Study On Extraction, Purification, Structure And Biological Activity Of Lentinan

In this dissertation, research of extraction, isolation, purification, physicochemical properties and structure of lentinan were made, a comparison of in vivo anti-HBV activity was also carried between three lentinan with different molecular weight. The purification methods of lentinan are complex at present, and the product is expensive. Aimed at the application prospect and development situation, in this thesis, the studied emphasis was to develop the technology of extraction and purification of high purity lentinan, in order to obtain the short process, simplicity and economic technology of high purification and recovery yield based on industrialization. The research expressed that the design of process route was reasonable, the method was proper and the purity and recovery yield of product reached higher lever. The main contents and conclusions are listed as follows:1. The extraction methods of lentinan were systematacially studied, the method of leaching with hot water, ultrasonic-assisted extraction, microwave extraction and enzymatic extraction were investigated respectively, and these processes were optimized. The results showed that: the extraction yield of lentinan extracted by hot water was 4.53%, the lentinan purity was 23.2%; the extraction yield by ultrasonic-assisted extraction was 4.48%, the lentinan purity was 21.5%,; the extraction yield by microwave extraction was 4.44%, the lentinan purity was 24.2%; the extraction yield by enzymatic extraction was 6.63%, the lentinan purity was 32.1%. Compared with the method of extracting by hot water, ultrasonic-assisted extraction could reduce the extraction temperature and time, microwave extraction could reduce the extraction time very much, enzymatic extraction could improve the extraction yield and purity of the product obviously. So enzymatic extraction mothod is the best extraction mothod.2. The lentinan extraction solution was settled with chitosan for the first time, the suspended particulate, gelatinoid, partial pigment and protein could be eliminated effectively, at the same time the solution clarification degree could be improved and the solution viscosity could be decreased. The process was optimized. The pigment in solution decreased about 26%, protein concentration decreased 35% and the solution viscosity decreased 15% about also.3. The ultrafiltration technology was used to isolate and classify the leached solution of lentinan for the first time. Two kinds of ceramic membrane of molecular weight cut-off 50kDa and 300kDa were selected, the lentinan was separated to three parts with different molecular weight: Le1, Le2, Le3, the purity of them were 70.5%, 52.3%, 26.3%. Le1 was low molecular weight lentinan with low content protein, Le2 was middle molecular weight lentinan including little protein, Le3 was macromolecular lentinan with high content protein. The optimized separation conditions of ultrafiltration were as follows: temperature 35℃, pH value 7.0, 0.15MPa pressure. The mechanism of membrane fouling and method of membrane cleaning were investigated. The membrance resistance of ultrafiltration of lentinan solution was analyzed with gel polarization model, the membrance resistance model could fit the gel polarization model well.4. The application of ion exchange resin and macroporous adsorption resin to purify lentinan was studied for the first time, it was found that macroporous adsorption resin DA201-C had the best decolorizing effect of lentinan, meanwhile, it could also remove protein at some degree. The technology process of lentinan isolation by DA201-C was optimized. The decolorizing ratio of lentinan was 87.7%, the removal ratio of protein in lentinan was 42%, and lentinan loss ratio was 15%. The purity of Le1, Le2 and Le3 after decolorizing by macroporous adsorption resin DA201-C were 85.3%, 72.9%, 49.6% separately.5. The application of ion exchange resin to removal protein was studied for the first time. The technology processing of reproteinization from lentinan by 717 anion- exchange resin was optimized, the acidity was the major factor influencing the adsorption effect, the best pH of lentinan solution was 8.8～9.2. The content of protein in Le1, Le2 and Le3 were 5.4%, 15.3% and 38.7% before removing protein by 717 anion-exchange resin, after removing protein, the content of protein in Le1, Le2 and Le3 were declined to 0.5%, 1.6% and 6.4%, and the content of lentinan were 95.1%, 89.5%, 86.4% separatery. The resin could regenerate by 0.5mol/L HCl solution, and the adsorption efficiency could return to 90% above.6. Adsorption dynamics research of protein indicated that adsorption rate of protein was governed by membrane diffusion rate, the adsorption isotherm of protein could be described with the equation of Langmuir, adsorption reaction was exothermic reaction at the same time.7. The physicochemical properties of three kinds of lentinan Le1, Le2 and Le3 were studied, the molecular weight of Le1, Le2 and Le3 was proved to be homogeneous by gel filtration chromatography of Sephadex G-200 and agargel electrophoresis. The polysaccharides component were determined by gas chromatography, three lentinans were composed of glucose (Glu), arabinose (Ara), xylose (Xyl), mannose (Man) and galactose (Gla). The neutral saccharide molar ratios Ara : Xyl : Man : Gal : Glu are as follows: Le1, 0.15 : 0.52 : 1.00 : 1.20 : 7.20; Le2, 0.21 : 0.68 : 1.00 : 1.02 : 11.56; Le3, 0.29 : 0.42 : 1.00 : 0.85 : 16.20. The average molecular weight (M_w) of the three polysaccharides Le1, Le2 and Le3 were determined to be 4.02×10⁴, 2.16×10⁵, 8.93×10⁵, respectively. The ¹HNMR, C¹³NMR and IR spectra confirmed that the sugar residue of Le1 wasα-glycosidically linked, the sugar residue of Le3 wasβ-glycosidically linked, Le2 containedα-glycosidically linked andβ-glycosidically linked component at the same time.8. The anti-HBV activity of Le1, Le2 and Le3 was determined in vivo. The results proved all these lentinan had anti-HBV activity, Le3 showed highest anti-HBV activity, and Le1 showed lowest anti-HBV activity, it demonstrated the bio-activity of lentinan had some connection with its molecular weight.