Structural Characterization And Antioxidant Activity Of Skate Chondroitin Sulfate And Its Degradation Products

Chondroitin Sulfate(CS)is a glycosaminoglycan commonly found in cartilage and connective tissue of animals.It has many biological activities,such as anticoagulation,anti-tumor,promoting coronary circulation,scavenging free radicals in vivo,alleviating arthritis and so on.It is widely used in food,medicine,cosmetic and other fields,and has a broad prospects for development.At present,the commercialized CSs in China are mostly derived from the cartilage tissues of pig,cattle and shark.However,because of the problems of religious ethics,infectious diseases and endangered species,it has brought great challenges to the further development and application of CS.Therefore,it is urgent to find new sources of cartilage to prepare harmless CS with stable quality.Skate(Rajiformes)is abundant in resources in China,but the effective use of its cartilage is almost blank.Therefore,the exploration of the value of skate cartilage resources will become an important way to realize industrial value-added and develop to intensive processing.In this study,skate cartilage was selected as the experimental material,and skate CS with high purity was prepared by screening method,single factor test and response surface design.Low molecular weight chondroitin sulfates(LMWCS)was obtained by various degradation methods.In addition,the structure of skate CS and LMWCSs were characterized in detail and the antioxidant activity in vitro were determined.The specific test methods and results are as follows:(1)Optimizing the preparation process of skate CS:The pretreated skate cartilage was extracted by dense alkali-salt method,dilute alkali-enzymatic method and ultrasound-assisted alkali-enzymatic method respectively.The best extraction method was selected and optimized by single factor test and response surface design test.The results showed that ultrasonic-assisted alkali-salt-enzymatic hydrolysis was the most suitable method for extracting skate CS.The concentration of alkali solution,extraction temperature and the amount of enzymatic addition had significant effects on the extraction rate and the content of glucuronic acid.The best purification conditions were determined by response surface test:alkali concentration 2.3%,extraction temperature 43℃,and enzyme addition 0.7%.Under these conditions,CS was prepared.The extraction rate was 43.32%.(2)Physicochemical properties and main quality indicators of skate CS:According to the standard requirements,the physical and chemical properties and main quality indicators of prepared skate CS were analyzed and evaluated.The results showed that the main quality indexes of the prepared skate CS samples met the standard requirements.The protein content was 1.35%,the glucuronic acid content was 28.20%,and the sulfate content was up to 17.18%.It was a highly sulfated pure chondroitin polysaccharide.(3)Structural characterization of skate CS:The GPC,UV,IR and 1H NMR spectroscopy were used to characterize the structure of CS and compare with CS from shark origin.The results showed that the molecular weight of skate CS was 40752 Da,and its UV spectrum overlapped shark CS with maximum absorption at 210-220 nm.IR spectroscopy and NMR spectroscopy,skate CS and shark CS had similar absorption peaks,and their main disaccharides were CS-C and CS-D.(4)Preparation and structural characterization of LMWCS:LMWCS was prepared by acid degradation,oxidation degradation and enzymatic degradation,respectively.The yield,appearance quality,purity,sulfate content and molecular weight of the products were compared.The structure changes of polysaccharides before and after degradation were analyzed by IR and NMR spectroscopy.The results showed that all the degradation methods prepared LMWCS with purity of 99%and molecular weight less than 10000 Da.However,the appearance quality of LMWCS-A products degraded by acid was poor,and the yield and sulfate content were the lowest,44.20%and 14.84%,respectively.The three degradation methods did not change the main disaccharide composition of CS,and had no significant effect on the main structure.They were all degraded by breaking the β-1,4 glycoside bond.However,oxidative degradation and acid degradation resulted in the fall-off of sulfate group,while enzymatic degradation and acid degradation resulted in the formation of Δ4,5-unsaturated bond at the non-reductive end.(5)Antioxidant capacity of skate CS and its degradation products in vitro:The antioxidant capacity of shark CS,skate CS and its degradation products in vitro was determined by DPPH·,·OH,O2-·scavenging capacity test and ORAC test,and the antioxidant capacity of each sample was evaluated and compared by calculating IC50 value.The results showed that besides ·OH and O2-scavenging capacity,skate CS exhibited stronger free radical scavenging capacity and higher ORAC value than shark CS.All three degradation treatments significantly enhanced the antioxidant capacity of skate CS,among which LMWCS-E obtained by enzymatic degradation was stronger,while LMWCS-A obtained by acid degradation was weaker.