with the increasing of peptides content. Scavenging effects of collagen peptides from S. japonicus on superoxide was the best with IC50 values of 2.18 mg/mL while that of collagen peptides from H. mexicana on hydroxyl radical was the best with IC50 values of 0.20 mg/mL among the three kind of sea cucumbers.6. Effect of collagen peptides from three kind of sea cucumbers on vascular endothelial cell (ECV-304) oxidative stress induced by oxidized low density lipoprotein (ox-LDL) suggested that collagen peptides could protect ECV-304 from oxidative injury induced by ox-LDL, decrease the content of MDA and increase the content of NO and activity of NOS significantly in concentration dependent fashion(P<0.05, P<0.01). However, S. japonicus had the best effects among the three kind of sea cucumbers.7. Antioxidation activity of collagen peptides in different concentration extracted from S. japonicus against chronic ultraviolet radiation-induced skin photoaging in hairless mice were studied in this chapter. The results showed that the content of hydroxyproline(in skin) and activity of SOD and GSH-PX increased while the content of MDA decreased (p<0.01) in serum and skin of skin photoaging-induced hairless mice given different doses of collagen peptide from S. japonicus. The effect of collagen peptide extracted from S. japonicus on the immune system capacity was also studied in this chapter. The results showed that collagen peptide could increase the indexes of thymus, the indexes of spleen and the transformation rate of lymphocytes and improve phagocytic activity of peritoneal macrophage(P<0.05,P<0.01).8. This chapter was mainly about regulation of tyrosinase activity and content of tyrosinase mRNA in B16F10 murine melanoma cells by collagen peptide extracted from S. japonicus. The results showed that collagen peptide could remarkly improve the proliferation of B16F10 and had no cytotoxic activity(P<0.05,P<0.01). However, collagen peptide exerted obvious inhibition on the activity of tyrosinase in concentration dependent fashion. The inhibition percentage on the activity of tyrosinase was about 21.33% after treated with 100μg/mL collagen peptide for 48 h(p<0.01). Furthermore, collagen peptide exhibited an inhibitory effect on the expression of tyrosinase mRNA. The expression content of tyrosinase mRNA decreased 36.79% and 69.69% (p<0.01) after treated with 25μg/mL and 100μg/mL collagen peptide for 48 h, respectively.