Spectrum-effect Relationships Between HPLC Fingerprints And The Antioxidant And Anti-inflammatory Activities Of Cartilage Collagen Peptides

Osteoarthritis?OA?,the most common degenerative joint disease worldwide found in elderly individuals,is a leading cause of physical disability.Although the etiology and underlying mechanisms of OA are complicated,much evidence has suggested that the progression of OA in patients is significantly associated with oxidative stress and the inflammatory factor network.Naturally derived type II collagen?CII?is thought to be a potential oral alternative for OA treatment.In virtue of the extreme complexity of CII,the components contributing to their effects are still unclear.Hence,it's necessary to develop an efficient method for screening the active components of CII by establishing spectrum-effect relationships,which will provide methodology reference and experimental basis for the therapeutic material basis of CII and the development of CII in drugs and functional foods.In this study,a total of 13 batches of cartilage type II collagen?CCII?samples were extracted,isolated,and purified from chicken sternal cartilage under various process parameters.The fingerprint profiles of 13 batches of CCII were established by high performance liquid chromatography?HPLC?.The in vitro antioxidant activities and anti-inflammatory activities of CCII were measured.Subsequently,the spectrum-effect relationships between HPLC fingerprints and the antioxidant and anti-inflammatory activities of CCII were investigated using grey relational analysis?GRA?.The main results of this study are appended below.?1?The physicochemical characteristics of 13 batches of CCII were investigated.The Kjeldahl method and bicinchoninic acid?BCA?method were used to determine the protein content.The amino acids were derivatized with phenylisothiocyanate?PITC?and then determined by RP-HPLC.The?KTA Purifier 100 system was used to measure the molecular weight distributions.CCII held a protein content of 71.93%⁷8.84%and20.45%³2.40%using Kjeldahl method and BCA method,respectively.CCII were rich in Gly,Ala,and Pro,which accounted for 23.98%,16.20%,and 9.64%of the amino acid residues,respectively,whereas HyP?0.24%?,HyL?0.51%?,His?0.52%?,and Tyr?0.73%?were relatively rare among the 18 quantified amino acids.All of the hydrolysates of the CCII were primarily composed of fractions II?500–1000 Da?and III?1000–3000 Da?,which accounted for approximately 64.80%of the sample after enzymatic treatment.?2?The fingerprint profiles of 13 batches of CCII were established by HPLC.HPLC analyses were performed using an Inertsil ODS-SP C18 column?4.6×250 mm,5?m?.A binary gradient elution system,comprising mobile phase?A?0.05%TFA/acetonitrile and mobile phase?B?0.1%TFA/water,was applied.Fifteen common peaks were obtained from the HPLC fingerprints of CCII.The similarities between the reference fingerprint and chromatographic profile of batches 1–13 were 0.777,0.936,0.972,0.963,0.977,0.976,0.969,0.977,0.972,0.967,0.961,0.953 and 0.711,respectively.?3?The in vitro antioxidant activities of CCII were measured using the 2,2?-Azinobis?3-ethylbenzothiazoline-6-sulphonic acid?diammonium salt?ABTS?assay,the2,2-diphenyl-1-picrylhydrazyl?DPPH?assay,the ferric-reducing antioxidant power?FRAP?assay and an assay of the oxidative damage induced by hydrogen peroxide?H₂O₂?in the degenerative cartilage cells from the knee joint of rat C518?C518 cell line?.The anti-inflammatory activities of CCII were assessed by measuring the inflammatory responses induced by lipopolysaccharides?LPS?in C518 cells.The results of these experiments suggested that S1,S7,S10 and S11 possessed good antioxidant efficacy while S1,S2 and S10 possessed good anti-inflammatory efficacy.S13 exhibited the lowest antioxidant capacity in the ABTS assay,DPPH assay,FRAP assay and an assay of the oxidative damage induced by H₂O₂ in the C518 cell line.?4?The spectrum-effect relationships between HPLC fingerprints and the antioxidant and anti-inflammatory activities of CCII were investigated using GRA.The correlation coefficient between the common characteristic peaks and the efficacy of CCII were above0.6,indicating that these components are related to the antioxidant and anti-inflammatory activities of CCII.Peaks 2 and 8 were the main components contributing to the antioxidant activity of CCII,whereas peak 14 was the main component contributing to the anti-inflammatory activity of the CCII.The components of peaks 8 and 14 were identified as GPRGPPGPVGP and VAIQAVLSLYASGR by UPLC-MS/MS.This work has provided experimental basis for the development of collagen peptides in drugs and functional foods.