CN104371036B - A kind of extraction preparation method and applications of sclerotia polysaccharide - Google Patents
A kind of extraction preparation method and applications of sclerotia polysaccharide Download PDFInfo
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Abstract
The invention discloses the preparation method of a kind of sclerotia polysaccharide: after sclerotia soak with ethanol, distilled water extraction, extracting solution concentrates, it is subsequently adding the ethanol precipitate with ethanol of 95% 100%, take the precipitate with ethanol component that ethanol final volume concentration is 60%, first use anion-exchange column separating-purifying, separate with gel filtration chromatography post again after gradient elution, prepare sclerotia polysaccharide.The sclerotia polysaccharide that the inventive method prepares is homogeneous polysaccharide, has antioxidant activity, can remove DPPH free radical, can be used for preparing antioxidant.
Description
Technical field:
The present invention relates to the preparation method and applications of a kind of sclerotia polysaccharide.
Background technology:
Polyporus (Polyporus umbellatus) is under the jurisdiction of Holobasidiomycetidae, aptychus Zoopagales, Polyporaceae, porous
Pseudomonas, is mainly made up of sclerotium and sporophore two parts.Sclerotia is the famousst and precious Chinese crude drug, in China existing 2000
Medicinal history for many years, has diuretic, eliminating dampness by diuresis, antipyretic effect, be commonly used for treat acute nephritis, gonorrhea, diabetes, whole body float
Swollen, urine is smooth, urgent micturition frequent micturition, urethra pain, suffer from heatstroke watery diarrhea and the disease such as acute hepatitis, acute gastritis.Modern medicine grinds
Study carefully discovery sclerotia water decoction extract and there is treatment chronic viral hepatitis and antineoplastic action.
Polysaccharide is the high molecular polymer by glycosidic bond link of the monosaccharide by more than 10, be nature content
One of abundant material, is also the class biomacromolecule being closely related with human lives.Compared with protein and nucleic acid, composition
The saccharide residue of polysaccharide is of a great variety, does not contain only different different head configurations and can connect different substituent groups；The most each
Sugar monomer has multiple link site, and these all make polysaccharide have extremely complex structure, the most also has loading abundant raw
The ability of thing information.Polysaccharide has become the 3rd milestone exploring life secret after protein and nucleic acids research, attracts
The interest of more and more scholars.The most many researchs are proved polysaccharide and have multiple biological activity and function, particularly to machine
The effect of body immunity function, these all make polysaccharide become the important component part in natural drug and health product research and development.And
And the existing multiple polysaccharide in the whole world or have the most respectively carried out the clinical examinations such as regular antitumor, anti-AIDS and treating diabetes
Test.In addition to bioactive research, the structure of polysaccharide is also one of main research focus of many correlational study persons, because
Polysaccharide structures decides its biological activity and function, and the elaboration of structure is to understand its activity and the base preferably developed
Plinth.And obtain the premise that homogeneous polysaccharide component is carbohydrate structure research, also it is one of difficult point and key point.
Polyporusum bellatus has immunostimulation, can improve body immunity, can significantly reduce the oxidation in liver and eliminate
Radical damage, for delaying, histiocyte is aging, protection body, anti-senility are highly beneficial, is the weight of Polyporus sclerotium
Want one of active component.The existing product such as ZHULINGDUOTANG ZHUSHEYE, polyporusum bellatus capsule of China comes out, and clinical and daily guarantor
In Jian widely used.Research about polyporusum bellatus at present focuses mostly in the activity of crude polysaccharides, and the research of homogeneous polysaccharide then rarely has
Report.Some are disclosed the most about the patent of polyporusum bellatus, and such as patent CN 102048769 A, to disclose a kind of Polyporus the most
The extraction process of sugar, this invention uses soxhlet extraction, ethanol precipitation to obtain polyporusum bellatus, mass fraction of polysaccharide about 20%；Specially
Profit CN 101602812 A discloses the preparation method of a kind of polyporusum bellatus, and the method uses soak by water, ethanol precipitate with ethanol, macropore tree
Fat adsorbs, it is thus achieved that the product that polyoses content is higher；Patent CN 101525389 A discloses taking off during a kind of polyporusum bellatus refines
Color method, the method uses hot water extraction, and ethanol precipitate with ethanol obtains crude polysaccharides, utilizes activated carbon that crude polysaccharides is carried out desolventing technology,
And determine the optimum process condition of decolouring.It is low that the Patents of current published polyporusum bellatus is crude polysaccharides, polyoses content,
Or be hetetopolysaccharide, do not relate to single Polyporus homogeneous polysaccharide component and structure thereof and activity.
Summary of the invention:
The invention provides a kind of sclerotia polysaccharide, this polysaccharide has the activity removing DPPH free radical, can be used for making
Standby antioxidant.
The invention provides the preparation method of a kind of sclerotia polysaccharide, said method comprising the steps of:
(1) sclerotia being dried, is grinding to obtain sclerotia powder, sclerotia powder volume fraction 95%-
The ethanol of 100% fully soaks, and filters, obtains filtering residue；
(2) filtering residue of step (1) gained adds distilled water, and 90-100 DEG C is extracted 2-4h, repeat to extract 3～4 times, filters, and closes
And all of supernatant, gained supernatant A is evaporated to the 1/10 of original volume, obtains concentrated solution A；
(3) in the concentrated solution A that step (2) obtains, the ethanol of volume fraction 95%-100% it is slowly added to, to ethanol eventually
Volumetric concentration is 30%, and at a temperature of 4 DEG C, standing 8～12h, centrifugation, be precipitated I and supernatant I, in supernatant I slowly
Add the ethanol of volume fraction 95%-100%, be 60% to ethanol final volume concentration, stand 8～12h at a temperature of 4 DEG C, be centrifuged
Separate, collect precipitation II, vacuum lyophilization, obtain precipitate with ethanol component；
(4) the precipitate with ethanol component that step (3) obtains adds distilled water dissolve, centrifugal, obtain supernatant B, be splined on anion and hand over
Change post, successively with water, 0.1mol/L sodium-chloride water solution, 0.2mol/L sodium-chloride water solution, 0.4mol/L sodium-chloride water solution
Carry out gradient elution, polyoses content in Phenol sulfuric acid procedure monitoring eluent, collect 0.2mol/L sodium-chloride water solution and elute
Eluent, be concentrated into sodium chloride concentration in gained concentrated solution B and reach saturated, concentrated solution B is splined on gel filtration chromatography post,
Using distilled water eluting, Phenol sulfuric acid procedure detects, and collects the eluent containing polysaccharide, prepares Polyporus after concentration, vacuum lyophilization
Sclerotium polysaccharide.
In described step (1), the volumetric usage of the ethanol of volume fraction 95%-100% is with the quality of sclerotia powder
It is calculated as 8-10L/kg.
In described step (1), described immersion is preferably soaked 3～4 times, soaks 12～16h every time.
In described step (2), 10-20 times of the quality that quality consumption is sclerotia powder of distilled water.
In described step (4), precipitate with ethanol component adds distilled water and dissolves, and the volumetric usage of described distilled water is with the matter of precipitate with ethanol component
Amount is typically calculated as 0.1～0.2mL/mg.
In described step (4), the filler of described ion exchange column is DEAE-Sepharose Fast Flow (diethylamino
Ethyl-agarose quickly flows) anion exchange resin, chromatographic column specification is 26cm internal diameter × 100cm height, applied sample amount 5-
15mL, flow velocity 3mL/min；
In described step (4), the program of described gradient elution is preferably: wash 1～2 column volume successively with water,
0.1mol/L sodium chloride solution eluting 1.5～3 column volumes, 0.2mol/L sodium chloride solution eluting 1.5～3 column volumes,
0.4mol/L sodium chloride solution eluting 1～3 column volumes, after eluting terminates, the aqueous sodium chloride of ion exchange column 1mol/L
Liquid regenerates, then 4-5 column volume of watering balance.
Further, in described step (4), the program of described gradient elution is more preferably: wash 1 cylinder successively with water
Long-pending, 2 column volumes of 0.1mol/L sodium chloride solution eluting, 2 column volumes of 0.2mol/L sodium chloride solution eluting, 0.4mol/L chlorine
Change 1 column volume of sodium solution eluting, after eluting terminates, the sodium-chloride water solution regeneration of ion exchange column 1mol/L, then use water
Balance 4-5 column volume；
In described step (4), the filler of described gel filtration chromatography post is that Sephacryl S-500 HR (gather by propylene Portugal
Sugar gel), chromatographic column specification is 16cm internal diameter × 100cm height, applied sample amount 2～8mL, and flow velocity 1mL/min typically washes with water
At least 1 column volume.
The present invention also provides for the sclerotia polysaccharide prepared according to the method for the invention, and described sclerotia polysaccharide is
Homogeneous polysaccharide, using glucose and glucuronic acid as main construction unit, the neutral fraction wherein constituted with glucose
Weight percentage is 60.7%, the percentage by weight of the acidic moiety constituted with glucuronic acid for 24.2%, the weight of total sugar
Amount percentage composition is 84.9%, without albumen and nucleic acid；This sclerotia polysaccharide is homogeneous polysaccharide, and mean molecule quantity is
290KDa, its structure be with 1 → 6 connect β-D-Glucose as main chain, with 1 → 3 connect β-D-Glucose, 1 → 4 connect
β-D-Glucose, 1 → 3 β-D-Glucose aldehydic acid connected, 1 → 4 β-D-Glucose aldehydic acid connected, end β-D-Glucose structure
The substitution in side chain become is in the O-3 position of main chain, and the residue being wherein replaced on main chain and unsubstituted residue ratio is for 1:1.
The sclerotia polysaccharide that the present invention provides can be used for preparing antioxidant.
Preparation method of the present invention is simple to operate, easily controllable, and agents useful for same is water and ethanol, safety non-toxic, low cost.This
The sclerotia polysaccharide that inventive method prepares has antioxidant activity, can remove DPPH free radical, and antioxidant activity is along with dense
Degree increase and increase.
Accompanying drawing illustrates:
The HPGPC figure of the PUP60S2 component that Fig. 1 embodiment 2 prepares.
The infrared spectrogram of the PUP60S2 component that Fig. 2 embodiment 2 prepares.
The DPPH clearance rate curve chart of the PUP60S2 component that Fig. 3 embodiment 2 prepares.
Detailed description of the invention:
With specific embodiment, technical scheme is described further below, but protection scope of the present invention is not
It is limited to this.
The extraction of embodiment 1 sclerotia polysaccharide PUP60
Weigh dry sclerotia, be grinding to obtain sclerotia powder (crossing 20 mesh sieves), take 1.0kg sclerotia powder
End, adds soak with ethanol 12h of volume fraction 96v% of 8.0L, repeats to soak 3 times, filters, abandon supernatant and obtain filtering residue after immersion；
The filtering residue of gained adds the distilled water of 20L, and 100 DEG C are extracted 3h, extract three times, filter, and merge all of supernatant, and supernatant reduces pressure
Concentrate the 1/10 of most original volume, obtain concentrated solution；The ethanol being slowly added to 96v% in concentrated solution to ethanol final volume concentration is
30%, after 4 DEG C stand overnight, centrifugation, collect precipitation I and supernatant I respectively.Supernatant I continues to be slowly added to 96v%
Ethanol be 60% to ethanol final volume concentration, after 4 DEG C stand overnight, centrifugation, collect precipitation II, precipitation II cold through vacuum
Freeze the 60% precipitate with ethanol component i.e. obtaining sclerotia crude polysaccharides after drying, be designated as PUP60, yield 5.1g.
Prepared by the purification of embodiment 2 sclerotia polysaccharide PUP60S2
Take 50mg PUP60 and be dissolved in the solution being made into 5mg/mL in 10mL distilled water, centrifugal, take supernatant and be splined on
DEAE-Sepharose Fast Flow ion exchange column (purchased from GE medical treatment) carries out column chromatography.With the flow velocity of 3mL/min successively
Wash 1 column volume, 2 column volumes of 0.1mol/L sodium-chloride water solution eluting, 0.2mol/L sodium-chloride water solution eluting 2 with water
Individual column volume, 1 column volume of 0.4mol/L sodium-chloride water solution eluting, Phenol sulfuric acid procedure detects, collects each elution requirement respectively
Under elution fraction.
After above-mentioned gradient elution completes, successively with sodium-chloride water solution regeneration pillar and the distilled water balance columns of 1mol/L
Son.Then according to above-mentioned steps, take PUP6050mg × 9 in batches, repeat above-mentioned column chromatography steps and do 9 times, merge 10 times altogether
The component of 0.2mol/mL sodium-chloride water solution eluting, is evaporated to sodium chloride concentration in concentrated solution and reaches saturated, take concentrated solution
Being splined on Sephacryl S-500 HR gel column (purchased from GE medical treatment), each applied sample amount 5mL, with distilled water with 1mL/ in batches
The flow velocity eluting of min, phenol sulphuric acid detects, and collects the eluent containing polysaccharide.By containing that gel filtration chromatography in batches obtains
The eluent of polysaccharide merges, and concentrating under reduced pressure i.e. obtains sclerotia polysaccharide, is designated as PUP60S2 component, yield after vacuum lyophilization
150mg。
The analysis of physical and chemical property of embodiment 3 PUP60S2
PUP60S2 component 25mg that Example 2 is obtained, adds distilled water and is made into 1mg/mL solution, takes 0.2mL and adds examination
Guan Zhong, then to adding the 0.0125mol/mL sodium tetraborate sulfuric acid solution adding 1.2mL in test tube, trash ice cools down, whirlpool shakes
Swing on device after mix homogeneously, in 100 DEG C of boiling water baths, react 5min, frozen water adds hydroxyl between 20 μ L 0.15% after cooling
The NaOH solution of biphenyl, after mixing, measures its light absorption value at 520nm.With D-GlcA as reference standards, record
In PUP60S2 component, glucuronic acid content is 24.2%.Take above-mentioned 1mg/mL PUP60S2 solution 10mL, add distilled water diluting
The solution of 0.1mg/mL, takes 1mL and adds in tool plug test tube, add 1mL distilled water, 1mL 6% phenol, 5mL concentrated sulphuric acid again,
Vibration mix homogeneously, boiling water bath reaction 15min, at 490nm, measure absorbance.With glucose as a standard reference substance, records
In PUP60S2 component, total sugar content is 84.9%.Ultraviolet full scan result shows in PUP60S2 without albumen and nucleic acid.
The purity of PUP60S2 component and molecular weight determination utilize high-efficient gel filtration chromatography (HPGPC) to measure.Condition determination
As follows: sample concentration is 1mg/mL, applied sample amount is 10 μ L, and chromatographic column is TSK-gel PWXLG4000, flowing is 0.1mol/L mutually
Sodium nitrate solution, flow velocity 1mL/min, detector is Waters 2414 differential instrument.Acquired results is shown in Fig. 1, for single symmetry
Peak, illustrates that PUP60S2 component is homogeneous polysaccharide.PUP60S2 component is through bent with the standard that Dextran series standard glucosan prepares
Line comparison show that molecular weight is 290KDa.
1mg PUP60S2 component, after pressing potassium bromide troche, uses the scanning of Nicolet 6700 infrared spectrometer, and gained is infrared
3400.0cm in spectrogram (Fig. 2)-1Strong and the wide absworption peak that left and right occurs is the hydroxyl stretching vibration peak associated,
2920.0cm-1Neighbouring peak is C-H stretching vibration peak, 1200-1000cm-1The absworption peak that peak is C-O-H and C-O-C of left and right,
The above is all the characteristic absorption peak of polysaccharide；1615.8cm-1And 1414.4cm-1For the characteristic absorption peak of carboxylate, explanation
Containing alduronic acid in PUP60S2 component；895.2cm-1One weak absworption peak occurs, and 840.0cm-1Neighbouring without absworption peak, explanation
Saccharide residue containing only beta comfiguration in PUP60S2.PUP60S2 component is the acidic polysaccharose being made up of the saccharide residue of beta comfiguration.
The structural analysis of embodiment 4 PUP60S2
Reference literature (Fau, T.R., Conrad, H.E., Stoichiometric depolymerization of
polyuronides and glycosaminoglycuronans to monosaccharides following
reduction of their carbodiimide-activated carboxyl groups.Biochemistry.1972,
11:1383-1388.) by the carboxyl reduction of PUP60S2 component, method particularly includes: 20.0mg PUP60S2 component is dissolved in 10.0mL
In distilled water, add 190.0mg EDC, utilize automatical potentiometric titrimeter to maintain pH 4.5 to react 2h with 0.1mol/mL HCL solution,
Then in 1h, it is slowly added to 20mL 2mol/L NaBH4Solution, and maintain pH 7.0 with 4mol/mL HCL solution simultaneously.Also
Product after former, after distilled water is dialysed, concentrates, vacuum lyophilization, obtains the component after the reduction of PUP60S2 component
PUP60S2R.Between PUP60S2R warp, the detection of xenol method is without alduronic acid.
Reference literature (Zhang, A.Q., et al.Purification and structural investigation
of a water-soluble polysaccharide from Flammulina velutipes.Carbohydrate
Polymers, 2012,87 (3): 2279-2283.), PUP60S2 and PUP60S2R before and after reduction is respectively through hydrolysis, reduction, second
After Xian Hua, use GC chromatography, method particularly includes: take 2mg PUP60S2 and PUP60S2R, add respectively 4mL 2mol/L TFA in
110 DEG C of hydrolysis 2h, add 3mL methanol concentrating under reduced pressure and are evaporated, be repeated 5 times；Above-mentioned be evaporated after product add 30mg NaBH4And it is molten
In 3mL distilled water, reduce 3h at room temperature, then with in glacial acetic acid and the NaBH of excess4, to solution at generation bubble it is not
Only, adding 3mL methanol, concentrating under reduced pressure is evaporated, and is repeated 5 times, and is placed in vacuum desiccator overnight.Next day, 110 DEG C of baking ovens heat
15min, fully goes out after the moisture of residual, adds 4mL acetic anhydride, 100 DEG C of reaction 1h, cooling, be subsequently adding 3mL toluene, pressurization
Concentration is evaporated, and is repeated 5 times, to remove unnecessary acetic anhydride.Above-mentioned acetylizad product 3mL chloroform dissolves, and adds a small amount of distillation
Water cleans, and is repeated 4 times.Chloroform layer is dried with appropriate anhydrous sodium sulfate, suitably dilutes with chloroform, and analyzes with GC.GC bar
Part: HP-5 capillary column, hydrogen flame ionization detector, nitrogen is carrier gas, column temperature be 120 DEG C maintain 1min, then with 10 DEG C/
Min is warming up to 240 DEG C, and maintains 6.5min.Injection port and detector temperature are 250 DEG C.GC result display PUP60S2 be by
Neutral fraction and the acidic moiety of alduronic acid composition that glucose is constituted form.
Reference literature (Ciucanu, I., Caprita, R. .Per-O-methylation of neutral
carbohydrates directly from aqueous samples for gas chromatography and mass
PUP60S2 and PUP60S2R is complete spectrometry analysis.Anal.Chim.Acta.2007,585 (1): 81-85)
After permethylated, method particularly includes: take 2mg PUP60S2 and PUP60S2R, add 1mLDMSO and minor amount of water respectively, ultrasonic dissolution,
After being cooled to room temperature, add 20mg NaOH powder and 50mg lamellar NaOH, vibration, then add 0.5mL CH3I reacts
10min, adds 2mL distilled water and terminates reaction, and the chloroform adding 3mL extracts, and discards upper strata aqueous phase, adds distillation washing 5 times, has
Machine is concentrating under reduced pressure at 40 DEG C.Repeat above-mentioned demethylation step to methylate once.IR detection methylates without the explanation of OH absworption peak
Completely.Polysaccharide after methylating is dissolved in the formic acid solution of 3mL 88%, at 100 DEG C after depolymerization 3h, adds methanol concentration and is evaporated, repeat
3 times.Polysaccharide after depolymerization is by above-mentioned acetylizad step process (i.e. hydrolyze, reduction, acetylation), and according to above-mentioned GC condition
Analyze with GC-MS.Analysis result is shown in Table 1.The saccharide residue understanding PUP60S2 in table 1 mainly has glucose end, 1 → 3 connection
Glucose, 1 → 4 glucose connected, 1 → 6 glucose connected, 1 → 3,6 glucoses connected, 1 → 4 glucose connected
Aldehydic acid and 1 → 3 glucuronic acid connected, and wherein alduronic acid is linked in 1 → 3, on 6 glucose residues.
30mg PUP60S2 component, adds D2After O lyophilizing three times repeatedly, it is dissolved in 0.5ml D2In O, use 500MHz ANANCE
III nuclear magnetic resonance analyser analysis.1The chemical shift of H NMR with HOD δ 4.78 when 25 DEG C as internal standard,13The chemical shift of C NMR is with full
It is external standard with the δ 0.00 at heavy aqueous solution (D2O+DSS) peak of 3-trimethyl silane sodium sulfonate.NMR spectra the results are shown in Table 2.Pass through
In conjunction with above-mentioned monosaccharide composition and methylated result, it is determined that the structure of PUP60S2 is that the β-D-Glucose connected with 1 → 6 is
Main chain, with 1 → 3 connect β-D-Glucose, 1 → 4 connect β-D-Glucose, 1 → 3 connect β-D-Glucose aldehydic acid, 1 →
The substitution in side chain that the 4 β-D-Glucose aldehydic acid connected, end β-D-Glucose are constituted is in the O-3 position of main chain, wherein on main chain
The residue being replaced and unsubstituted residue are than for 1:1.
The result that methylates of table 1 PUP60S2 and PUP60S2R
Table 2 PUP60S21H and13The chemical shift of C
The Antioxidative Activity Determination of embodiment 5 PUP60S2
Take 0.10,0.25,0.50,1.00, the PUP60S2 solution 2mL of 2.00mg/mL in test tube, be separately added into 1mL
The DPPH ethanol solution of 0.2mol/L, seals, mixing, is placed in lucifuge reaction 1h in 25 DEG C of constant temperature, then at 517nm
Measure light absorption value Ai；The distilled water of same volume replaces polysaccharide as blank, is designated as Ac；Same volume methanol replaces DPPH conduct
Sample controls group, is denoted as Aj, to eliminate the impact of polysaccharide solution self light absorption value.The computing formula of clearance rate R% is as follows:
R%=[1-(Ai-Aj)/Ac] × 100%
Ai: the absorbance of sample
Aj: methanol replaces the absorbance that DPPH records
Ac: the absorbance of blank
The result measured is as it is shown on figure 3, result shows that PUP60S2 component has antioxidant activity, and activity is along with concentration
Increase and increase.
Claims (6)
1. the preparation method of a sclerotia polysaccharide, it is characterised in that described sclerotia polysaccharide is homogeneous polysaccharide, averagely
Molecular weight is 290KDa, structure be with 1 → 6 connect β-D-Glucose as main chain, with 1 → 3 connect β-D-Glucose, 1 → 4
β-the D-Glucose of connection, 1 → 3 β-D-Glucose aldehydic acid connected, 1 → 4 β-D-Glucose aldehydic acid connected, end β-D-Portugal
The substitution in side chain that grape sugar is constituted is in the O-3 position of main chain, and the residue being wherein replaced on main chain with unsubstituted residue ratio is
1:1；
Said method comprising the steps of:
(1) sclerotia being dried, is grinding to obtain sclerotia powder, sclerotia powder volume fraction 95%-100%
Ethanol fully soak filtration, obtain filtering residue；
(2) filtering residue of step (1) gained adds distilled water, and 90-100 DEG C is extracted 2-4h, repeat to extract 3～4 times, filters, and merges institute
Some supernatant, gained supernatant A is evaporated to the 1/10 of original volume, obtains concentrated solution A, the quality consumption of described distilled water
For the quality of sclerotia powder 10-20 times；
(3) in the concentrated solution A that step (2) obtains, the ethanol of volume fraction 95%-100% it is slowly added to, to ethanol final volume
Concentration is 30%, and at a temperature of 4 DEG C, standing 8～12h, centrifugation, be precipitated I and supernatant I, be slowly added in supernatant I
The ethanol of volume fraction 95%-100%, is 60% to ethanol final volume concentration, standing 8～12h at a temperature of 4 DEG C, centrifugation,
Collect precipitation II, vacuum lyophilization, obtain precipitate with ethanol component；
(4) the precipitate with ethanol component that step (3) obtains adding distilled water dissolve, the volumetric usage of described distilled water is with the matter of precipitate with ethanol component
Amount is calculated as 0.1～0.2mL/mg, centrifugal, obtains supernatant B, is splined on anion-exchange column, successively with water, 0.1mol/L sodium chloride
Aqueous solution, 0.2mol/L sodium-chloride water solution, 0.4mol/L sodium-chloride water solution carry out gradient elution, and Phenol sulfuric acid procedure monitoring is washed
Polyoses content in de-liquid, collects the eluent that 0.2mol/L sodium-chloride water solution elutes, is concentrated into chlorine in gained concentrated solution B
Changing na concn and reach saturated, concentrated solution B is splined on gel filtration chromatography post, use distilled water eluting, Phenol sulfuric acid procedure detects, and receives
The collection eluent containing polysaccharide, prepares sclerotia polysaccharide after concentration, vacuum lyophilization.
2. the method for claim 1, it is characterised in that in described step (1), the ethanol of volume fraction 95%-100%
Volumetric usage be calculated as 8-10L/kg with the quality of sclerotia powder.
3. the method for claim 1, it is characterised in that in described step (1), described immersion is immersion 3～4 times, every time
Soak 12～16h.
4. the method for claim 1, it is characterised in that in described step (4), the filler of described anion-exchange column
For DEAE-Sepharose Fast Flow anion exchange resin.
5. the method for claim 1, it is characterised in that in described step (4), the program of described gradient elution is: successively
Wash 1～2 column volume, 0.1mol/L sodium chloride solution eluting 1.5～3 column volumes, 0.2mol/L sodium chloride solution with water
Eluting 1.5～3 column volumes, 0.4mol/L sodium chloride solution eluting 1～3 column volumes.
6. the method for claim 1, it is characterised in that in described step (4), filling out of described gel filtration chromatography post
Material is Sephacryl S-500HR.
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