CN104262500B - It is a kind of that there is immunocompetent novel red support dictyophora fungus polysaccharide and its preparation method and application - Google Patents
It is a kind of that there is immunocompetent novel red support dictyophora fungus polysaccharide and its preparation method and application Download PDFInfo
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Abstract
The present invention discloses a kind of with immunocompetent novel red support dictyophora fungus polysaccharide and its preparation method and application, methods described, with Dictyophora rubrovalvata as raw material, through the preparation of following method：Hot water extraction, Savege method removing proteins, ethanol precipitation, are obtained after then being isolated and purified using ion-exchange chromatography, gel filtration chromatography, ultra-filtration and separation, four step separation of gel filtration chromatography.The preparation technology of the present invention is rationally simple, and is easily achieved industrialized production.Novel red support dictyophora fungus polysaccharide prepared by the present invention has good immunologic enhancement, can be as the adjuvant therapy medicaments of disease under the immunologic functions such as tumour or as the functional health-care food with enhance immunity effect or food.
Description
Technical field
The present invention relates to a kind of immunocompetence polysaccharide and its preparation method and application, and in particular to a kind of that there is immunocompetence
Novel red support dictyophora fungus polysaccharide and its preparation method and application.
Background technology
Dictyophora phalloidea（Dictyophora indusiata）, call " bamboo fungus ", be the general name for belonging to fungi to a big class dictyophora phalloidea, be subordinate to
Belong to Eumycota（Eumycota）, Basidiomycotina（BasidiomycotinA), Gasteromycete（Gasteromycetes）, Phallus
Mesh（Phallales）, Phallaceae（Phallaceae）.
Dictyophora phalloidea is a kind of hidden colored mushroom for colonizing in withered bamboo root, and slightly like netted extra dry white wine snakeskin, it has bottle-green shape
Cap, the columned stem of snowy white, peach egg type volva have on stem top and enclose
Careful pure white netted skirt is spread out from cap downwards, is known as " snow skirt fairy maiden ", " flower of mountain delicacy ", " fungi
Flower ", " queen in bacterium ".Dictyophora phalloidea can be divided into mycelium and fructification two parts, and the trophosome of dictyophora phalloidea is mycelium, the son of dictyophora phalloidea
Entity point volva, stem, four part of indusium and cap, it has often been said that dictyophora phalloidea just refer to its fructification.During Dictyophora rubrovalvata is
The dictyophora phalloidea species of state's special product, is a kind of precious medicine-food two-purpose fungi.Wild Dictyophora rubrovalvata be distributed mainly on Chinese yunnan, four
On corruption soil under the bamboo grove of the provinces and regions such as river, Zhejiang, Guizhou province.Large-scale artificial cultivation is carried out at present.
Dictyophora phalloidea is nutritious, aromatic flavour, and flavour is delicious, is just classified as one of " careless eight delicacies " from ancient times.According to Historical Data Data About, early in
618 years Christian eras, China begin to cultivate and eat dictyophora phalloidea, and using it as a kind of medicinal fungi come treat inflammation, stomach trouble and
Neurogenic disease etc..Dictyophora phalloidea has good nutritive value, is such as beneficial to eyes and cardiovascular system；Also there is pharmacological action
Such as mental-tranquilization, antitumor, anti-oxidant, anti-inflammatory, anticoagulation etc..Dictyophora phalloidea thalline contain rich in protein and amino acid into
Point, such as Dictyophora echino-volvata Zane thalline contains 21 kinds of amino acid, and 8 kinds are human body institute essential amino acid.Dictyophora phalloidea is also containing abundant dimension
Raw element E, beta carotene, thiamine, riboflavin, nicotinic acid, vitamin C, calcium and phosphate etc..
Dictyophora phalloidea has hypotensive, blood sugar, antitumor, strengthens immunologic function and the medical value such as antibacterial, medicinal to dictyophora phalloidea to grind
Study carefully be concentrated mainly on that dictyophora fungus polysaccharide, dictyophora phalloidea agglutinin, dictyophora phalloidea be antibacterial and some small molecule active compositions in terms of.Dictyophora fungus polysaccharide is wide
It is general to be present in the cell membrane of fructification, it is with highly active macromolecular substances, in antitumor, anticoagulation, anti-inflammatory, stimulation
Immune and hypoglycemic aspect has certain curative effect.
Hua Yanglin reports dictyophora phalloidea fructification Jing after extraction with aqueous solution, and filter residue adopts alkali carries, 1M HCl solutions to extract, filtrate
Dictyophora phalloidea Thick many candies Di-1 is obtained after concentrated in vacuo；Filter residue is extracted with 1% NaOH solution again, obtains dictyophora phalloidea after filter vacuum concentration
Thick many candies Di-2.Immune animal experiment shows：Di-1 and Di-2 play the role of different degrees of enhancing immunity.（Hua Yang
Woods, South China Science ＆ Engineering University Ph.D. Dissertation, 2011）.Wang Beibei reports Dictyophora rubrovalvata Thick many candies to superoxide anion freedom
Base, hydroxyl radical free radical, DPPH free radicals have certain scavenging action, to bacterium（Staphylococcus aureus, bacillus subtilis
Bacterium, Escherichia coli）, fungi（Brewer's yeast, apple mould）There is bacteriostasis（Wang Beibei, Shaanxi Normal University's master's degree
Paper, 2012）.Tian Tian reports Dictyophora echino-volvata Zane cap polysaccharide and has certain antioxidation activity in vitro, and to saccharomycete, green grass or young crops
Mould, staphylococcus albus, Bacillus cercus and Escherichia coli have certain inhibitory action（Tian Tian, Shaanxi Normal University
Master thesis, 2012）.Xu Jingjing reports the sulfuric ester and phosphate derivative pair of dictyophora phalloidea fructification water-insoluble polysaccharide
The growth of two kinds of tumour cells of B16 and MCF-7 has stronger inhibitory action, wherein being respectively 40.07% He to the inhibiting rate of B16
28.96%, 25% is less than to the inhibiting rate of MCF-7（Xu Jingjing, Southern Yangtze University's master thesis, 2012）.Wang Jiatang is reported
The isolation and purification method of the water-soluble polysaccharide PD3 that dictyophora phalloidea 80 DEG C of hot water are carried, chemical composition, molecular structure and three spiral structures
As feature（Wang Jiatang, Wuhan University of Technology's master thesis, 2009）.
Patent application publication number discloses " a kind of preparation method of dictyophora fungus polysaccharide ", publication date for the patent of CN1165148
Phase is 1997-11-19, applies for artificial Dalian Inst of Chemicophysics, Chinese Academy of Sciences.Cap, volva of the method using dictyophora phalloidea
With dictyophora phalloidea processing offal or submerged fermentation technology production dictyophora phalloidea vegetative mycelium be raw material, preprocessed, hot-water extraction,
The master operations such as alcohol analysis, dialysis and washing make Thick many candies, and wherein hot-water extraction operation is soaked with water at 80 ~ 100 DEG C
Bubble raw material 1 ~ 10 hour, extractive process can be repeated 2 ~ 5 times, Thick many candies can be made solution use to obtain high purity product
DEAE- cellulose columns carry out chromatography, and collection is obtained high-purity dictyophora fungus polysaccharide containing saccharide portion.
Patent application publication number is disclosed for the patent of CN101029087 " to be had in Dictyophora rubrovalvata volva and suppresses tumour thin
The separation method of the polysaccharide of intracellular growth ", publication date are 2007-09-05, apply for artificial Zhejiang University.
After the method the drying of Dictyophora rubrovalvata volva, will be crushed, water extract-alcohol precipitation separates precipitate and simultaneously dries；By the analysis of drying
Go out thing and be made into the aqueous solution, with alcohol analysis being carried out after the de- albumen of Savege methods, separate precipitate and the water of 12 mg/mL is made into after drying
Solution, is separated in chromatographic column, obtains Dictyophora rubrovalvata volva polysaccharide DRVP1.Dictyophora rubrovalvata volva polysaccharide DRVP1 is to swollen
The growth of oncocyte has obvious inhibitory action.
Patent application publication number discloses a kind of " extraction side of triple helix Dictyophora phalloidea polysaccharide for the patent of CN101503477
Method ", publication date are 2009-08-12, apply for artificial Wuhan University of Technology.The method is specifically：First by crushing after natural bamboo
The degreasing in apparatus,Soxhlet's of sweet-smelling grass organic solvent, then by the residue successively water extraction under 20 ~ 80 DEG C of gradient temperatures, filters
Filtrate, repeats three times, and centrifuging and taking supernatant obtains extract；Extract Jing Sevage methods take off albumen and use H2O2Oxidizing process
After decolouring, respectively with clear water and redistilled water dialysis to remove small molecule, and Jing after DEAE posts remove acidic polysaccharose, solution is cold
It is lyophilized dry, obtain the triple helix Dictyophora phalloidea polysaccharide that form is flocculent white.
Patent application publication number discloses " application of triple spiral Dictyophora phalloidea polysaccharide ", publication date for the patent of CN101502532
Phase is 2009-08-12, applies for artificial Wuhan University of Technology.The triple spiral Dictyophora phalloidea polysaccharide is extracted from natural dictyophora phalloidea and is obtained, point
Son amount is 30 ~ 1,000,000, and with triple helix ordered structure, the monose of triple spiral Dictyophora phalloidea polysaccharide consists of glucose.The invention
Find that triple spiral Dictyophora phalloidea polysaccharide has obvious inhibitory action to S-180 tumour cells by cell experiment, it is swollen with lentinan
Knurl inhibitory action is suitable.
For the patent of CN103044566A, patent application publication number discloses that " antioxidant is more in a kind of dictyophora phalloidea water extraction residue
The preparation method of sugar ", publication date is 2013-04-17, applies for artificial HeFei University of Technology.The method includes that dictyophora phalloidea water extraction is residual
The preparation of slag, the configuration of polysaccharide extraction liquid, the extraction of polysaccharide in dictyophora phalloidea water extraction residue, water-insoluble α in polysaccharide extraction liquid-(1 →
3) removal of-d- glucans, decolourizes, the removal of small molecular weight impurity, and the purifying of polysaccharide is dialysed and freeze-drying, antioxidation activity
Detection etc..
Patent application publication number discloses " a kind of preparation method of dictyophora phalloidea proteoglycan " for the patent of CN103360504A,
Publication date is 2013-10-23, applies for artificial village Asia people etc..The method with dry dictyophora phalloidea as raw material, through coarse crushing, hot water extraction,
Coarse filtration, clarification refined filtration, decolouring, concentrated in vacuo, alcohol precipitation, alcohol hypostasis is dried, crushes, sieving can be prepared by dictyophora phalloidea proteoglycan.Institute
The dictyophora phalloidea proteoglycan of production has an obvious inhibitory action to mouse S-180 tumour cells, and inhibiting rate is 48.12% ~
62.25%。
Japan Patent JP2004262905 discloses a kind of " Claritin or food ", inventor Nakasugi
Toru etc., publication date are 2004-09-24.The raw material of the Claritin is by Pistacia weinmannifolia, dictyophora phalloidea, Radix Glycyrrhizae, snow tea, pepper, sieve
Shop sign in the form of a streamer, cardamom, bolete, bloom bolete, honeysuckle, one or more composition of emblic.The patent is using dictyophora phalloidea as system
A kind of raw material of standby Claritin.
United States Patent (USP) US20080199488A1 discloses the preparation method of a kind of " anti-AIDS drug ", inventor
Tani Michio, publication date are 2009-08-21.The method is with tomonea, kapur, cluster life from pleat umbrella, dictyophora phalloidea, Pu'er
One or more of tea, peppermint and STEVIA REBAUDIANA prepare a kind of anti-AIDS drug for raw material.The patent is anti-as preparing using dictyophora phalloidea
A kind of raw material of AIDS-treating medicine.
United States Patent (USP) US8617567 discloses one kind " to be had the fungi polysaccharide composition of immunological enhancement and its answers
With ", inventor Tang Jian etc., publication date is 2013-12-13.The present invention relates to a kind of with strengthening immunization
The fungi polysaccharide of compound, composition of raw materials is：Mushroom 8 ~ 100, Poria cocos 15 ~ 100, dictyophora phalloidea 10 ~ 200, white fungus 15 ~ 80, bat moth
Paecilomycerol filament 2 ~ 50.The patent is using dictyophora phalloidea as a kind of raw material of the preparation with enhancing immunization health food.
In sum, egg is removed with regard to Dictyophora rubrovalvata fructification as raw material, sequentially passing through hot water extraction, Savege methods at present
In vain, ethanol precipitation, then using " ion-exchange chromatography-gel filtration chromatography-ultra-filtration and separation-gel filtration chromatography " four
Step separation is obtained after being isolated and purified, and carries out mouse macrophage immunity work with the Dictyophora rubrovalvata polysaccharide after isolating and purifying
Property experiment report there is no.And the invention provides a kind of preparation method with immunocompetent novel red support dictyophora fungus polysaccharide and
Its application, is that the deep processing of dictyophora phalloidea and efficient utilization have established theoretical foundation and technical foundation.
The content of the invention
The technical problem to be solved is the polysaccharide in Dictyophora rubrovalvata fructification to be carried out effectively to extract and is divided
From purifying, and to isolating and purifying after the immunocompetence of Dictyophora rubrovalvata polysaccharide study, there is provided it is a kind of have it is immunocompetent
Novel red support dictyophora fungus polysaccharide and its preparation method and application.
A kind of preparation method with immunocompetent novel red support dictyophora fungus polysaccharide, with Dictyophora rubrovalvata as raw material, Jing Guoru
It is prepared by lower section method：Hot water extraction, Savege method removing proteins, ethanol precipitation, then using ion-exchange chromatography, solvent resistant column
Chromatography, ultra-filtration and separation, four step separation of gel filtration chromatography are obtained after isolating and purifying.Methods described comprises the following steps：
（1）By Dictyophora rubrovalvata fructification in 40 ~ 70 DEG C of 0.5 ~ 4 h of drying, then crushed with powder beater；
（2）1 ~ 4 h of hot water extraction, solid-liquid ratio is 1: 20 ~ 1 :40 g/mL, temperature are 60 ~ 100 DEG C, leaching
Number of times is carried for 1 ~ 3 time；
（3）De- albumen is carried out using Sevage methods to process 6 ~ 10 times to hot water extraction's thing, upper strata polysaccharide solution is collected right
After concentrated and be dried, obtain the Thick many candies solution after deproteination；
（4）Ethanol solution is added toward the Thick many candies solution after deproteination, is stood overnight at 0 ~ 4 DEG C, 4000 ~
8000 r/min are centrifuged 10 ~ 20 min, collect sediment, Dictyophora rubrovalvata Thick many candies are obtained after vacuum freeze drying；
（5）Isolated and purified using four step separations：Purification procedures are：Initially with ion-exchange chromatography pair
Step（4）The Dictyophora rubrovalvata Thick many candies for obtaining are purified, and the NaCl solution of deionized water and 0.05 ~ 2 mol/L is carried out
Gradient elution, collects eluent, freeze-drying after dialysis, concentration；Then gel filtration chromatography is adopted, is washed with deionized water
De- agent, freeze-drying after dialysis, concentration；Purified using Ultrafiltration Membrane again, collected permeate and trapped fluid respectively, it is dense
Freeze-drying after contracting；Gel filtration chromatography is finally adopted, eluant, eluent is made with deionized water, freeze-drying after dialysis, concentration is obtained
To Dictyophora rubrovalvata polysaccharide.
In said method, step（4）In, the volume fraction of used ethanol is 65% ~ 95%（It is preferred that 80% ~ 95%）
Or absolute ethyl alcohol is used, the addition of ethanol is 1 ~ 5 times of Thick many candies liquor capacity.
In said method, step（5）In, using ion-exchange chromatography, gel filtration chromatography, ultra-filtration and separation, gel
Filter four step separation of column chromatography to be isolated and purified, the embodiment of four step separations is the one kind in following manner：
Mode one, using DEAE-52 or DEAE Sepharose FF anion exchange chromatographies, Sephadex G-
200 or Sephadex G-150 gel filtration chromatographies, ultra-filtration and separation, Sephadex G-100 gel filtration chromatographies；
Mode two, using 52 anion exchange chromatography of DE 52 or QA, Sephadex G-200 or Sephadex G-
150 gel filtration chromatographies, ultra-filtration and separation, Sephadex G-100 gel filtration chromatographies；
Mode three, DEAE-52 or Sepharose XL anion exchange chromatographies, Sephadex G-200 or
Sepharose Fast Flow gel filtration chromatographies, ultra-filtration and separation, Superose 12 or Bio-Gel P-4 gel filtrations
Column chromatography；
Mode four, Sepharose XL or CM52 anion exchange chromatographies, Sepharose 2B or Sepharose 4B
Gel filtration chromatography, ultra-filtration and separation, Sepharose 6B or Sephacryl S-400 HR gel filtration chromatographies.Dialysis bar
Part is 0 ~ 4 DEG C, 48 ~ 60 h.
In said method, step（5）Ultra-filtration and separation in, the retention relative molecular mass of the milipore filter for being adopted for 20 ~
1000 kDa。
It is a kind of that there is immunocompetent novel red support dictyophora fungus polysaccharide, by galactolipin, three kinds of monose groups of glucose and mannose
Into molecular weight is homogeneous, purity is high and good stability.Cell experiment proves that the novel red support dictyophora fungus polysaccharide can be by activation
Nitric oxide in mouse macrophage RAW264.7（NO）, tumor necrosis factor-alpha（TNF-α）And interleukin-6（IL-6）'s
Expression, so as to improve the immunocompetence of body
A kind of medicine system that immune deficiency class disease and tumour are applied to immunocompetent novel red support dictyophora fungus polysaccharide
It is standby.
It is a kind of to be applied to prepare the food or the health care that improve immunologic function with immunocompetent novel red support dictyophora fungus polysaccharide
Food.
The novel red support dictyophora fungus polysaccharide that obtained of the present invention has the advantages that molecular weight is homogeneous, purity is high, and also has
Good immunologic enhancement, can be used for the auxiliary treatment of immune deficiency class disease and tumour, it can also be used to improve immunologic function
Food or health food.The preparation process is simple of the present invention is reasonable, environmental protection, and is easily achieved industrialized production.
Description of the drawings
Fig. 1 is Dictyophora rubrovalvata polysaccharide ion-exchange chromatography elution curve.
Fig. 2 is Dictyophora rubrovalvata polysaccharide DP1 gel filtration chromatography elution curves.
Fig. 3 is Dictyophora rubrovalvata polysaccharide DP2 gel filtration chromatography elution curves.
Infrared spectrograms of the Fig. 4 for Dictyophora rubrovalvata polysaccharide DP1.
Infrared spectrograms of the Fig. 5 for Dictyophora rubrovalvata polysaccharide DP2.
Monose composition chromatography of ions figure of the Fig. 6 for Dictyophora rubrovalvata polysaccharide DP1.
Monose composition chromatography of ions figure of the Fig. 7 for Dictyophora rubrovalvata polysaccharide DP2.
Fig. 8 is impact block diagrams of the variable concentrations Dictyophora rubrovalvata polysaccharide DP1 to mouse macrophage NO secretory volumes.
Fig. 9 is impact block diagrams of the variable concentrations Dictyophora rubrovalvata polysaccharide DP2 to mouse macrophage NO secretory volumes.
Figure 10 is impact block diagrams of the variable concentrations Dictyophora rubrovalvata polysaccharide DP1 to mouse macrophage TNF-α secretion amount.
Figure 11 is impact block diagrams of the variable concentrations Dictyophora rubrovalvata polysaccharide DP2 to mouse macrophage TNF-α secretion amount.
Figure 12 is impact block diagrams of the variable concentrations Dictyophora rubrovalvata polysaccharide DP1 to mouse macrophage IL-6 secretory volumes.
Figure 13 is impact block diagrams of the variable concentrations Dictyophora rubrovalvata polysaccharide DP2 to mouse macrophage IL-6 secretory volumes.
Specific embodiment
It is described further below in conjunction with enforcement of the specific embodiment to the present invention, but the enforcement not limited to this of the present invention.
Embodiment 1：
（1）100 g Dictyophora rubrovalvatas fructifications are dried into 2 h at 40 DEG C, are then crushed with powder beater；
（2）100 DEG C of 1 h of hot water extraction, solid-liquid ratio is 1:40 g/mL, extracting times are 1 time；4000 r/min are centrifuged
15 min, collect supernatant rotary evaporation reduced pressure concentration at 60 DEG C.
（3）De- albumen is carried out using Sevage methods to process to hot water extraction's thing, the compound method of Sevage reagents be chloroform,
N-butanol by volume 4:1 mixing；The Deproteinated method of Sevage methods is polysaccharide and Sevage reagent by volume 1:1 mixes
Closing, shaking 20 min, 3000 r/min are centrifuged 10 min, take out upper strata polysaccharide solution, dictyophora phalloidea Thick many candies are obtained after being dried；De- egg
White process is repeated 10 times.
（4）The absolute ethyl alcohol of 3 times of volumes is added toward the Thick many candies solution after deproteination, is stood overnight at 4 DEG C；
Then 20 min are centrifuged under 4000 r/min, collect sediment, after vacuum freeze drying, obtain dictyophora phalloidea Thick many candies.
（5）Take 100 mg steps（4）The Thick many candies for obtaining are dissolved in 10 mL deionized waters again, using DEAE-52 anion
Exchange column chromatography to be purified, successively using deionized water, the NaCl solution of 0.05,0.1,0.2,0.3 and 0.5 mol/L
Gradient elution is carried out, eluent flow rate is 0.5 mL/min；The component of wherein deionized water wash-out is named as DP1 components, uses
The component of the NaCl solution wash-out of 0.05 mol/L is named as DP2 components；Dialyse at 4 DEG C 48 h, then carries out freezing dry
It is dry.
（6）20 mg steps are taken respectively（5）Dictyophora rubrovalvata polysaccharide DP1, DP2 component for obtaining, be dissolved in respectively 4 mL go from
In sub- water, purified using Sephadex G-200 gel filtration chromatographies, using deionized water as eluant, eluent, flow velocity is
0.5 mL/min, freeze-drying after concentration obtain just separating dictyophora fungus polysaccharide.
（7）Using Ultrafiltration Membrane to step（6）The polysaccharide component for obtaining is separated, and it is super that DP1 components are adopted
The retention relative molecular mass of filter membrane is 1000 kDa, collects trapped fluid, carries out next step purifying after concentration, freeze-drying；DP2
The retention relative molecular mass of the milipore filter adopted by component is collected permeate, is carried out down after concentration, freeze-drying for 20 kDa
One step is purified.
（8）20 mg steps are taken respectively（7）The Dictyophora rubrovalvata polysaccharide DP1 and DP2 component for obtaining, is dissolved in 4 mL respectively and goes
In ionized water, purified using Sephadex G-100 gel filtration chromatographies, using deionized water as eluant, eluent, flow velocity
For 0.5 mL/min, freeze-drying is carried out after concentration and obtains Dictyophora rubrovalvata polysaccharide finished product.Dictyophora rubrovalvata polysaccharide ion exchange column layer
Analysis elution curve is shown in Fig. 1；Dictyophora rubrovalvata polysaccharide DP1 gel filtration chromatography elution curves are shown in Fig. 2；Dictyophora rubrovalvata polysaccharide DP2 coagulates
Glue Filter column chromatography elution curves are shown in Fig. 3.
Embodiment 2：
（1）100 g Dictyophora rubrovalvatas fructifications are dried into 0.5 h at 70 DEG C, are then crushed with powder beater；
（2）90 DEG C of 2 h of hot water extraction, solid-liquid ratio is 1:20 g/mL, extracting times are 3 times；4000 r/min are centrifuged
15 min, collect supernatant rotary evaporation reduced pressure concentration at 60 DEG C.
（3）De- albumen is carried out using Sevage methods to process to hot water extraction's thing, the compound method of Sevage reagents be chloroform,
N-butanol by volume 4:1 mixing；The Deproteinated method of Sevage methods is polysaccharide and Sevage reagent by volume 1:1 mixes
Closing, shaking 20 min, 3000 r/min are centrifuged 10 min, take out upper strata polysaccharide solution, dictyophora phalloidea Thick many candies are obtained after being dried；De- egg
White process is repeated 10 times.
（4）The absolute ethyl alcohol of 3 times of volumes is added toward the Thick many candies solution after deproteination, is stood overnight at 4 DEG C；
Then 10 min are centrifuged under 8000 r/min, collect sediment, after vacuum freeze drying, obtain dictyophora phalloidea Thick many candies.
（5）Take 100 mg steps（4）The Thick many candies dissolving for obtaining in 10 mL deionized waters, is handed over using 52 anion of QA again
Change column chromatography to be purified, successively using deionized water, the NaCl solution of 0.05,0.1,0.5,1 mol/L carries out gradient and washes
De-, eluent flow rate is 0.5 mL/min；The component of wherein deionized water wash-out is named as DP1 components, with 0.1 mol/L's
The component of NaCl solution wash-out is named as DP2 components；Dialyse at 4 DEG C 48 h, then carries out freeze-drying.
（6）20 mg steps are taken respectively（5）Dictyophora rubrovalvata polysaccharide DP1, DP2 component for obtaining, be dissolved in respectively 4 mL go from
In sub- water, purified using Sephadex G-150 gel filtration chromatographies, using deionized water as eluant, eluent, flow velocity is
0.5 mL/min, carries out freeze-drying and obtains just separating dictyophora fungus polysaccharide after concentration.
（7）Using Ultrafiltration Membrane to step（6）The polysaccharide component for obtaining is separated, and it is super that DP1 components are adopted
The retention relative molecular mass of filter membrane is 1000 kDa, collects trapped fluid, carries out next step purifying after concentration, freeze-drying；DP2
The retention relative molecular mass of the milipore filter adopted by component is collected permeate, is carried out down after concentration, freeze-drying for 30 kDa
One step is purified.
（8）20 mg steps are taken respectively（7）The Dictyophora rubrovalvata polysaccharide DP1 and DP2 component for obtaining, is dissolved in 4 mL respectively and goes
In ionized water, purified using Sephadex G-100 gel filtration chromatographies, using deionized water as eluant, eluent, flow velocity
For 0.5 mL/min, freeze-drying is carried out after concentration and obtains Dictyophora rubrovalvata polysaccharide finished product（Dictyophora rubrovalvata polysaccharide ion exchange column layer
Analysis elution curve can be found in Fig. 1；Dictyophora rubrovalvata polysaccharide DP1 gel filtration chromatography elution curves can be found in Fig. 2；Dictyophora rubrovalvata is more
Sugared DP2 gel filtration chromatographies elution curve can be found in Fig. 3）.
Embodiment 3：
（1）100 g Dictyophora rubrovalvatas fructifications are dried into 3 h at 60 DEG C, are then crushed with powder beater；
（2）100 DEG C of 4 h of hot water extraction, solid-liquid ratio is 1:30 g/mL, extracting times are 3 times；8000 r/min are centrifuged
10 min, collect supernatant rotary evaporation reduced pressure concentration at 60 DEG C.
（3）De- albumen is carried out using Sevage methods to process to hot water extraction's thing, the compound method of Sevage reagents be chloroform,
N-butanol by volume 4:1 mixing；The Deproteinated method of Sevage methods is polysaccharide and Sevage reagent by volume 1:1 mixes
Closing, shaking 20 min, 3000 r/min are centrifuged 10 min, take out upper strata polysaccharide solution, dictyophora phalloidea Thick many candies are obtained after being dried；De- egg
White process is repeated 10 times.
（4）The absolute ethyl alcohol of 3 times of volumes is added toward the Thick many candies solution after deproteination, is stood overnight at 4 DEG C；
Then 15 min are centrifuged under 4000 r/min, collect sediment, after vacuum freeze drying, obtain dictyophora phalloidea Thick many candies.
（5）Take 100 mg steps（4）The Thick many candies for obtaining are dissolved in 10 mL deionized waters again, using Sepharose XL
Anion exchange chromatography is purified, successively using deionized water, 0.05,0.1,0.2,0.3 and 0.5 mol/L's
NaCl solution carries out gradient elution, and eluent flow rate is 0.5 mL/min；The component of wherein deionized water wash-out is named as DP1
Component, the component eluted with the NaCl solution of 0.05 mol/L are named as DP2 components；Dialyse at 4 DEG C 48 h, then carries out
Freeze-drying.
（6）20 mg steps are taken respectively（5）Dictyophora rubrovalvata polysaccharide DP1, DP2 component for obtaining, be dissolved in respectively 4 mL go from
In sub- water, purified using Sepharose Fast Flow gel filtration chromatographies, using deionized water as eluant, eluent,
Flow velocity is 0.5 mL/min, carries out freeze-drying and obtain just separating dictyophora fungus polysaccharide after concentration.
（7）Using Ultrafiltration Membrane to step（6）The polysaccharide component for obtaining is separated, and it is super that DP1 components are adopted
The retention relative molecular mass of filter membrane is 1000 kDa, collects trapped fluid, carries out next step purifying after concentration, freeze-drying；DP2
The retention relative molecular mass of the milipore filter adopted by component is collected permeate, is carried out down after concentration, freeze-drying for 30 kDa
One step is purified.
（8）20 mg steps are taken respectively（7）The Dictyophora rubrovalvata polysaccharide DP1 and DP2 component for obtaining, is dissolved in 4 mL respectively and goes
In ionized water, purified using Bio-Gel P-4 gel filtration chromatographies, using deionized water as eluant, eluent, flow velocity is
0.5 mL/min, carries out freeze-drying and obtains Dictyophora rubrovalvata polysaccharide finished product after concentration（Dictyophora rubrovalvata polysaccharide ion-exchange chromatography
Elution curve can be found in Fig. 1；Dictyophora rubrovalvata polysaccharide DP1 gel filtration chromatography elution curves can be found in Fig. 2；Dictyophora rubrovalvata polysaccharide
DP2 gel filtration chromatography elution curves can be found in Fig. 3）.
The Dictyophora rubrovalvata polysaccharide as obtained in above example 1 carries out Structural Identification and activity analysis by the following method, real
The result for applying example 2 and 3 is similar to Example 1.
1st, the infrared spectrum analysis of Dictyophora rubrovalvata polysaccharide
Each 2.0 mg of Dictyophora rubrovalvata polysaccharide DP1 and DP2 is taken, the appropriate KBr powder being dried is separately added into, is well mixed,
The uniform rear transparent compressing tablet for adding compressing tablet in press mold, making that about 1 mm of thickness, diameter are about 10 mm or so is ground in agate mortar.
Use FT-IR（FTIS, model Nexus, Thermo Nicolet companies of the U.S.）Respectively to DP1 and
DP2 compressing tablets are in 400 ~ 4000 cm-1Interval is scanned, and gathers the infrared absorption pattern of sample, uses Nexus systems soft wares
Process is analyzed to collection of illustrative plates.The infrared absorption spectroscopy of dictyophora fungus polysaccharide DP1 and DP2 is respectively as shown in Figure 4 and Figure 5.
From the infrared spectrogram of Fig. 4 Dictyophora rubrovalvata polysaccharide DP1, in 3335.69 cm-1Nearby have one it is strong and wide
Absworption peak, this is belonging to the stretching vibration characteristic absorption peak of hydroxyl（3600 ~ 3200 cm-1）；In 2926.33 cm-1Neighbouring
Absworption peak is belonging to C-H stretching vibration absworption peaks（2950 ~ 2850 cm-1）；In 1425.46 cm-1With 1374.38 cm-1It is attached
Near absworption peak is belonging to C-H in-plane bending vibration absworption peaks（1470 ~ 1430 cm-1；1380 ~ 1360 cm-1）；By with
Upper several groups of absworption peaks can be with the preliminary judgement material as saccharide compound；1727.93 cm-1Neighbouring absworption peak is belonging to aldehyde, ketone
The stretching vibration absworption peak of C=O double bonds（1750 ~ 1710 cm-1）, show containing aldehyde radical or ketone carbonyl；1648.01 cm-1It is attached
Near absworption peak is belonging to the stretching vibration absworption peak of C=C double bonds（1680 ~ 1620 cm-1）, can speculate that the carbohydrate contains alkene two
Alcohol structure；1074.32 cm-1With 1040.25 cm-1Neighbouring absworption peak is belonging to C-O stretching vibration absworption peaks（1080 ~
1030 cm-1）, these absworption peaks are the characteristic absorption peaks of the C-O keys of the ehter bond C-O-C and alcohols on pyranoid ring；1750 ~
1700 cm-1Without absworption peak, show that the polysaccharide does not contain uronic acid.
From the infrared spectrogram of Fig. 5 Dictyophora rubrovalvata polysaccharide DP2, in 3374.38 cm-1Nearby have one it is strong and wide
Absworption peak, this is belonging to the stretching vibration characteristic absorption peak of hydroxyl（3600 ~ 3200 cm-1）；In 2928.90 cm-1Neighbouring
Absworption peak is belonging to C-H stretching vibration absworption peaks（2950 ~ 2850 cm-1）；In 1405.93 cm-1Neighbouring absworption peak is category
In C-H in-plane bending vibration absworption peaks；By more than, three groups of absworption peaks can be with the preliminary judgement material as saccharide compound；
1640.93 cm-1Neighbouring absworption peak is belonging to the stretching vibration absworption peak of C=C double bonds（1680 ~ 1620 cm-1）, can push away
Survey the carbohydrate and contain enediol structure；1250 ~ 950 cm-1Neighbouring absworption peak is belonging to C-O stretching vibration absworption peaks, these
Absworption peak is the characteristic absorption peak of the C-O keys of the ehter bond C-O-C and alcohols on pyranoid ring；In 1750 ~ 1700 cm-1Without absorption
Peak, shows that the polysaccharide does not contain uronic acid.
2nd, the monosaccharide composition analysis of Dictyophora rubrovalvata polysaccharide
The monose composition of Dictyophora rubrovalvata polysaccharide DP1 and DP2 is determined using ion chromatograph, 20.0 mg dictyophora phalloidea are weighed respectively
Polysaccharide DP1 and DP2, are dissolved in 4.0 mL trifluoroacetic acids respectively, react 6 h at 105 DEG C.Unnecessary three are removed with rotary evaporation
Fluoroacetic acid.Then residue is re-dissolved in methyl alcohol, is concentrated with rotary evaporation.Finally residue is dissolved in ultrapure
In water, ion chromatograph is used（Dionex ICS-3000, Dai An companies of the U.S.）It is measured.Chromatography of ions condition is：Column temperature 30
℃；Chromatographic column:Analytical column is CarboPac PA 20（2 × 250 mm）；Detector：Pulsed amperometry, gold electrode；
Leacheate is constituted：A. ultra-pure water；B.0.2 mol/L NaOH；C.0.5 mol/L NaAC；D.0.02 mol/L NaOH.Wash-out
Program：0 ~ 2 min leacheates consist of 100% B；2 ~ 20 min are 10% D+90% A；20 ~ 40 min are 100%.By
The monose composition chromatography of ions figure of Fig. 6 Dictyophora rubrovalvata polysaccharide DP1 knows that the Dictyophora rubrovalvata polysaccharide is by galactolipin, glucose and sweet dew
Three kinds of monose compositions of sugar, the mol ratio of three is galactolipin：Glucose：Mannose=1: 3.97: 2.05.By the red support bamboos of Fig. 7
The monose composition chromatography of ions figure of sweet-smelling grass polysaccharide DP2 knows that the Dictyophora rubrovalvata polysaccharide is by galactolipin, three kinds of monose of glucose and mannose
Composition, the mol ratio of three is galactolipin：Glucose：Mannose=1: 4.73: 1.64.
3rd, the immunocompetence of Dictyophora rubrovalvata polysaccharide is determined
Experimental cell：Mouse macrophage RAW264.7, purchased from medical college of Zhongshan University.
Experiment reagent：The primary section's minimum essential medium of Dole（DMEM）, streptomysin and penicillin it is public purchased from U.S. Gibco
Department；Hyclone is purchased from Zhejiang Tian Hang bio tech ltd；Lipopolysaccharides（LPS）Purchased from Sigma Co., USA；NO detection examinations
Agent box builds up Bioengineering Research Institute purchased from Nanjing；Mouse IL-6 ELISA kits, mouse TNF-α ELISA kit are purchased from
Xin Bosheng bio tech ltd.
3.1 cell culture
The mouse macrophage primary section's minimum essential medium of Dole（DMEM）Cultivated, and added 10% tire ox
Serum（FBS）, 100 μ g/mL streptomysin and 100 U/mL penicillin, be put into 37 DEG C, 5% CO2Quiescent culture in incubator.
Take the logarithm growth period mouse macrophage RAW264.7, and cell concentration is adjusted to 1 × 10 by dilution6Individual/mL, is inoculated in 96
In orifice plate, 24 h of incubation change a nutrient solution.
The measure of 3.2 NO, tumor necrosis factor TNF-alpha and Interleukin-6 secretory volume
Test is set to zeroing group, experimental group, control group and positive controls.Wherein zeroing group is not added with cell, adds 100 μ
L nutrient solutions；Experimental group, control group and positive controls, add 100 μ L cell suspending liquids per hole.Experimental group is separately added into 125,
The 100 μ L of Dictyophora rubrovalvata polysaccharide solution of 250,500,1,000 tetra- concentration gradients of μ g/mL, add 50 μ in positive controls
The lipopolysaccharides of g/mL（LPS）100 μ L of liquid（Liquid and positive control are prepared with not serum-containing media）, each different groups
Sample is divided to set parallel 6 hole.To prevent microbiological contamination, with the sealed membrane Jing after alcohol wipe seal flat board place into 37 DEG C, 5%
CO224 h are incubated in incubator.According to the detection method that kit manufacturer provides, respectively with NO detection kits, mouse TNF-α
NO, TNF-α, the secretory volume of IL-6 in ELISA kit, mouse IL-6 ELISA kits detection cell supernatant.For red
Support dictyophora fungus polysaccharide DP1 samples, NO in supernatant, TNF-α, IL-6 content respectively as shown in Fig. 8, Figure 10, Figure 12.For red support
Dictyophora fungus polysaccharide DP2 samples, NO in supernatant, TNF-α, IL-6 content respectively as shown in Fig. 9, Figure 11, Figure 13.As seen from the figure,
Dictyophora fungus polysaccharide DP1 and DP2 in the range of 125 ~ 1000 μ g/mL, NO in cell supernatant, TNF-α, IL-6 content it is notable
Higher than control group（Control）, and dosage effect is presented, with the rising of polysaccharide concentration, NO, TNF-α, the content of IL-6
Raise.Therefore DP1 and DP2 can significantly improve NO, TNF-α, the content of IL-6 in mouse macrophage RAW264.7 supernatants,
And the immunocompetence of DP2 components is better than DP1 components.
Claims (6)
1. a kind of preparation method with immunocompetent Dictyophora rubrovalvata polysaccharide, it is characterised in that with Dictyophora rubrovalvata as raw material, Jing
Cross following method to prepare：Hot water extraction, Savege method removing proteins, ethanol precipitation, then using ion-exchange chromatography, gel mistake
Filter column chromatography, ultra-filtration and separation, four step separation of gel filtration chromatography are obtained after isolating and purifying；
Methods described comprises the following steps：
（1）By Dictyophora rubrovalvata fructification in 40 ~ 70 DEG C of 0.5 ~ 4 h of drying, then crushed with powder beater；
（2）1 ~ 4 h of hot water extraction, solid-liquid ratio is 1: 20 ~ 1 :40 g/mL, temperature are 60 ~ 100 DEG C, extraction time
Number is 1 ~ 3 time；
（3）De- albumen is carried out using Sevage methods to process 6 ~ 10 times to hot water extraction's thing, upper strata polysaccharide solution is collected and then is entered
Row is concentrated and is dried, and obtains the Thick many candies after deproteination；
（4）Ethanol solution is added toward the solution of the Thick many candies after deproteination, is stood overnight at 0 ~ 4 DEG C, 4000 ~
8000 r/min are centrifuged 10 ~ 20 min, collect sediment, Dictyophora rubrovalvata Thick many candies are obtained after vacuum freeze drying；
（5）Isolated and purified using four step separations：Purification procedures are：Initially with ion-exchange chromatography to step
（4）The Dictyophora rubrovalvata Thick many candies for obtaining are purified, and the NaCl solution of deionized water and 0.05 ~ 2 mol/L carries out gradient
Wash-out, collects the eluent DP2 of the eluent DP1 and NaCl solution of deionized water, freeze-drying after dialysis, concentration respectively；So
After gel filtration chromatography is respectively adopted, eluant, eluent is made with deionized water, freeze-drying after dialysis, concentration；It is respectively adopted again super
Filter isolation technics is purified, and DP1 collects trapped fluid, and DP2 collects freeze-drying after permeate concentration；Gel is respectively adopted finally
Column chromatography is filtered, eluant, eluent is made with deionized water, freeze-drying after dialysis, concentration obtains Dictyophora rubrovalvata polysaccharide；
Step（5）In, using ion-exchange chromatography, gel filtration chromatography, ultra-filtration and separation, four step of gel filtration chromatography point
Isolated and purified from method, the embodiment of four step separations is the one kind in following manner：
Mode one, using DEAE-52 or DEAE Sepharose FF anion exchange chromatographies, Sephadex G-200 or
Sephadex G-150 gel filtration chromatographies, ultra-filtration and separation, Sephadex G-100 gel filtration chromatographies；
Mode two, using 52 anion exchange chromatographies of QA, Sephadex G-200 or Sephadex G-150 solvent resistant columns
Chromatography, ultra-filtration and separation, Sephadex G-100 gel filtration chromatographies；
Mode three, DEAE-52 or Sepharose XL anion exchange chromatographies, Sephadex G-200 or Sepharose
Fast Flow gel filtration chromatographies, ultra-filtration and separation, Superose 12 or Bio-Gel P-4 gel filtration chromatographies；
Mode four, Sepharose XL or CM52 anion exchange chromatographies, Sepharose 2B or Sepharose 4B gels
Filter column chromatography, ultra-filtration and separation, Sepharose 6B or Sephacryl S-400 HR gel filtration chromatographies.
2. preparation method according to claim 1, it is characterised in that step（4）In, the volume fraction of used ethanol
65% ~ 95% or to use absolute ethyl alcohol, 1 ~ 5 times for Thick many candies liquor capacity of the addition of ethanol.
3. preparation method according to claim 1, it is characterised in that step（5）Ultra-filtration and separation in, the ultrafiltration for being adopted
The retention relative molecular mass of film is 20 ~ 1000 kDa.
4. the preparation method by described in any one of claim 1-3 prepare with immunocompetent Dictyophora rubrovalvata polysaccharide.
5. the Dictyophora rubrovalvata polysaccharide described in claim 4 is applied to the medicine preparation of immune deficiency class disease and tumour.
6. the Dictyophora rubrovalvata polysaccharide described in claim 4 is applied to prepare the food for improving immunologic function.
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