Isolation Purification And Mechanism Analysis Of Anti-Inflammatory Peptides From Yak Bone

Yak is a precious animal husbandry breed in China,mainly living in high altitude areas such as the Qinghai-Tibet Plateau.It is a rare species with low temperature resistance and strong tolerance.Yak bone collagen peptides（YBCPs）have attracted much attention due to their diverse physiological activities.Intensifying functional research on YBCPs can increase the added value of yak meat processing,increase herdsmen’s income,and promote social and economic development in pastoral areas.In this study,yak bone was used as the research object to prepare YBCPs using enzymatic hydrolysis technology,and their physicochemical properties,antioxidant and anti-inflammatory activities were characterized;Then,using anti-inflammatory activity as an indicator,the anti-inflammatory active peptides in YBCPs were isolated,purified,and identified by ultrafiltration,high-performance liquid chromatography,and reverse liquid chromatography tandem mass spectrometry;Combining bioinformatics and molecular simulation docking technology to predict the potential biological activity of identified peptides;Finally,the function of the target peptide segment was validated in vitro through an antioxidant and anti-inflammatory evaluation system;Detect the phosphorylation level of related signal pathway proteins in inflammatory cell models,and analyze the mechanism of anti-inflammatory active peptides.The main research results obtained are as follows:（1）YBCPs with a relative molecular weight distribution of<3 kDa account for approximately 91.13%of the total;The total protein content of YBCPs is about 97.80±0.31%,the polypeptide content is about 92.19±0.35%,and the ash content is about 2.21±0.11%.The content of aliphatic amino acids in YBCPs is relatively high,accounting for about 59.79%of the total amino acid content,while hydrophobic amino acids account for about 30.08%of the total amino acid content.（2）YBCPs have good antioxidant activity,and the-OH clearance rate,ABTS⁺clearance rate,and Fe²⁺chelation rate of YBCPs at a concentration of 30 g·L^-1 are 99.83%,99.22%,and68.05%,respectively;In addition,YBCPs also have anti-inflammatory activity,The inhibition rates with a concentration of 1.11 g·L^-1 of YBCPs affecting IL-1β,IL-6、TNF-αand NO were67.98%,84.67%,58.88%,and 36.28%,respectively.（3）The inhibitory rates of YBCPs with a relative molecular weight<3 kDa affecting IL-1βIL-6,TNF-αand NO were 73.48%,96.43%,71.01%,and 47.06%,respectively.This component was selected for high performance liquid phase separation.After liquid phase separation,F2,F5,F7,and F8 components with good anti-inflammatory activity were selected for mass spectrometry identification,and 105 polypeptides were selected.A total of 12polypeptides were obtained by selecting polypeptides with a mass spectrometry reliability score of over 100 and a biological activity of over 0.5 in combination with toxicity,sensitization,water solubility,free radical clearance,ion chelating power,and anti-inflammatory activity.After that,six polypeptides were screened by molecular simulation docking technology:GPAGPSGPAGK（Seq3）,GPAGPSGPAGKDGR（Seq5）,GPSGPQGIR（Seq6）,GPAGPQGPR（Seq7）,GEAGPAGPAGPAGPR（Seq8）,and GEGGPQGPR（Seq10）,and were chemically synthesized.（4）The target peptide segments have strong-OH clearance,ABTS⁺clearance,and Fe²⁺chelation rates,and,they can also effectively increase the content of catalase（CAT）,glutathione（GSH）,superoxide dismutase（SOD）,and reduce the content of malondialdehyde（MDA）in cells,showing strong antioxidant capacity.The Fe²⁺chelation rate,-OH clearance rate,CAT growth rate,GSH growth rate,and SOD growth rate of Seq8 are 57.75%,50.44%,64.94%,87.28%,and 78.20%,respectively;The-OH clearance rate,CAT growth rate,GSH growth rate,and SOD growth rate of Seq10 were 44.97%,68.14%,95.33%,and 66.86%,respectively.The target peptides with anti-inflammatory activity can also effectively inhibit IL-1β,IL-6,TNF-αand NO content,in which inhibition rate IL-1β,TNF-αand NO inhibition rate of Seq3 were 88.72%,85.80%,and 60.22%,respectively.（5）The inhibition rates of IκB phosphorylation by Seq10 and Seq8 were 87.11%and59.80%,respectively,and the inhibition rate of p65 phosphorylation by Seq6 was 45.61%,which can effectively regulate NF-κB signaling pathway.Secondly,the target peptide reduced the production of NO by inhibiting the expression level of iNOS.The inhibition rates of Seq3and Seq5 on the expression level of iNOS were 88.26%and 85.08%,respectively.These results suggest that the target peptide plays an inflammatory role by inhibiting the NF-κB signaling pathway and the production of NO.The above research indicates that both YBCPs and the purified target peptide segments have antioxidant and anti-inflammatory activities,which can not only expand the utilization range of yak bones and obtain high-quality protein resources,but also increase the added value of yak product processing,increase the income of Tibetan herdsmen,and reduce environmental pollution.Meanwhile,this study provides a theoretical basis for the application of YBCPs as a functional bioactive component.