Purification,Structural Characteristics And Conformation In Solution Of Polysaccharide From Sclerotia Of Polyporus Umbellatus

Polyporus umbellatus is a valuable and rare medicinal fungus and has been used in traditional Chinese medicine for 2000 years.The sclerotia are its main medicinal parts,and polysaccharides are one kind of their dominant bioactive ingredients.Large amounts of activities have been reported for polysaccharides from P.umbellatus sclerotia,such as antitumor,immunoregulation,antioxidant,hepatoprotective and so on,while the structure playing important roles in function have limited reports.This thesis focused on P.umbellatus sclerotia polysaccharides from the following parts: optimization of extraction parameters;separation and purification of homogeneous fractions;physicochemical properties,structure elucidation and conformation in solution of these homogeneous fractions.1.Optimization of extraction parametersUltrasound-assisted extraction parameters of polysaccharides from P.umbellatus sclerotia were optimized by response surface methodology using a Box–Behnken design based on the results of single-factor experiments.Besides the independent effects of ultrasonic power,extraction temperature,extraction time and water-to-raw material ratio,the following interactions were also found to have a significant impact on the extraction yield: between extraction temperature and ultrasonic power or water-to-raw material ratio,between water-to-raw material ratio and ultrasonic power or extraction time.The maximum yield of 2.48% were obtained under the following optimal extraction parameters set: temperature of 72.3 oC,ultrasonic power of 304.2 W,water-to-raw material ratio of 21.7 mL/g and extraction time of 63.6 min.2.Separation and purification,and physicochemical properties of homogeneous polysaccharide fractionsThree crude polysaccharide fractions（PUPF30,PUPF60 and PUPF80）were recovered from extract by fractional precipitation with adding alcohol successively to bring final alcohol concentration to 30,60 and 80 vol.%.Given the water-soluble and the results of antioxidant activities,PUPF60 and PUPF80 were selected to prepare the homogeneous polysaccharide fractions with further purification.DEAE-Sepharose Fast Flow and Sephacryl S series gel column chromatography were then carried out for purification of crude fractions.After purification,PUPF60 and PUPF80 separately generated three fractions,PUPF60 W,PUPF60S1,PUPF60S2,along with PUPF80 W,PUPF80S1,PUPF80S2,the average molecular weight of which were 2.09 × 10⁴ Da,2.27 × 10⁴ Da,2.44 × 10⁴ Da,0.65 × 10⁴ Da,0.88 × 10⁴ Da,0.60 × 10⁴ Da,respectively.These fractions showed the characteristic absorptions of polysaccharides and contained little protein except that PUPF80S2 possessed more than 8% protein.Uronic acid were found to be presented in the four fractions prepared by elution with salt solutions: PUPF60S1,PUPF60S2,PUPF80S1 and PUPF80S2,and content were 3.75%,24.20%,10.02% and 14.23%,respectively.3.Structure elucidation of homogeneous polysaccharide fractionsAfterwards,the primary structure characteristics of the six fractions were elucidated by chemical methods（including monosaccharide compositions analysis,methylation analysis,carboxyl reduction,etc.）combined with technologies of instrumental analysis like gas chromatography,combination of gas chromatography with mass spectrum,1D and 2D NMR spectra,etc.The structure properties were obtained including monosaccharide compositions,types and sequences of linkages,anomeric configuration,etc.（1）The structure properties of PUPF60WPUPF60W was a high branched polysaccharide with a degree of branching of 74.7%,composed mainly of fucose,glucose and galactose with a molar ratio of 0.82:1.00:12.25.As shown below,its backbone was constructed mainly with α-1,6-D-Galp of which about two third were substituted at position O-2 with non-reducing terminal α-D-Galp.In addition,there may also exist in the structure little of other residues like terminal α-L-Fucp,α-1,2,3-D-Galp,α-1,3-D-Glcp,etc.（2）The structure properties of PUPF60S1PUPF60S1 was a branched polysaccharide with a degree of branching of 41.2%.Besides little of glucuronic acid and fucose,its main monosaccharide units were glucose and galactose with a molar ratio of 2.02:1.00.Its backbone comprised mainly of α-1,6-D-Galp,α-1,4-D-Glcp,β-1,6-D-Glcp and β-1,3-D-Glcp,among which α-1,6-D-Galp were partially substituted at position O-2 with non-reducing terminal α-D-Galp and β-1,6-D-Glcp were partially substituted at position O-3 by side chain including non-reducing terminal β-D-Glcp,β-1,4-D-Glcp,β-1,3-D-Glcp.The probable structure was proposed as: Where x:y:m:n has an approximate value of 11:5:2:8.Moreover,there also existed in PUPF60S1 little of other residues like terminal α-L-Fucp,α-1,2,3-D-Galp,α-1,4,6-D-Glcp.（3）The structure properties of PUPF60S1PUPF60S2 was a polysaccharide with a degree of branching of 41.2% and composition sugar units of glucose and glucuronic acid.It had a backbone composed primarily of β-1,6-D-Glcp,of which every second was linked at position O-3 by side chain by side chain including non-reducing terminal β-D-Glcp,β-1,4-D-Glcp,β-1,3-D-Glcp,β-1,4-D-GlcpA,β-1,3-D-GlcpA.The proposed structure was shown below: Where β-1,4-D-GlcpA and β-1,3-D-Glcp may be partially displaced by β-1,4-D-Glcp and β-1,3-D-GlcpA,respectively.（4）The structure properties of PUPF80WPUPF80W consisted mainly of glucose and galactose with a molar ratio of 2.02:1.00,and had a small degree of branching of 28.5%.Its backbone was comprised primarily of α-1,6-D-Galp,α-1,4-D-Glcp,β-1,6-D-Glcp and β-1,3-D-Glcp,among which α-1,6-D-Galp were partially substituted at position O-2 with non-reducing terminal α-D-Galp and β-1,6-D-Glcp were partially substituted at position O-3 by side chain including non-reducing terminal β-D-Glcp,β-1,3-D-Glcp,α-1,3-D-Glcp.The structure was proposed as follows: Where x,y,z,l,m and n has a ratio about 6:3:7:2:10:6.In the structure,there may also little of 1,2,3-D-Galp and β-1,4-D-Glcp.（5）The structure properties of PUPF80S1PUPF80S1 consisted also mainly of glucose and glucuronic acid,but its degree of branching was only 22.8%.In addition to little of β-1,3-D-Glcp,the backbone was composed mainly of β-1,6-D-Glcp which was partially substituted at position O-3 by side chain including non-reducing terminal β-D-Glcp,β-1,4-D-Glcp,β-1,3-D-Glcp.A part of β-1,4-D-Glcp and β-1,3-D-Glcp in the structure was displaced by their counterparts β-1,4-D-GlcpA and β-1,3-D-Glcp A.The following structure was proposed for PUPF80S1: Where β-1,4-D-GlcpA and β-1,3-D-GlcpA partially displaced their neutral counterparts β-1,4-D-Glcp and β-1,3-D-Glcp in the structure,respectively.（6）The structure properties of PUPF80S2PUPF80S2 had a degree of branching of 22.8%,and consisted mainly of glucose,glucuronic acid and galactose.It contained mainly residues such as terminal D-Glcp,1,3-D-Glcp,1,4-D-Glcp,1,4-D-GlcpA,1,6-D-Glcp,1,3,6-D-Glcp,etc.NMR analyses were demanded for more information on its structure after preparing more quantities.4.Conformation in solution of homogeneous polysaccharide fractionsFinally,multidetection gel permeation chromatography and atomic force microscope were applied for analysis of conformation in solution of PUPF60 W,PUPF60S1,PUPF60S2,PUPF80 W and PUPF80S1.The results revealed that PUPF60 W,PUPF60S1,PUPF80 W and PUPF80S1 occurred in spherical chain conformation in solution,and their chain density were 0.44,0.28,0.43 and 0.38 g/mL,respectively.PUPF60S2 occurred in flexible chain conformation in solution.The molecular weight per unit contour length,persistence length and chain diameter were 408 nm^-1,1.58 nm and 0.76 nm,respectively.