Study On The Extraction, Purification,Structure And Antioxidation Of Glycoprotein From Sepia Pharaonis Muscle

Sepia pharaonis (S.pharaonis) is a macro-cephalopod, which distributed in the South China Sea area and is a very promising species with respect to culture potential due to tasting delicious, high feeding rate, good environmental adaptive ability, and high growth rate. S.pharaonis is rich in protein and high in EPA/DHA ratio, which is of important nutrition values and pharmacological effects. The extracted materials from squid ink and cuttlebone, including polysaccharides, protein complexes and other substances, have good anti-tumor activity. And the extractions from marine mollusks which is similar to cuttlefish, including polysaccharides, proteins, peptides and terpenes, have anti-tumor, anti-aging, immune regulation, anti-hypertensive, anti-oxidation and so on. In order to make better use of these active substances, it is essential to carry out in-depth research on extraction, separation, structure analysis and biological activity. In this study, we chose S.pharaonis muscle as experimental materials and study the extraction glycoprotein, removal of protein, purification, structures and antioxidant activity. The major results obtained in this study were as follows:Response surface methodology (RSM) was employed to optimize the extraction process of glycoprotein from S.pharaonis muscle (SPMG). Four independent variables, including sodium chloride (NaCl) concentration, liquid-solid ratio, extraction temperature and extraction time in relation to the yield of SPMG, were studied, and combined effects of extraction conditions on glycoprotein yield were also studied by using a three-level four-factor Box-Behnken design based on the results of single-factor test. The results showed that the highest yield of SPMG could present under the most optimal condition of NaCl concentration3.8%, liquid-solid ratio20.78mL/g, extraction time1.48h and extraction temperature66.03℃. Under this condition, the predictive yield of SPMG was7.93%.Optimum conditions of deproteinization of SPMG using Sevage method were studied by orthogonal test. And the optimal saturation of ammonium sulfate precipitation SPMG and its isoelectric point were also studied. The optimum conditions were obtained as follows:chloroform and sevage ratio was2:1, chloroform and1-butanol ratio was6:1, shaking time was15min, standing time was40min, and the deproteinized rate can reach67.97%under4times protein removal at the optimum conditions. The optimal saturation of ammonium sulfate precipitation SPMG was80%and its isoelectric point was5.6. SPMG-Ⅰ and SPMG-Ⅱ were obtained from SPMG by DEAE-Cellulose52ion-exchange chromatography and Sephadex G-100molecular sieve chromatography. The purities of SPMG-Ⅰ and SPMG-Ⅱ were verified by High Performance Gel Permeation Chromatography (HPGPC) and Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), the results indicated SPMG-Ⅰ and SPMG-Ⅱ were homogeneous components. SDS-PAGE showed that the molecular weight of SPMG-Ⅰ and SPMG-Ⅱ were42.5kDa and36.3kDa, respectively.UV spectrum of SPMG-Ⅰ and SPMG-Ⅱ absorbtions before and after β-elinination showed that they all contained O-glycopeptide linkage. From the FT-IR, SPMG-Ⅰ and SPMG-Ⅱ were glycoproteins and contained pyranoid glycoside bond. Amino acid analysis showed that SPMG-Ⅰ and SPMG-Ⅱ were rich in acidic amino acids, the percentage of aspartic and glutamic in total amino acids were approximately40.20%and36.75%. The most abundant amino acid in SPMG-Ⅰ and SPMG-Ⅱ were glutamic, the contents were28.04%and20.07%, respectively. GC-MS detected the monosaccharide composition of SPMG-Ⅰ and SPMG-Ⅱ mainly fucose, glucose and mannose. The most aboundant monosaccharide in SPMG-Ⅰ and SPMG-Ⅱ was glucose, which can reach57.18%and59.02%, respectively. Antioxidant activity of SPMG was evaluated by its superoxide anion, hydroxyl free radical, hydrogen peroxide scavenging abilities and the reducing power. The results showed the scavenging effects or antioxidant activity in four assays (scavenging effects of superoxide and hydroxyl radicals and the reducing power) significantly increased with increasing concentration, which indicated SPMG was of potential utility as a source of natural antioxidants.