Isolation,Purification And Antiosteoporosis Activity Of Donkey Bone Collagen And Its Antioxidant Peptides

Osteoporosis is a systemic disease caused by various factors,characterized by the destruction of bone microstructure,decreased bone density and bone quality,increased bone fragility,and extreme susceptibility to fracture.The level of oxidative stress in patients with osteoporosis is generally higher than normal,and reducing the level of oxidative stress may improve osteoporosis.Related studies have shown that donkey bone collagen has the effect of improving osteoporosis in rats and antioxidants.This project mainly explores the impact of donkey bone collagen in improving osteoporosis from the perspective of oxidative stress.Donkey bone collagen(DC)was extracted by mixing hot water,alkali,and enzyme.We found that DC has the highest UV absorption peak at 228 nm,and the molecular weight is diffuse between 5 kDa and 245 kDa,containing hydrogen bonds between molecules and showing some orderliness.At a concentration of 500 μg/mL,DC showed some antioxidant activity with a scavenging rate of 17.89%against 1,1-diphenyl-2-picrylhydrazyl(DPPH).The model of oxidative damage to osteoblasts was established with H2O2.DC at the concentration of 100 μg/mL could increase the cell survival rate by 27.31%and reduce the intracellular ROS level by 52.48%.In addition,osteoblasts treated with 50 μg/mL DC differentiation showed a 62.65%increase in alkaline phosphatase expression compared to the model group.It can be shown that DC can protect osteoblasts from damage caused by hydrogen peroxide,significantly reduce the level of peroxides in cells,and promote osteoblastic differentiation.A mouse osteoporosis model with oxidative stress was established by feeding D-galactose with a low-calcium diet,and serum,bones,and organs were collected to explore the intervention of each group of drugs on mice.It was found that after gavage of 2.4 g/kg DC in mice,the content of superoxide dismutase in the serum increased by 19.17%,the content of malondialdehyde decreased by 38.95%,and the bone mineral density increased by 27.27%,the trabecular bone became dense from sparse,and the abnormal structure of the mouse cancellous bone was repaired.The result indicates that DC can reduce oxidative stress,increase bone density and improve bone microarchitecture in mice.In order to screen the active components produced by oral DC,DC was degraded by simulating gastrointestinal digestion in vitro.The enzymatic hydrolysis products were separated and purified,and finally,the antioxidant component DCHcA3-α was obtained.LCMS/MS determined that the composition included 7 peptides of ANLGPA,HYFL,PAEGPA,KLLHA,HVPNA,GGWFL,and GWFK.Solid-phase synthesis of 7 peptides was performed to detect the scavenging activities of the peptides on DPPH,superoxide anion,ABTS cations,and hydroxyl radicals to evaluate their antioxidant activity,the results showed that 7 peptides had good antioxidant activity and the peptide ANLGPA had a more comprehensive antioxidant activity.The oxidative damage model of osteoblasts was established with H2O2,and the three peptides of ANLGPA,GGWFL,and GWFK can protect osteoblasts from hydrogen peroxide damage,significantly reduce the level of intracellular peroxides,and promote the differentiation of osteoblasts.In conclusion,this study lays an experimental foundation for the pharmacological effect of donkey bone collagen against osteoporosis and the application of antioxidant peptides derived from collagen in donkey bone.