Extraction, Purification, Structural Characterization And Activity Evaluation Of Glycosaminoglycan From Swim Bladder

The swim bladder,also known as fish bubble,is an important organ in the fish that is used to maintain balance.It is a traditional medicine and food widely used in China's coastal areas.Traditional medicine holds that it has a high medicinal value.However,its actions and function factors remain unclear.China is a big country with abundant aquatic resources,including fish swim bladder resources.However,the current research and utilization of swim bladder was only with simple method,most are dried products,single variety and backward processing method technology.Meanwhile,the deep processing and basic research of swim bladder was obviously insufficient.The research on pharmaceutical value of swim bladder is an important direction for high-valued of swim bladder.It is a key scientific issue to discuss the material basis and mechanism of glycosaminoglycan from swim bladder.From this starting point,the extraction and purification was studied,and the basic physicochemical and structural characteristics were investigated.On the basis of this,some functional activities were preliminarily evaluated.This study could provide theoretical basis for exploiting and utilizing swim bladder.The main research content and results are as follows:?1?Optimize the extraction method of the glycosaminoglycan from the swim bladder.On the basis of the single factor experiment,the enzyme dosage,the enzyme temperature and pH were studied with the Box-Behnken response surface design method and their interaction on the yield of glycosaminoglycan were also studied.The quadratic regression model was developed by Design Expert 8.05 software.This model can better analyze and predict the best method extraction of glycosaminoglycan.The optimum conditions were as follows:enzyme dosage 7%,enzymolysis pH 8.0,enzymolysis temperature 50?,and on these conditions,the yield of glycosaminoglycan from fish swim bladder was 0.227%.?2?S0 was extracted by the optimal conditions.S1 was obtained by Sevag method,and S2 was obtained by ethanol precipitation method.S3 and S4 were isolated on the ion-exchange column?DEAE C-52?,the recovery rate was 35.6%and 14.2%,respectively.Two different components of S3-1 and S4-1 were obtained by Sephadex G-300 column,and the recoveries were 70%and 64.5%,respectively.?3?The basic physicochemical of different components of glycosaminoglycan from swim bladder were measured.The results showed that the content of polysaccharides in S0,S2,S3-1 and S4-1 was 26.3%,36.7%,27.81%and 31.46%,glycosaminoglycan was 20.9%,40.7%,53.06%,65.38%;protein was 60.7%,21.3%,9.26%and 2.82%;SO₄^-was 1.81%,2.83%,1.56%and 1.18%;uronic acid was 12.5%,0.7%,10.56%and 11.88%;total hexosamine was 20.68%,12.71%,11.67%,11.84%;lactosamine was 18.75%,7.29%,10.21%,11.05%;glucosamine was 1.93%,5.42%,1.46%,0.79%.?4?The relative molecular weight and structure characteristics of glycosaminoglycan were analyzed.The results showed that the relative molecular weight of S3-1 was 3.14 KDa and S4-1 was 2.98 to 6.26 KDa.The IR indicated that S3-1 was contained acetylamino and sulfate,and the glycosidic bond type was alpha glycosidic bond;S4-1 was contained acetylamino,carboxyl and sulfate,and the glycosidic bond type also was alpha glycosidic bond.The sorts of S3-1 and S4-1 were identified by cellulose acetate electrophoresis.The electrophoretic mobility of S3-1 was lower than CS,and it contains two different Polysaccharides.S4-1 has a single band,and the electrophoretic mobility was aligned with chondroitin sulfate?CS?.The monosaccharide compositions of S3-1 were glucose,galactose,galacturonic acid,xylose,rhamnose,arabinose,mannose and glucuronic acid.The monosaccharide compositions of S4-1 were glucose,rhamnose,glucuronic acid,galactose and galacturonic acid.¹H NMR and ¹³C NMR indicated that S3-1 and S4-1 were composed of various monosaccharides,and they were alpha-pyranose.?5?The hygroscopicity and moisture retention of glycosaminoglycan from swim bladder were measured.The results showed that S2 has excellent capacities of hygroscopicity and moisture retention,which were superior to those of the conventional humectants,such as chitosan and sodium alginate.The anti complement activity of S4-1 were studied by erythrocyte hemolysis method.The results showed that the complement activity of the classic pathway was inhibited in a concentration range of 0¹ mg/m L of S2 and S4-1,and the maximal inhibition rate was17.27%,and the anti complement activity of glycosaminoglycan was decreased with the concentration increased.Under simulating human gastrointestinal pH environment,the cholate-binding ability of S4-1 active components in vitro was tested in order to evaluate the blood lipids reducing function.The results showed that S4-1 had faintly sodium glycochlate and sodium cholic capacity under the concentration range of 2⁴ mg/mL,and had substantially no taurocholic acid sodium capacity.