Extraction,Purification And Antioxidant Activity Studying Of Chondroitin Sulfate From Oncorhynchus Keta

Cartilage is a hyaline connective tissue specifically belongs to animals and is rich in chondroitin sulfate.The physiological function of chondroitin sulfate has been studied and applied in areas of food,drug,cosmetics and clinical application.Numerous studies have demonstrated that chondroitin sulfate from different sources and parts had different structures and functions.There exists significant difference in species between chondroitin sulfate of livestock poultry and fishes. Only extracting chondroitin sulfate from pig,cattle and chicken was limited to single species.With increasing focus on chondroitin sulfate appearing a lot of reports about chondroitin sulfate from various kinds of fish.Indicating that the study of chondroitin sulfate was more comprehensive and in-depth.This paper treated oncorhynchus keta nasal cartilage as material.From extraction to separation and purification to antioxidative activity research, adopted response surface methodology to optimize extraction process, ethanol precipitation-ion exchange column chromatography combined method to separate and purify chondroitin sulfate,spectroscopy to analyze absorbance of functional groups,high-performance liquid chromatography to detect the composition of monosaccharide, determination of free radical scavenging rate to study antioxidative activity.The experimental results are as follows:1. The two-step method of alkaline extraction-enzyme hydrolysis was suitable for extracting chondroitin sulfate from oncorhynchus keta nasal cartilage.Its optimal solutions respectively were:first step conducting under the condition of temperature was 53.1℃,time was 3.6h,ratio of material to solvent was 1:43.4;Second step conducting under the condition of the amount of added alkaline protease was 0.24%,p H was 9.0,temperature was 55℃,time was 2h,the final CS yield was about 35%.2. Using ethanol precipitation to purify the crude chondroitin sulfate extracts. the optimal ethanol precipitation parameters were : ethanol concentration was 60%,sodium acetate mass concentration was 4%,p H was 6.0,under the above the chondroitin sulfate recovery rate and the protein removal rate could simultaneously achieve a good balance point to satisfy the purpose of purification. It turned out that the ethanol precipitated sample A-02 and E-02 were typical C type chondroitin sulfate by infrared spectroscopy.3. Using ion exchage chromatography to separate the ethanol precipitated sample into different species.Analyzing their monosaccharide composition by high-performance liquid chromatography.We found that among three fractions of sample A-02 pure chondroitin sulfate were A-02-02 and A-02-03,A-02-01 was a mixture of chondroitin sulfate and glucosamine. Two fractions of sample E-02,the E-02-01 was a mixture of glucosamine and lactose, E-02-02 was pure chondroitin.The above results showed that the polysaccharide of oncorhynchus keta nasal cartilage mostly were chondroitin sulfate,in addition possibly also contained a certain amount of chitosan.4. Conducting antioxidant activity in vitro experiment object to the ethanol precipitated chondroitin sulfate,the crude chondroitin sulfate extracts and the acid degradation products of chondroitin sulfate.Experimental results concluded as follows:(1) The ethanol precipitated chondroitin sulfate had no obvious scavenging effects on DPPH· while the crude chondroitin sulfate extracts had a certain scavenging effects on DPPH·.Maybe the peptide in crude extracts had scavenging activity on DPPH·.The acid degradation products of chondroitin sulfate had strong scavenging effect on DPPH·.(2) The ethanol precipitated chondroitin sulfate,the crude chondroitin sulfate extracts and the acid degradation products of chondroitin sulfate all had obvious scavenging effects on ·OH.Due to different ectraction methods,the sample A-01 had higher ·OH scavenging rate than E-01,A-02 than E-02.Theses differences may be associated with the composition of crude extracts and the structures of chondroitin sulfte were different.There was no difference between A-03 and E-03,probably because A-03 and E-03 all changed into small molecular chondroitin sulfate oligosaccharides through acid degradation breaking down their complete polysaccharide chain.The produced unsaturated uronic acid structure maybe the key factor affect the antioxidant capacity.(3) The ethanol precipitated chondroitin sulfate and the crude chondroitin sulfate extracts had no scavenging effects on O2-· but the acid degradation products of chondroitin sulfate had strong scavenging effects on O2-·.