Extraction, Isolation, Purification And Biological Activity Research On Sparassis Crispa Polysaccharide

Sparassis crispa is a kind of edible fungi, with the functional activities such as anticancer, anti-tumor, antivirus, to improve metabolism, decrease the body blood fat and balance the blood pressure, improve the hematopoietic function and immunorregulation. Polysaccharides in Sparassis crispa is one of the most importantly active ingredient. The content of ?-glucan goes up to 50%-60% in Sparassis crispa polysaccharides. The polysaccharide extracted from Sparassis crispa poses a sensational commercial potential. So far, the cultivation and processing on Sparassis crispa has increased progressly, whereas the study in the field polysaccharide extraction, it's product development and clinical application remains in blank. In this thesis, the polysaccharide extraction from Sparassis crispa with compound enzyme, protein detaching from the crude polysaccharide, the antioxidation activity and the structural identification of polysaccharide extracted from Sparassis crispa were implemented to provide available theory basis for further development and application of Sparassis crispa.Enzymes such as cellulose, pectase and papain are compounded to extract polysaccharide from Sparassis crispa. The ratio of the compound enzymes and the enzymolysis parameters were optimized through the orthogonal experiment and the response surface method respectively. Then, the deproteinization, dialysis treatment, column chromatography treatment were employed to separate and purify the Sparassis crispa polysaccharide, Appraisal the extracorporal antioxidation activity of the deproteinized component, uniform component and crude polysaccharide; Analysised the polysaccharide structure through IR spectrum and NMR spectrum.(1) The extraction polysaccharide from Sparassis crispa with compound enzymesThe extracting conditions polysaccharide form Sparassis crispa with compound enzymes was optimized as follows:cellulose 0.6%, pectase 0.4%and papain 0.6%, enzymolysis pH 4.16, time 3.41h, temperature 53.73?,the ratio of solid vs liquid was 1:15.63. And under this condition the polysaccharide extraction rate was up to 14.3%.(2) The deprotein technics for Sparassis crispa polysaccharide Enzyme, calcium chloride, hydrochloric acid, TCA and sevage methods were used respectively to remove the protein from Sparassis crispa polysaccharide and to evaluate the deproteinizated rate and the polysaccharide retention rate. The results indicated that TCA method was proved to be the optimum. Under the condition namely the concentration of TCA15%, the TCA addition 30%, the oscillation time 15min, the hold-times 25 min, the deproteinizated rate increased up to 90.64%and polysaccharide retention rate was 80.59%.(3)The comparsions of extracorporal antioxidation activities of different contents of Sparassis crispa polysaccharideThe test kits were applied to compare the extracorporal antioxidation activities among the SC1-1, SC2-1 single component polysaccharide, deproteinizated polysaccharide and crude polysaccharide such as the ability to scavenge hydroxyl radical ("OH), superoxide anion radical (C2-2·),1,1-diphenyl-2-picrylhydrazyl radical (DPPH-) and total antioxidant capacity. The resoultes showed that the scavenge ability of the single component SC and SCI deproteinizated are better than that of their crude polysaccharide respectively. And it also indicated that different components of the Sparassis crispa polysaccharide affected synergisticly on the scavenge activity. The abilities of deproteinized liquid and puritied polysaccharide are stronger than crude polysaccharide in inhibit the superoxide anion radical (O2·-).The total antioxidant capacity of the acid component of polysaccharide was significant(P<0.05) than that of neutral polysaccharide. The deproteinized liquid could scavenge 96.25% of the 1,1-diphenyl-2-picrylhydrazyl radical (DPPH·) when the concentration was 5mg/mL. Thus,the polysaccharide concentration and the extracorporal antioxidation activity has a significant(P<0.05) dose-response ralationships.(4)Structural analysis for polysaccharideThe structural analysis through the infra-red spectrum showed that polysaccharide component:SC1-1,SC2-1 both have the functional groups such as-OH,-COO-, C-O-C, C=C,-CH3,-CH2,-CH. And the polysaccharide component was a heteropolysaccaride with a-glucans and ?-glucans, and dominated by ?-glucans. The NMR spectrum test showed that SC1-1, SC2-1 was composed by ?-(1?3)-linked D-glucan and ?-(1?6)-linked D-glucan. The conclusions could be drawed that the biological activity of the polysaccharide component may be responsibled for the ?-(1?>6)-linked D-glucopyranose. Since the molecules structure of SCI-1 was similar to SC2-1, it may be their cooporation and synergistic effecets who promoted the biological activity to some extent.