| Auricularia auricular is a nutritious and has medicinal value of food with a long history. With the depth study of the biological activity of the Auricularia auricular, the sale of Auricularia auricular increase gradually at home and abroad year after year which liked by people. AAP is the main activated composition of Auricularia auricular, which have biological activity of anti-tumor, anti-oxidation, regulating immunity, anti-aging, anti-fatigue, lowering blood pressure and other activity in the literature. The main contents of this issue have ultrasonic extraction of black fungus polysaccharides, depigmentation, deprotein, ion exchange column purification, structural characterization of the polysaccharide and anticoagulant activity in vitro. To obtain high anticoagulant activity of AAP by trying simplify the extraction and purification process. The main results of the study are as follows.1. Ultrasonic Extraction and Optimization of CAAPUltrasonic extraction of CAAP by single factor experiment and response surface experiments, determine the optimum extraction conditions of ultrasonic extraction of CAAP by SAS software analysis. Response surface analysis of variance, the lack of fit test item analysis, p value was 0.0613, the correlation coefficient (R2) was 0.9537, the optimum extraction conditions were ultrasonic frequency 70.17KHz, ultrasonic bath temperature 50.99℃, ultrasonic time 23.02min, liquid-solid ratio 35.78mL/g, theoretical maximum of CAAP was 6.39%. Under optimum conditions be verified experimentally, CAAP yield was 6.32%.2. Decolorization and deproteinization of CAAPCAAP was decolorized by hydrogen peroxide, dialyze to remove small molecules, whose color is from brown to pale yellow.Three deproteinization methods of Sevag, TCA and polyamide adsorption were contrast. Sevag method was a traditional method of removing protein, relatively mild, polysaccharide retention rate was the highest, deproteinized better. TCA have the highest rate of removing protein, but TCA is able to degradate polysaccharides, retention rate of polysaccharide is the lowest among them. Polyamide adsorption is better to remove protein, but adsorbed more polysaccharides, retention rate of polysaccharide is the lower than Sevag method. Sevag have the rate of removal protein 73.31%, the rate of polysaccharide retention 55.89%.3. Purification of Auricularia auricula polysaccharidesAAP was obtained by decoloration and deprotein and purfied by the ion exchange column DEAE Sepharose CL-6B. Four major fractions respectively were eluted with distilled water,0.1,0.2,0.5mol/L sodium chloride solution, collected and then concentrated. The four components (AAPⅠ, AAPⅡ, AAPⅢ and AAPⅣ) were obtained. The yields were 5.21%,39.36%,22.47% and 2.35%, total recovery polysaccharide was 69.39%.4. The characterization of AAP(1) Qualitative and quantitative experiments of AAP. In qualitative tests, UV-visible spectrum and FT-IR were used. About 280nm, the absorbance value of AAP decreased than CAAP, protein removal was good.260-280nm, AAPI and AAPII had no significant absorption peak, indicating AAPI and AAPII had almost no nucleic acids and proteins. AAP CAAP, AAP, AAPI and AAPII, had characteristic absorption peak of polysaccharide (3429.09cm-1 and 2939.07cm-1), which had similar infrared spectral features. In quantitative experiments, total sugar, protein and uronic acid of CAAP, AAP, AAPI and AAPII were tested.(2) High performance gel permeation chromatography (HPGPC) analysis of AAP AAPI, AAPII and AAPIIa determined molecular weights and molecular weight distribution. AAPI had two major components, whose weight average molecular weights (MW) were 60.7×104 and 1.425×104Da. AAPII contained two components, whose MW were 155×104 and 1.419×104Da. AAPIIa is homogeneous polysaccharide component, whose weight MW is 59.8×104Da.(3) Monosaccharide composition of AAP AAPⅠ, AAPⅡ and AAPⅡa were assayed by high performance anion exchange chromatography (HPAEC). AAPⅠ and AAPⅡ contain xylose, mannose and galactose. AAP AAPⅡ and AAPⅡa have mannose as the main ingredient, AAPⅡa contains uronic acid. The monosaccharide composition of AAPⅡa has Fuc, Rha, Ara, GalN, Gal, Glu, Xyl, Man and GlcA, whose molar percentage is 0.59:0.30:1.56:1.56:1.16:6.05:22.49:56.80:10.83.5. Anticoagulant activity of Auricularia auricula polysaccharides in vitro Crude Auricularia auricular polysaccharides(CAAP), decolorization and deproteinization polysaccharides AAP, AAPⅠ, AAPⅡ, AAPⅡa and AAPⅢ of anticoagulant activity were study in vitro, APTT, PT, TT as an indicator. Auricularia auricula polysaccharides have no influence on the TT. AAPI and AAPⅡ have no effect on APTT and PT. AAP has a significant influence on APTT and PT. CAAP, AAPⅡ and AAPⅡa have significant effects. With concentration gradually increased, AAPⅡa is significant on APTT and PT. CAAP, AAPⅡ and AAPⅡa can activate the endogenous and exogenous coagulation system coagulation system, which have no effect on the common coagulation pathway. |
PDF Full Text Request