Hypoglycemic Activity And Structure Identification Of Polysaccharides From Inonotus Obliquus

Objective Inonotus obliquus as raw material, extraction polysaccharides form Inonotus obliquus(IOP), purification and separation obtained homogenous IOP. Screening composition of higher hypoglycemic activity.To analyze its molecular weight and monosaccharides composition, then study its structure elucidation, and investigated the biological activity of antioxidant, bacteriostat and immunoregulition.Methods(1) Using water extraction and ethyl alcohol method obtained polysaccharides form Inonotus obliquus, the optimum conditions of extraction technology is based on the yield index; Phenol-sulfuric acid method was used to detect the polysaccharide content of polysaccharides form Inonotus obliquus; coomassie blue staining method was used to detect the protein content of polysaccharides form Inonotus obliquus.(2) Used the column chromatography to puruficate and separate the crude the polysaccharides, purification and separation of polysaccharides form Inonotus obliquus was used macroporous adsorptive resin AB-8 and DEAE-52 column.(3) Purified polysaccharides were identified by high performance gel permeation chromatography-evaporative light scattering detector(HPGPC-ELSD), used PMP derivative method to determination monosaccharides of polysaccharides form Inonotus obliquus.(4) Used the Hep G2 cells as receptor model in vitro, to study the impact of different concentrations of IOP1,IOP2,IOP3,IOP4 and IOP5 on glucose consumption of Hep G2 and its poisoning effect, screening composition of higher hypoglycemic.(5) The structure of homogenous IOP wsa analyzed by high performance liquid chromatography(HPLC), periodate oxidation, Smith degradation, methylation analusis, gas chromatography-mass spectrometer(GC-MS), infrared spectroscopy(IR) and nuclear magnetic resonance(NMR).(6)Determination Inonotus obliquus polysaccharides and Vc scavenging superoxide anion radical,hydroxyl radical, DPPH radical, to prove its antioxidation activity in vitro.(7) Used susceptibility paper method to detect bacteriostatic activity of Inonotus obliquus polysaccharides on Staphylococcus aureus, Bacillus coli, Bacillus subtilis and Pseudomonasaeruginosa, used dilution method to detect its minimum inhibitory concentration and minimum bactericidal concentration.(8) By detected the impact of Inonotus obliquus polysaccharides on the proliferation of mousee spleen cells in vitro, to discussed the function of Inonotus obliquus polysaccharides on immune regulation.Results(1) Based on the single factor test, to optimize the homogenate extraction of polysaccharide from Inonotus obliquus based on response surface methodology, the optimum condition was: solid-liquid ratio 1:22, extraction time 2.5 h, and extraction temperature 90 ?.The extraction rate was 13.43%, the polysaccharide content of Inonotus obliquus polysaccharides was 19.43%, the protein content was 9.70%.(2) Five homogenous polysaccharides are purified and isolated through macroporous adsorptive resin AB-8 and DEAE Cellulose 52, there are IOP1,IOP2, IOP3, IOP4 and IOP5, respectively.(3) The molecular weights of five homogenous IOP are 3.39�105 Da?7.90�105 Da?4.26�105 Da?1.82�106 Da and 9.66�105 Da, respectively.IOP1 was consist of Man, Rha, Glc A, Gal A, Glc, Gal, Xyl and Ara, its molar ratio was 5.37 :3.61 : 1.00 : 1.13 : 8.78 : 14.83 : 3.41 : 1.91; IOP2 was consist of Man, Rha, Glc A, Glc, Gal, Xyl and Ara, its molar ratio was 3.63 : 1.00 : 1.79 : 1.90 : 6.38 : 3.26 : 3.79; IOP3 was consist of Man,Rha, Gal A, Gal, Xyl and Ara, its molar ratio was 4.47 : 1.71 : 2.98 : 2.31 : 1.24 : 1.00; IOP4 was consist of Man, Rha, Glc A, Gal A, Glc and Xyl, its molar ratio was 6.41 : 3.45 : 1.00 : 1.20 :10.21 : 11.51; IOP5 was consist of Man, Rha, Gal A, Gal, Xyl and Ara, its molar ratio was 3.70 :1.00 : 1.44 : 1.48 : 1.23 : 1.62.(4) Different concentrations of IOP1, IOP2, IOP3, IOP4 and IOP5 all could promote, and the effect of IOP1 was the best. When its concentration was 0.25 mg�m L-1,the effect was the best. When the concentration was 0.25 mg�m L-1, IOP1, IOP2, IOP3, IOP4 and IOP5 all could promote glucose consumption of insulin resistance of Hep G2, the glucose consumption of Hep G2 was gradually increased with the extension of time, and the effect of IOP1 was the best.(5) The structure of homogenous IOP wsa analyzed by periodate oxidation,Smith degradation, methylation analusis, gas chromatography-mass spectrometer(GC-MS) and infrared spectroscopy(IR), obtained the linked ways of IOP1 sugar residues, and the main chain of IOP1 was mainly composed of Glc and Gal.(6)Inonotus obliquus polysaccharides had obvious scavenging free radical effects in a dose dependent manner.When Inonotus obliquus polysaccharides was in a higher concentration, its ability of clearing hydroxyl radicals and DPPH is better than superoxide anion.(7) The bacteriostatic activity of different concentrations Inonotus obliquus polysaccharides on Staphylococcus aureus, Bacillus coli, Bacillus subtilis andPseudomonas aeruginosa was Staphylococcus aureus > Bacillus subtilis > Bacillus coli >Pseudomonas aeruginosa. The minimum bactericidal concentration of Staphylococcus aureus was 12.5 mg�m L-1, the minimum bactericidal concentration of Bacillus subtilis, Bacillus coli and Pseudomonas aeruginosa was 25 mg�m L-1.(8) High and medium dosage of Inonotus obliquus polysaccharides could promote Con A to induce the proliferation reaction of splenocyte, low dosage of Inonotus obliquus polysaccharides could inhibition Con A to induce the proliferation reaction of splenocyte; high and medium dosage of Inonotus obliquus polysaccharides could promote LPS to induce the proliferation reaction of splenocyte, low dosage of Inonotus obliquus polysaccharides could inhibition LPS to induce the proliferation reaction of splenocyte.Conclusions The extraction rate of Inonotus obliquus polysaccharides was obvious improved after using response surface methodology to optimize the homogenate extraction of polysaccharide from Inonotus obliquus. Screening composition of higher hypoglycemic activity in vitro, and identificated its structure. The Inonotus obliquus polysaccharides also had good antioxidant, bacteriostat and immunoregulition activity.