Effect Of Collagen Hydrolyastes And Zinc-chelating Complex From Scallop Mantle On Osteoporosis

The marine bivalve mollusk Pinctada martensii, is an important aquaculture SHecies used to cultivate sea water pearls worldwide. The mantle and other processing wastes of P.martensii with the increasing aquaculture of sea water pearls are merely discarded. Tilapia,a warm water fish SHecies, is the most widely cultured fish globally that are known as one of the major sources of animal protein in the future. The scallop mantle, fish scale, fish bone and other processing wastes of scallop and tilapia are merely discarded during the processing. Therefore,the efficient use of scallop mantle by-products as a sustainable source of collagens for various purposes, including value-added foods, is an interesting topic. In this study, scallop mantle and tilapia fish scale were selected as raw material to extracted collagen. This thesis explored the effect of collagen hydrolysates and zinc-chelating complex from scallop mantle on bone formation, tilapia fish scale were used as control. The main research contents and results were as follows:1 The preparation and characterized properties of collagen hydrolysates from scallop mantle and tilapia fish scaleThe contents of crude protein in scallop mantle and tilapia fish scale were 59.79% and 63.92% (dry weight basis) by Kjeldahl method, reSHectively. The extraction rate of collagen was 7.063% and 8.507%, reSHectively. The different properties of scallop mantle and tilapia fish scale collagen were determined the SDS-PAGE, UV SHectroscopy and amino acid composition analysis. Through contrasting research, the results showed that scallop mantle collagen is type V-like collagen, tilapia fish scale collagen is type I collagen.Then the collagen hydrolysates of scallop mantle (SH) and tilapia fish scale (TH) were prepared by trypsin. HPLC analysis showed that the molecular weight of SH was mainly distributed between 500 Da and 10 KDa, while the molecular weight of TH was mainly between 500 Da and 3 KDa.2 The effect of collagen hydrolysate from scallop mantle on osteoblastic proliferation and differentiationThis study explored the effect of SH on the proliferation and differentiation of the osteoblastic cell line MC3T3-E1, TH as a control. The proliferation of MC3T3-E1 cells was determined by Methyl thiazolyl tetrazolium (MTT) assay, the activity of alkaline phoSHhatase (ALP) was assessed by p-nitrophenyl phoSHhate (p-NPP), the secretion of collagen type (COL I ),and osteocalcin (OCN) were detected by enzyme-linked immunosorbent assay (ELISA) and the mineralized nodule of MC3T3-E1 cells was evaluated by staining with Alizarin red S dye. In comparison to the control, results showed that both of SH and TH could not stimulated osteoblastic proliferation. However,SH and TH upregulated osteoblastic ALP activity, Type ? collagen (COL ?), osteocalcin (OCN)levels as well as promoted bone formation. And TH extered a better effect on osteoblast differentiation as compared to SH. 800 ?g/mL SH and TH significantly increased alkaline phoSHhatase (ALP) activity by 151.74% and 194.67%, the bone nodules formation was increased 156.63% and 197.95% in osteoblastic cells, compared with the control.3 The effect of collagen hydrolysate from scallop mantle on osteoclastic differentiationTo investigate the effect of SH on the activity of osteoclasts, the murine osteoclast model was differentiation by RAW264.7 cells by receptor activator for nuclear factor-?B ligand (RANKL) in vitro, TH as control. The viability of RAW264.7 was determined by MTT assay. The effects of SH on the differentiation from RAW264.7 to osteoclast were carried out by tartrate resistant acid phoSHhatase (TRAP) staining, cell apotosis were detected by flow cytometry. MTT results showed that both of SH and TH had no cytotoxicity on the cells at any concentrations. Dose of 400, 800 ?g/mL SH significantly suppressed the differentiation of RAW264.7 cells, and TH had a beret effect. Treatment with 400, 800 ?g/mL SH and TH induced apoptosis in osteoclast-like cells. The results suggest that SH suppresses osteoclastogenesis via the inhibition of osteoclast differentiation and the induction of apoptosis in osteoclasts.4 The effect of zinc-chelating complex on osteoblastic proliferation and differentiationSH and TH were used to prepared zinc-chelating complex (SH-Zn and TH-Zn). The structure of the SH- Zn and TH- Zn was detected by ATR-FTIR. Atomic absorption SHectra assay results showed that the chelating activity of SH-Zn and TH-Zn 4.86 and 1.62 ?g/mg, reSHectively. Then this paper investigate the effects of SH-Zn and TH-Zn on the proliferation and differentiation of the osteoblastic cell line MC3T3-E1. In comparison to the control, Methyl thiazolyl tetrazolium (MTT) assay results showed that SH-Zn and TH-Zn significantly stimulated osteoblastic proliferation to 142.31% and 126.93%. In addition, doses of 50 ?g/mL SH-Zn and TH-Zn increased alkaline phoSHhatase (ALP)activity by 1.56- and 1.24-folds in osteoblastic cells, compared with the control. At the same time, 12.5-50 ?g/mL of SH-Zn exerted a strong effect than that of TH-Zn on the proliferation and differentiation of MC3T3-E1.