Extraction, Purification, Bioactivity And Structure Of Polysaccharides From Hyriopsis Cumingii

Hyriopsis cumingii, commonly called pearl mussel, belongs to freshwater bivalve mussel and has many applying value. Although Hyriopsis cumingii is endemic to China, there are extensively distribution and abundant resources of Hyriopsis cumingi in China. Thus, how to develop and utilize this precious resource scientifically and reasonably become a project deserving to research deeply.Hyriopsis cumingii has been one of the animals used as medicine and food simultaneously since ancient times. However, little information about Hyriopsis cumingii polysaccharides (HCPS) is available at present. In this paper, HCPS was studied systemically, including optimization of extraction parameters, isolation and purification, physicochemical characterization, antioxidation activity, immunomodulation activity and structure of HCPS. Main results are listed as follows:1 Optimization of extraction parameters of HCPSHCPS was extracted by using methods of hot water lixiviation, ethanol precipitation, Sevag's deproteination and ethanol precipitation again. Extraction yield of HCPS was in the range of 1.30-3.60%.Extraction temperature, extraction time, ratio of water to raw material and extraction times were selected in single-factor experiments. Based on the single-factor experiments, combination of the extraction parameters (time, temperature and ratio of water to raw material) was optimized by using three-factor-three-level Box-Behnken design (BBD). The optimum conditions were extracting temperature 80℃, extracting time 4.5 h, ratio of water to raw material 8 ml/g and extraction times 2.Five optimized extraction conditions and five other extraction conditions were selected to certify applicability of model equation. There was no significant difference between experimental value and estimated value from the model equation. The optimized extraction condition was practicable.2 Isolation and purification of HCPSThe crude HCPS was separated firstly through DEAE-cellulose 52 chromatography column and further purified through chromatography column of Sephadex G-100. Three fractions, named as HCPS-1, HCPS-2 and HCPS-3, were obtained. The recovery rates of HCPS-1, HCPS-2 and HCPS-3 based on the amount of crude HCPS were 26.91%,30.18% and 3.93% respectively.Purity of HCPS-1, HCPS-2 and HCPS-3 was further confirmed by using HPLC, and results showed that HCPS-1, HCPS-2 and HCPS-3 were purified polysaccharides respectively.3 Basic physicochemical characterization of HCPSBasic physicochemical characterization of crude HCPS, HCPS-1, HCPS-2 and HCPS-3 were determined by using the methods of chemistry and physics.As to crude HCPS, HCPS-1, HCPS-2 and HCPS-3, polysaccharides content was 76.43%,98.88%,96.60% and 80.06% respectively. Protein content was 1.36%, not detected, not detected and 9.42% respectively. Uronic acid content was 13.17%,16.66%, 16.37% and 17.25% respectively. Sulfuric radical content was 1.38%,0.38%,0.60% and 6.29% respectively. Relative viscosity was 1.18,1.16,1.16 and 1.31 respectively. The crude HCPS was composed of rhamnose, arabinose, fucose, mannose, glucose and galactose with a molar ratio of 5.71:2.21:1.69:3.40:82.21:4.78. HCPS-1 was composed of only arabinose and glucose with a molar ratio of 2.12:97.88. HCPS-2 was composed only of glucose. HCPS-3 was composed of rhamnose, fucose, mannose, glucose and galactose with a molar ratio of 13.80:4.51:7.70:64.92:9.07. The relative molecular weight of HCPS-1, HCPS-2 and HCPS-3 was 432.2kDa,457.9kDa and 503.1kDa respectively.In FTIR spectrum, characteristic absorptions of polysaccharides, carboxyl group and pyranose ring were observed in crude HCPS, HCPS-1, HCPS-2 and HCPS-3. Absorption peaks of amino group and sulfuric acid radicals in HCPS-3 were stronger than that in crude HCPS, HCPS-1 or HCPS-2.4 Antioxidant activities of HCPSAntioxidant activities in vitro of crude HCPS, HCPS-1, HCPS-2 and HCPS-3 were measured by using the chemical methods. Results showed that crude HCPS, HCPS-1, HCPS-2 and HCPS-3 could scavenge H2O2, scavenge free radicals of O2·and DPPH·, chelate Fe2+ and reduce Fe3+ in vitro. Crude HCPS, HCPS-1, HCPS-2 and HCPS-3 had some antioxidant activities in vitro. HCPS-3 exhibited much higher antioxidant activities than crude HCPS, HCPS-1 and HCPS-2.Antioxidant activities in vivo of crude HCPS were determined by using the animal model experiments. Results indicted that administration of low dose crude HCPS could overcome oxidant injury induced by D-Gal, and treatment of high dose crude HCPS could inhibit significantly the formation of MDA, enhance significantly the activities of antioxidant enzymes (SOD, CAT and GSH-Px) and increase significantly the value of TAOC in livers and serums in a dose-dependent manner. Crude HCPS could exhibit some antioxidant activities in vivo.5 Immunomodulating activities of HCPSImmunomodulating activities in vitro of crude HCPS, HCPS-1, HCPS-2 and HCPS-3 were evaluated by using the cell model experiments. Results showed that crude HCPS, HCPS-1, HCPS-2 and HCPS-3 could promote proliferation of spleen cell, enhance activity of acid phosphatase in peritoneal macrophages and increase ability of devouring neutral red of peritoneal macrophages in vitro. Crude HCPS, HCPS-1, HCPS-2 and HCPS-3 had some immunoenhancing activities in vitro. HCPS-3 exhibited much higher immunoenhancing activities than crude HCPS, HCPS-1 and HCPS-2.Immunomodulating activities in vivo of crude HCPS were measured by using the animal model experiments. Results showed that administration of low dose crude HCPS could recovery weakened immune function induced by CPA, and treatment of high dose crude HCPS could increase significantly the index of spleen and thymus, the swelling rate of ear in DTH and the concentration and activity of LZM in serums in a dose-dependent manner. Crude HCPS could exhibit some immunoenhancing activities in vivo.6 Structure of HCPSSpectra of FTIR and NMR implied that there were pyranose rings in HCPS-1, HCPS-2 and HCPS-3. HCPS-1 and HCPS-2 showed feature of a-glycosidic bond, and HCPS-3 exhibited character of bothα-andβ-glycosidic bond.Results of periodate oxidation, Smith degradation, methylation analysis, GC/MS and NMR suggested that Glc in HCPS-1, HCPS-2 and HCPS-3 were linked to produce Glc backbone chain mainly by 1→4 glycosidic bond. Glc backbone chain were branched by 1→4,6 and 1→3,4 glycosidic bond in HCPS-1, HCPS-2 and HCPS-3. Ara in HCPS-1 and Rha, Fuc, Man and Gal in HCPS-3 were linked to Glc backbone chain or side chain mainly by 1→3 glycosidic bond.Congo red experiments suggested that there were not three-strand spiral structure in HCPS-1, HCPS-2 and HCPS-3. Linkage-bond between sugar chain and peptide chain, detected by using the method of alkaline hydrolysis, was O-glycopeptide linking bond in HCPS-3. Thirteen species of amino acid (Asp, Glu, Ser, Gly, Thr, Ala, Val, Tyr, Met, Phe, Ile, Leu and Lys) were detected by applying the method of acid hydrolysis-derivation-HPLC in HCPS-3. Content percentage of Gly to total amino acid was highest (25.18%), and that of Phe was lowest (3.13%).