CN104262500A - Novel Dictyophora rubrovolvata polysaccharide with immunocompetence, and preparation method and application thereof - Google Patents
Novel Dictyophora rubrovolvata polysaccharide with immunocompetence, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a novel Dictyophora rubrovolvata polysaccharide with immunocompetence, and a preparation method and application thereof. The method uses Dictyophora rubrovolvata as the raw material and comprises the following steps: leaching with hot water, removing proteins by a Savege process, carrying out ethanol precipitation, and carrying out separation and purification by ion-exchange column chromatography, gel filtration chromatography, ultrafiltration separation and gel filtration chromatography. The preparation technique is reasonable and simple, and can easily implement industrialized mass production. The novel Dictyophora rubrovolvata polysaccharide has favorable immunological enhancement action, and can be used as an auxiliary therapeutic drug for tumors and other immunocompromised diseases or as a functional health food or food capable of enhancing immunity.
Description
Technical field
The present invention relates to a kind of immunocompetence polysaccharide and its preparation method and application, be specifically related to one and there is immunocompetent Novel red holder dictyophora fungus polysaccharide and its preparation method and application.
Background technology
Dictyophora phalloidea (
dictyophora indusiata), another name " bamboo fungus ", be a general name large class dictyophora phalloidea being belonged to fungi, be under the jurisdiction of Mycophyta (
eumycota), Basidiomycotina (
basidiomycotina), Gasteromycete (
gasteromycetes), Phallales (
phallales), Phallaceae (
phallaceae).
Dictyophora phalloidea is the hidden colored mushroom of one colonizing in withered bamboo root, and shaped slightly is like netted extra dry white wine snakeskin, and it has bottle-green cap, the columned stem of snowy white, and peach egg type volva has on stem top and encloses
Careful pure white netted skirt spreads out downwards from cap, is called " snow skirt fairy maiden ", " flower of mountain delicacy ", " flower of fungi ", " in bacterium queen " by people.Dictyophora phalloidea can be divided into mycelium and sporophore two portions, and the nourishing body of dictyophora phalloidea is mycelium, and the sporophore of dictyophora phalloidea divides volva, stem, indusium and cap four parts, and the dictyophora phalloidea that it has often been said just refers to its sporophore.Dictyophora rubrovalvata is the dictyophora phalloidea kind of special product of China, is a kind of medicine-food two-purpose fungi of preciousness.Wild Dictyophora rubrovalvata is mainly distributed on the corruption soil under the provinces and regions bamboo groves such as Chinese yunnan, Sichuan, Zhejiang, Guizhou province.Carry out large-scale artificial culture 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, as far back as 618 years Christian eras, China just starts cultivation and edible dictyophora phalloidea, and it is treated inflammation, stomach trouble and nervous system disease etc. as a kind of medicinal fungi.Dictyophora phalloidea has good nutritive value, is such as of value to eyes and cardiovascular systems; Also there is pharmacological action as tranquilizing by nourishing the heart, antitumor, anti-oxidant, anti-inflammatory, anticoagulation etc.Dictyophora phalloidea thalline contains rich in protein and aminoacid component, and such as Dictyophora echino-volvata Zane thalline contains 21 seed amino acids, and 8 kinds is human body institute indispensable amino acid.Dictyophora phalloidea is also containing abundant vitamin-E, β-carotene, VitB1, riboflavin, nicotinic acid, vitamins C, calcium and phosphoric acid salt etc.
Dictyophora phalloidea has hypotensive, blood sugar, antitumor, strengthens immunologic function and the pharmaceutical use such as antibacterial, mainly concentrates on antibacterial and some the small molecule active composition aspects of dictyophora fungus polysaccharide, dictyophora phalloidea lectin, dictyophora phalloidea to dictyophora phalloidea medicinal study.Dictyophora fungus polysaccharide is extensively present in the cell walls of sporophore, is to have highly active macromolecular substance, antitumor, anticoagulation, anti-inflammatory, immune stimulatory and hypoglycemic in have certain curative effect.
Hua Yanglin reports dictyophora phalloidea sporophore after extraction with aqueous solution, and filter residue adopts alkali to carry, and 1M HCl solution extracts, and obtains dictyophora phalloidea Crude polysaccharides Di-1 after filter vacuum is concentrated; Filter residue extracts by 1% NaOH solution again, obtains dictyophora phalloidea Crude polysaccharides Di-2 after filter vacuum is concentrated.Immune animal experiment shows: Di-1 and Di-2 all has the effect of enhancing immunity in various degree.(Hua Yanglin, South China Science & Engineering University Ph.D. Dissertation, 2011).Wang Beibei reports Dictyophora rubrovalvata Crude polysaccharides and has certain scavenging(action) to ultra-oxygen anion free radical, hydroxyl radical free radical, DPPH free radical, bacteriostatic action (Wang Beibei is all had to bacterium (streptococcus aureus, subtilis, intestinal bacteria), fungi (cereuisiae fermentum, apple mould), Shaanxi Normal University's master thesis, 2012).Tian Tian reports Dictyophora echino-volvata Zane cap polysaccharide and has certain antioxidation activity in vitro, and all there is certain restraining effect (Tian Tian to yeast, Penicillium notatum, Staphylococcus albus, bacillus cereus and intestinal bacteria, Shaanxi Normal University's master thesis, 2012).Xu Jingjing reports the sulfuric ester of dictyophora phalloidea sporophore water-insoluble polysaccharide and the growth of phosphate derivative to B16 and MCF-7 two kinds of tumour cells has stronger restraining effect, wherein 40.07% and 28.96% are respectively to the inhibiting rate of B16, to the inhibiting rate of MCF-7 lower than 25%(Xu silently, Southern Yangtze University's master thesis, 2012).Wang Jiatang reports the separation purification method of the water-soluble polysaccharide PD3 that dictyophora phalloidea 80 DEG C of hot water are carried, chemical constitution, molecular structure and triple helical conformational characteristic (Wang Jiatang, Wuhan University of Technology's master thesis, 2009).
Patent application publication number is patent discloses " a kind of preparation method of dictyophora fungus polysaccharide " of CN1165148, and publication date is 1997-11-19, and application people is Dalian Inst of Chemicophysics, Chinese Academy of Sciences.The method utilizes the cap of dictyophora phalloidea, volva and the offal of dictyophora phalloidea processing or the dictyophora phalloidea vegetative mycelium of submerged fermentation technology production to be raw material, analyse through pre-treatment, hot-water extraction, alcohol, to dialyse and the master operation such as washing makes Crude polysaccharides, wherein hot-water extraction operation carries out immersion raw material 1 ~ 10 hour with water at 80 ~ 100 DEG C, extractive process can carry out 2 ~ 5 times repeatedly, Crude polysaccharides can be made solution DEAE-cellulose column carry out chromatographic separation for obtaining high purity product, collect and can obtain high-purity dictyophora fungus polysaccharide containing saccharide portion.
Patent application publication number is patent discloses " separation method in Dictyophora rubrovalvata volva with the polysaccharide of inhibition tumor cell growth " of CN101029087, and publication date is 2007-09-05, and application people is Zhejiang University.
The method is by after the drying of Dictyophora rubrovalvata volva, pulverizing, and water extract-alcohol precipitation, is separated precipitate and also dries; The precipitate of oven dry being made into the aqueous solution, analysing with carrying out alcohol after Savege method deproteinated, be separated the aqueous solution being made into 12 mg/mL after precipitate is also dried, be separated in chromatography column, obtain Dictyophora rubrovalvata volva polysaccharide DRVP1.This Dictyophora rubrovalvata volva polysaccharide DRVP1 growth to tumour cell has obvious restraining effect.
Patent application publication number is patent discloses " a kind of extracting method of triple helix Dictyophora phalloidea polysaccharide " of CN101503477, and publication date is 2009-08-12, and application people is Wuhan University of Technology.The method is specifically: first by the natural dictyophora phalloidea organic solvent degreasing in apparatus,Soxhlet's after pulverizing, then by residue successively water extraction under 20 ~ 80 DEG C of gradient temperatures, filter to get filtrate, repeat three times, centrifuging and taking supernatant liquor, obtains extracting solution; This extracting solution is through Sevage method deproteinated and use H
2o
2after oxidation style decolouring, dialyse with clear water and redistilled water to remove small molecules respectively, and after DEAE post removing acidic polysaccharose, solution lyophilize, obtains the triple helix Dictyophora phalloidea polysaccharide that form is flocculent white.
Patent application publication number is patent discloses " application of triple spiral Dictyophora phalloidea polysaccharide " of CN101502532, and publication date is 2009-08-12, and application people is Wuhan University of Technology.This triple spiral Dictyophora phalloidea polysaccharide extracts and obtains from natural dictyophora phalloidea, and molecular weight is 30 ~ 1,000,000, has triple helix ordered structure, and the monose of triple spiral Dictyophora phalloidea polysaccharide consists of glucose.By cell experiment, this invention finds that triple spiral Dictyophora phalloidea polysaccharide has obvious restraining effect to S-180 tumour cell, suitable with the tumor inhibition effect of lentinan.
Patent application publication number is patent discloses of the CN103044566A preparation method of antioxidant polysaccharide " in a kind of dictyophora phalloidea water extraction residue ", and publication date is 2013-04-17, and application people is HeFei University of Technology.The method comprises the preparation of dictyophora phalloidea water extraction residue, the configuration of polysaccharide extraction liquid, the extraction of polysaccharide in dictyophora phalloidea water extraction residue, the removal of water-insoluble α in polysaccharide extraction liquid-(1 → 3)-d-dextran, decolouring, the removal of small molecular weight impurity, the purifying of polysaccharide, dialysis and lyophilize, anti-oxidant activity detection etc.
Patent application publication number is patent discloses " a kind of preparation method of dictyophora phalloidea protein-polysaccharide " of CN103360504A, and publication date is 2013-10-23, the sub-people in the application Ren Wei village etc.The method with dry dictyophora phalloidea for raw material, through coarse crushing, hot water extraction, coarse filtration, clarification essence filter, decolouring, vacuum concentration, alcohol precipitation, alcohol hypostasis drying, pulverize, sieving to obtain dictyophora phalloidea protein-polysaccharide.The dictyophora phalloidea protein-polysaccharide produced has obvious restraining effect to mouse S-180 tumour cell, and inhibiting rate is 48.12% ~ 62.25%.
Japanese Patent JP2004262905 discloses one " Claritin or food ", and invention people is Nakasugi Toru etc., and publication date is 2004-09-24.The raw material of this Claritin by Pistacia weinmannifolia, dictyophora phalloidea, Radix Glycyrrhizae, snow tea, pepper, tamarind, Elettaria cardamomum (L.) Maton, bolete, bloom bolete, honeysuckle, phyllanthus emblica one or more form.This patent using dictyophora phalloidea as a kind of raw material preparing Claritin.
US Patent No. 20080199488A1 discloses the preparation method of one " anti-AIDS drug ", and invention people is Tani Michio, and publication date is 2009-08-21.The method with Herba Melastomatis Candii, Dipterocarpus pilosus, cluster from pleat umbrella, dictyophora phalloidea, Leaf of Assam Tea, peppermint and sweet Stevia one or more prepare a kind of anti-AIDS drug for raw material.This patent using dictyophora phalloidea as a kind of raw material preparing anti-AIDS drug.
US Patent No. 8617567 discloses one and " has fungus polysaccharide composition and the application thereof of immuno-potentiation ", and invention people is Tang Jian etc., and publication date is 2013-12-13.The present invention relates to a kind of fungus polysaccharide with the compound strengthening immunization, composition of raw materials is: mushroom 8 ~ 100, Poria cocos 15 ~ 100, dictyophora phalloidea 10 ~ 200, white fungus 15 ~ 80, peacilomyce hepiahi bacterium filament 2 ~ 50.Dictyophora phalloidea is had as preparation a kind of raw material strengthening immunization protective foods by this patent.
In sum, at present about with Dictyophora rubrovalvata sporophore for raw material, successively through hot water extraction, Savege method removing protein, alcohol settling, then adopt " ion-exchange chromatography-gel filtration chromatography-ultra-filtration and separation-gel filtration chromatography " four step separations to obtain after carrying out separation and purification, and there is no with the report that the Dictyophora rubrovalvata polysaccharide after separation and purification carries out the experiment of mouse macrophage immunocompetence.And the invention provides a kind of preparation method and the application thereof with immunocompetent Novel red holder dictyophora fungus polysaccharide, for the deep processing of dictyophora phalloidea and efficiency utilization have established theoretical basis and technical foundation.
Summary of the invention
Technical problem to be solved by this invention is to carry out effective Extraction and separation purifying to the polysaccharide in Dictyophora rubrovalvata sporophore, and the immunocompetence of the Dictyophora rubrovalvata polysaccharide after separation and purification is studied, provide one to have immunocompetent Novel red holder dictyophora fungus polysaccharide and its preparation method and application.
A kind of preparation method with immunocompetent Novel red holder dictyophora fungus polysaccharide, take Dictyophora rubrovalvata as raw material, prepare through following method: hot water extraction, Savege method removing protein, alcohol settling, obtain after then adopting ion-exchange chromatography, gel filtration chromatography, ultra-filtration and separation, the separation and purification of gel filtration chromatography four step separation.Described method comprises the following steps:
(1) by Dictyophora rubrovalvata sporophore in 40 ~ 70 DEG C of drying 0.5 ~ 4 h, then pulverize with powder beater;
(2) hot water extraction 1 ~ 4 h, solid-liquid ratio is 1: 20 ~ 1: 40 g/mL, and temperature is 60 ~ 100 DEG C, and extracting times is 1 ~ 3 time;
(3) adopt Sevage method to carry out deproteinated process 6 ~ 10 times to hot water extraction's thing, collect upper strata polysaccharide soln and then carry out concentrated and dry, obtain the Crude polysaccharides solution after deproteination;
(4) in the Crude polysaccharides solution after deproteination, add ethanolic soln, hold over night at 0 ~ 4 DEG C, centrifugal 10 ~ 20 min of 4000 ~ 8000 r/min, collecting precipitation thing, obtains Dictyophora rubrovalvata Crude polysaccharides after vacuum lyophilization;
(5) four step separations are adopted to carry out separation and purification: purification procedures is: first to adopt ion-exchange chromatography to carry out purifying to the Dictyophora rubrovalvata Crude polysaccharides that step (4) obtains, gradient elution is carried out by the NaCl solution of deionized water and 0.05 ~ 2 mol/L, collect elutriant, dialysis, concentrated postlyophilization; Then adopt gel filtration chromatography, make eluent with deionized water, dialysis, concentrated postlyophilization; Adopt Ultrafiltration Membrane to carry out purifying again, collect permeate and trapped fluid respectively, concentrated postlyophilization; Finally adopt gel filtration chromatography, make eluent with deionized water, dialysis, concentrated postlyophilization, obtain Dictyophora rubrovalvata polysaccharide.
In aforesaid method, in step (4), the volume fraction of used ethanol is 65% ~ 95%(preferably 80% ~ 95%) or use dehydrated alcohol, the add-on of ethanol is 1 ~ 5 times of Crude polysaccharides liquor capacity.
In aforesaid method, in step (5), adopt ion-exchange chromatography, gel filtration chromatography, ultra-filtration and separation, gel filtration chromatography four step separation to carry out separation and purification, the embodiment of four step separations is as the one under type:
Mode one, adopts DEAE-52 or DEAE Sepharose FF anion exchange chromatography, Sephadex G-200 or Sephadex G-150 gel filtration chromatography, ultra-filtration and separation, Sephadex G-100 gel filtration chromatography;
Mode two, adopts DE 52 or QA 52 anion exchange chromatography, Sephadex G-200 or Sephadex G-150 gel filtration chromatography, ultra-filtration and separation, Sephadex G-100 gel filtration chromatography;
Mode three, DEAE-52 or Sepharose XL anion exchange chromatography, Sephadex G-200 or Sepharose Fast Flow gel filtration chromatography, ultra-filtration and separation, Superose 12 or Bio-Gel P-4 gel filtration chromatography;
Mode four, Sepharose XL or CM52 anion exchange chromatography, Sepharose 2B or Sepharose 4B gel filtration chromatography, ultra-filtration and separation, Sepharose 6B or Sephacryl S-400 HR gel filtration chromatography.Dialysis condition is 0 ~ 4 DEG C, 48 ~ 60 h.
In aforesaid method, in the ultra-filtration and separation of step (5), the relative molecular mass that retains of the ultra-filtration membrane adopted is 20 ~ 1000 kDa.
One has immunocompetent Novel red holder dictyophora fungus polysaccharide, and be made up of semi-lactosi, glucose and seminose three kinds of monose, molecular weight is homogeneous, purity is high and good stability.Cell experiment proves, this Novel red holder dictyophora fungus polysaccharide by activating the expression amount of nitrogen protoxide (NO), tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) in mouse macrophage RAW264.7, thus can improve the immunological competence of body
A kind of have the medicine preparation that immunocompetent Novel red holder dictyophora fungus polysaccharide is applied to immune deficiency class disease and tumour.
A kind of have food or the protective foods that immunocompetent Novel red holder dictyophora fungus polysaccharide is applied to preparation raising immunologic function.
The Novel red holder dictyophora fungus polysaccharide that the present invention obtains has the advantage that molecular weight is homogeneous, purity is high, and there is good immunologic enhancement, can be used for the assisting therapy of immune deficiency class disease and tumour, also can be used for the food or the protective foods that improve immunologic function.Preparation technology's advantages of simple of the present invention, environmental protection, and be easy to realize industrialized production.
Accompanying drawing explanation
Fig. 1 is Dictyophora rubrovalvata polysaccharide ion-exchange chromatography elution curve.
Fig. 2 is Dictyophora rubrovalvata polysaccharide DP1 gel filtration chromatography elution curve.
Fig. 3 is Dictyophora rubrovalvata polysaccharide DP2 gel filtration chromatography elution curve.
Fig. 4 is the infrared spectrogram of Dictyophora rubrovalvata polysaccharide DP1.
Fig. 5 is the infrared spectrogram of Dictyophora rubrovalvata polysaccharide DP2.
Fig. 6 is the monose composition chromatography of ions figure of Dictyophora rubrovalvata polysaccharide DP1.
Fig. 7 is the monose composition chromatography of ions figure of Dictyophora rubrovalvata polysaccharide DP2.
Fig. 8 is that different concns Dictyophora rubrovalvata polysaccharide DP1 affects histogram to mouse macrophage NO secretory volume.
Fig. 9 is that different concns Dictyophora rubrovalvata polysaccharide DP2 affects histogram to mouse macrophage NO secretory volume.
Figure 10 is that different concns Dictyophora rubrovalvata polysaccharide DP1 affects histogram to mouse macrophage TNF-α secretory volume.
Figure 11 is that different concns Dictyophora rubrovalvata polysaccharide DP2 affects histogram to mouse macrophage TNF-α secretory volume.
Figure 12 is that different concns Dictyophora rubrovalvata polysaccharide DP1 affects histogram to mouse macrophage IL-6 secretory volume.
Figure 13 is that different concns Dictyophora rubrovalvata polysaccharide DP2 affects histogram to mouse macrophage IL-6 secretory volume.
Embodiment
Below in conjunction with specific embodiment, enforcement of the present invention is described further, but enforcement of the present invention is not limited thereto.
Embodiment 1:
(1) by 100 g Dictyophora rubrovalvata sporophores dry 2 h at 40 DEG C, then pulverize with powder beater;
(2) 100 DEG C of hot water extraction 1 h, solid-liquid ratio is 1: 40 g/mL, and extracting times is 1 time; Centrifugal 15 min of 4000 r/min, collect supernatant liquor rotary evaporation concentrating under reduced pressure at 60 DEG C.
(3) adopt Sevage method to carry out deproteinated process to hot water extraction's thing, the compound method of Sevage reagent is 4: 1 mixing by volume of chloroform, propyl carbinol; To be polysaccharide to mix with Sevage reagent the Deproteinated method of Sevage method by volume at 1: 1, and centrifugal 10 min of jolting 20 min, 3000 r/min, take out upper strata polysaccharide soln, obtain dictyophora phalloidea Crude polysaccharides after drying; Deproteinated process repeats 10 times.
(4) in the Crude polysaccharides solution after deproteination, the dehydrated alcohol of 3 times of volumes is added, hold over night at 4 DEG C; Then centrifugal 20 min under 4000 r/min, collecting precipitation thing, obtains dictyophora phalloidea Crude polysaccharides after vacuum lyophilization.
(5) getting the Crude polysaccharides that 100 mg steps (4) obtain dissolves in 10 mL deionized waters again, DEAE-52 anion exchange chromatography is adopted to carry out purifying, successively adopt deionized water, 0.05,0.1, the NaCl solution of 0.2,0.3 and 0.5 mol/L carries out gradient elution, and eluent flow rate is 0.5 mL/min; Wherein use the component called after DP1 component of deionized water wash-out, by the component called after DP2 component of the NaCl solution wash-out of 0.05 mol/L; Dialyse 48 h at 4 DEG C, then carries out lyophilize.
(6) Dictyophora rubrovalvata polysaccharide DP1, DP2 component that 20 mg steps (5) obtain is got respectively, be dissolved in 4 mL deionized waters respectively, Sephadex G-200 gel filtration chromatography is adopted to carry out purifying, adopt deionized water as eluent, flow velocity is 0.5 mL/min, and concentrated postlyophilization obtains just being separated dictyophora fungus polysaccharide.
(7) adopt Ultrafiltration Membrane to be separated the polysaccharide fraction that step (6) obtains, the relative molecular mass that retains of the ultra-filtration membrane that DP1 component adopts is 1000 kDa, collects trapped fluid, carries out next step purifying after concentrated, lyophilize; The relative molecular mass that retains of the ultra-filtration membrane that DP2 component adopts is 20 kDa, collects permeate, carries out next step purifying after concentrated, lyophilize.
(8) Dictyophora rubrovalvata polysaccharide DP1 and DP2 component that 20 mg steps (7) obtain is got respectively, be dissolved in 4 mL deionized waters respectively, Sephadex G-100 gel filtration chromatography is adopted to carry out purifying, adopt deionized water as eluent, flow velocity is 0.5 mL/min, carries out lyophilize and obtain Dictyophora rubrovalvata polysaccharide finished product after concentrated.Dictyophora rubrovalvata polysaccharide ion-exchange chromatography elution curve is shown in Fig. 1; Dictyophora rubrovalvata polysaccharide DP1 gel filtration chromatography elution curve is shown in Fig. 2; Dictyophora rubrovalvata polysaccharide DP2 gel filtration chromatography elution curve is shown in Fig. 3.
Embodiment 2:
(1) by 100 g Dictyophora rubrovalvata sporophores dry 0.5 h at 70 DEG C, then pulverize with powder beater;
(2) 90 DEG C of hot water extraction 2 h, solid-liquid ratio is 1: 20 g/mL, and extracting times is 3 times; Centrifugal 15 min of 4000 r/min, collect supernatant liquor rotary evaporation concentrating under reduced pressure at 60 DEG C.
(3) adopt Sevage method to carry out deproteinated process to hot water extraction's thing, the compound method of Sevage reagent is 4: 1 mixing by volume of chloroform, propyl carbinol; To be polysaccharide to mix with Sevage reagent the Deproteinated method of Sevage method by volume at 1: 1, and centrifugal 10 min of jolting 20 min, 3000 r/min, take out upper strata polysaccharide soln, obtain dictyophora phalloidea Crude polysaccharides after drying; Deproteinated process repeats 10 times.
(4) in the Crude polysaccharides solution after deproteination, the dehydrated alcohol of 3 times of volumes is added, hold over night at 4 DEG C; Then centrifugal 10 min under 8000 r/min, collecting precipitation thing, obtains dictyophora phalloidea Crude polysaccharides after vacuum lyophilization.
(5) getting the Crude polysaccharides that 100 mg steps (4) obtain dissolves in 10 mL deionized waters again, adopts QA 52 anion exchange chromatography to carry out purifying, successively adopts deionized water, 0.05,0.1,0.5, the NaCl solution of 1 mol/L carries out gradient elution, and eluent flow rate is 0.5 mL/min; Wherein use the component called after DP1 component of deionized water wash-out, by the component called after DP2 component of the NaCl solution wash-out of 0.1 mol/L; Dialyse 48 h at 4 DEG C, then carries out lyophilize.
(6) Dictyophora rubrovalvata polysaccharide DP1, DP2 component that 20 mg steps (5) obtain is got respectively, be dissolved in 4 mL deionized waters respectively, Sephadex G-150 gel filtration chromatography is adopted to carry out purifying, adopt deionized water as eluent, flow velocity is 0.5 mL/min, carries out lyophilize and obtain just being separated dictyophora fungus polysaccharide after concentrated.
(7) adopt Ultrafiltration Membrane to be separated the polysaccharide fraction that step (6) obtains, the relative molecular mass that retains of the ultra-filtration membrane that DP1 component adopts is 1000 kDa, collects trapped fluid, carries out next step purifying after concentrated, lyophilize; The relative molecular mass that retains of the ultra-filtration membrane that DP2 component adopts is 30 kDa, collects permeate, carries out next step purifying after concentrated, lyophilize.
(8) Dictyophora rubrovalvata polysaccharide DP1 and DP2 component that 20 mg steps (7) obtain is got respectively, be dissolved in 4 mL deionized waters respectively, Sephadex G-100 gel filtration chromatography is adopted to carry out purifying, adopt deionized water as eluent, flow velocity is 0.5 mL/min, carries out lyophilize and obtain Dictyophora rubrovalvata polysaccharide finished product (Dictyophora rubrovalvata polysaccharide ion-exchange chromatography elution curve can see Fig. 1 after concentrated; Dictyophora rubrovalvata polysaccharide DP1 gel filtration chromatography elution curve can see Fig. 2; Dictyophora rubrovalvata polysaccharide DP2 gel filtration chromatography elution curve can see Fig. 3).
Embodiment 3:
(1) by 100 g Dictyophora rubrovalvata sporophores dry 3 h at 60 DEG C, then pulverize with powder beater;
(2) 100 DEG C of hot water extraction 4 h, solid-liquid ratio is 1: 30 g/mL, and extracting times is 3 times; Centrifugal 10 min of 8000 r/min, collect supernatant liquor rotary evaporation concentrating under reduced pressure at 60 DEG C.
(3) adopt Sevage method to carry out deproteinated process to hot water extraction's thing, the compound method of Sevage reagent is 4: 1 mixing by volume of chloroform, propyl carbinol; To be polysaccharide to mix with Sevage reagent the Deproteinated method of Sevage method by volume at 1: 1, and centrifugal 10 min of jolting 20 min, 3000 r/min, take out upper strata polysaccharide soln, obtain dictyophora phalloidea Crude polysaccharides after drying; Deproteinated process repeats 10 times.
(4) in the Crude polysaccharides solution after deproteination, the dehydrated alcohol of 3 times of volumes is added, hold over night at 4 DEG C; Then centrifugal 15 min under 4000 r/min, collecting precipitation thing, obtains dictyophora phalloidea Crude polysaccharides after vacuum lyophilization.
(5) getting the Crude polysaccharides that 100 mg steps (4) obtain dissolves in 10 mL deionized waters again, Sepharose XL anion exchange chromatography is adopted to carry out purifying, successively adopt deionized water, 0.05,0.1, the NaCl solution of 0.2,0.3 and 0.5 mol/L carries out gradient elution, and eluent flow rate is 0.5 mL/min; Wherein use the component called after DP1 component of deionized water wash-out, by the component called after DP2 component of the NaCl solution wash-out of 0.05 mol/L; Dialyse 48 h at 4 DEG C, then carries out lyophilize.
(6) Dictyophora rubrovalvata polysaccharide DP1, DP2 component that 20 mg steps (5) obtain is got respectively, be dissolved in 4 mL deionized waters respectively, Sepharose Fast Flow gel filtration chromatography is adopted to carry out purifying, adopt deionized water as eluent, flow velocity is 0.5 mL/min, carries out lyophilize and obtain just being separated dictyophora fungus polysaccharide after concentrated.
(7) adopt Ultrafiltration Membrane to be separated the polysaccharide fraction that step (6) obtains, the relative molecular mass that retains of the ultra-filtration membrane that DP1 component adopts is 1000 kDa, collects trapped fluid, carries out next step purifying after concentrated, lyophilize; The relative molecular mass that retains of the ultra-filtration membrane that DP2 component adopts is 30 kDa, collects permeate, carries out next step purifying after concentrated, lyophilize.
(8) Dictyophora rubrovalvata polysaccharide DP1 and DP2 component that 20 mg steps (7) obtain is got respectively, be dissolved in 4 mL deionized waters respectively, Bio-Gel P-4 gel filtration chromatography is adopted to carry out purifying, adopt deionized water as eluent, flow velocity is 0.5 mL/min, carries out lyophilize and obtain Dictyophora rubrovalvata polysaccharide finished product (Dictyophora rubrovalvata polysaccharide ion-exchange chromatography elution curve can see Fig. 1 after concentrated; Dictyophora rubrovalvata polysaccharide DP1 gel filtration chromatography elution curve can see Fig. 2; Dictyophora rubrovalvata polysaccharide DP2 gel filtration chromatography elution curve can see Fig. 3).
The Dictyophora rubrovalvata polysaccharide obtained by above embodiment 1 carries out Structural Identification and activation analysis by the following method, and the result of embodiment 2 and 3 is similar to Example 1.
1, the Infrared spectroscopy of Dictyophora rubrovalvata polysaccharide
Get each 2.0 mg of Dictyophora rubrovalvata polysaccharide DP1 and DP2, add appropriate dry KBr powder respectively, mix, in agate mortar, add compressing tablet in press mold after grinding evenly, make thick about 1 mm, transparent compressing tablet that diameter is about about 10 mm.Use FT-IR(Fourier transform infrared spectrometer, model is Nexus, Thermo Nicolet company of the U.S.) respectively to DP1 and DP2 compressing tablet at 400 ~ 4000 cm
-1interval is scanned, the infrared absorption pattern of collected specimens, carries out analyzing and processing with Nexus system software to collection of illustrative plates.The infrared absorption spectrum of dictyophora fungus polysaccharide DP1 and DP2 respectively as shown in Figure 4 and Figure 5.
From the infrared spectrogram of Fig. 4 Dictyophora rubrovalvata polysaccharide DP1, at 3335.69 cm
-1near have a strong and wide absorption peak, this is stretching vibration charateristic avsorption band (3600 ~ 3200 cm belonging to hydroxyl
-1); At 2926.33 cm
-1neighbouring absorption peak belongs to C-H stretching vibration absorption peak (2950 ~ 2850 cm
-1); At 1425.46 cm
-1with 1374.38 cm
-1neighbouring absorption peak belongs to C-H in-plane bending vibration absorption peak (1470 ~ 1430 cm
-1; 1380 ~ 1360 cm
-1); Can this material of preliminary judgement be saccharide compound by above several groups of absorption peaks; 1727.93 cm
-1neighbouring absorption peak is stretching vibration absorption peak (1750 ~ 1710 cm belonging to aldehyde, ketone C=O double bond
-1), show containing aldehyde radical or ketone carbonyl; 1648.01 cm
-1neighbouring absorption peak is stretching vibration absorption peak (1680 ~ 1620 cm belonging to C=C double bond
-1), can infer that this carbohydrate contains enediol structure; 1074.32 cm
-1with 1040.25 cm
-1neighbouring absorption peak belongs to C-O stretching vibration absorption peak (1080 ~ 1030 cm
-1), these absorption peaks are charateristic avsorption bands of the C-O key of ehter bond C-O-C on pyranoid ring and alcohols; At 1750 ~ 1700 cm
-1without absorption peak, show this polysaccharide not containing uronic acid.
From the infrared spectrogram of Fig. 5 Dictyophora rubrovalvata polysaccharide DP2, at 3374.38 cm
-1near have a strong and wide absorption peak, this is stretching vibration charateristic avsorption band (3600 ~ 3200 cm belonging to hydroxyl
-1); At 2928.90 cm
-1neighbouring absorption peak belongs to C-H stretching vibration absorption peak (2950 ~ 2850 cm
-1); At 1405.93 cm
-1neighbouring absorption peak belongs to C-H in-plane bending vibration absorption peak; Can this material of preliminary judgement be saccharide compound by above three groups of absorption peaks; 1640.93 cm
-1neighbouring absorption peak is stretching vibration absorption peak (1680 ~ 1620 cm belonging to C=C double bond
-1), can infer that this carbohydrate contains enediol structure; 1250 ~ 950 cm
-1near absorption peak be belong to C-O stretching vibration absorption peak, these absorption peaks are charateristic avsorption bands of the C-O key of ehter bond C-O-C on pyranoid ring and alcohols; At 1750 ~ 1700 cm
-1without absorption peak, show this polysaccharide not containing uronic acid.
2, the monosaccharide composition analysis of Dictyophora rubrovalvata polysaccharide
Adopt ion chromatograph to measure the monose composition of Dictyophora rubrovalvata polysaccharide DP1 and DP2, take 20.0 mg dictyophora fungus polysaccharide DP1 and DP2 respectively, be dissolved in 4.0 mL trifluoroacetic acids respectively, at 105 DEG C of reaction 6 h.Unnecessary trifluoroacetic acid is removed with rotary evaporation.Then residuum is dissolved in methyl alcohol again, concentrates with rotary evaporation.Finally residuum is dissolved in ultrapure water, measures with ion chromatograph (Dionex ICS-3000, Dai An company of the U.S.).Chromatography of ions condition is: column temperature 30 DEG C; Chromatographic column: analytical column is CarboPac PA 20(2 × 250 mm); Detector: pulsed amperometry, gold electrode; Leacheate forms: A. ultrapure water; B.0.2 mol/L NaOH; C.0.5 mol/L NaAC; D.0.02 mol/L NaOH.Elution program: 0 ~ 2 min leacheate consists of 100% B; 2 ~ 20 min are 10% D+90% A; 20 ~ 40 min are 100%.Form chromatography of ions figure by the monose of Fig. 6 Dictyophora rubrovalvata polysaccharide DP1 to know, this Dictyophora rubrovalvata polysaccharide is made up of semi-lactosi, glucose and seminose three kinds of monose, and the mol ratio of three is semi-lactosi: glucose: seminose=1: 3.97:2.05.Form chromatography of ions figure by the monose of Fig. 7 Dictyophora rubrovalvata polysaccharide DP2 to know, this Dictyophora rubrovalvata polysaccharide is made up of semi-lactosi, glucose and seminose three kinds of monose, and the mol ratio of three is semi-lactosi: glucose: seminose=1: 4.73:1.64.
3, the immunocompetence of Dictyophora rubrovalvata polysaccharide measures
Experimental cell: mouse macrophage RAW264.7, purchased from medical college of Zhongshan University.
Experiment reagent: Dole uncle section's minimum essential medium (DMEM), Streptomycin sulphate and penicillin purchased from American Gibco company; Foetal calf serum is purchased from Tian Hang bio tech ltd, Zhejiang; Lipopolysaccharides (LPS) purchased from American Sigma company; NO detection kit builds up Bioengineering Research Institute purchased from Nanjing; Mouse IL-6 ELISA kit, mouse TNF-α ELISA kit are purchased from Xin Bosheng bio tech ltd.
3.1 cell cultures
Mouse macrophage is cultivated with Dole uncle section's minimum essential medium (DMEM), and adds the foetal calf serum (FBS) of 10%, the Streptomycin sulphate of 100 μ g/mL and 100 U/mL penicillin, puts into 37 DEG C, 5% CO
2quiescent culture in incubator.Take the logarithm mouse macrophage RAW264.7 in vegetative period, by dilution, cell concn is adjusted to 1 × 10
6individual/mL, is inoculated in 96 orifice plates, hatches 24 h and changes a nutrient solution.
The mensuration 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 does not add cell, adds 100 μ L nutrient solutions; Experimental group, control group and positive controls, every hole adds 100 μ L cell suspending liquids.Experimental group adds 125 respectively, 250,500, the Dictyophora rubrovalvata polysaccharide soln 100 μ L of 1000 μ g/mL, tetra-concentration gradients, lipopolysaccharides (LPS) the liquid 100 μ L(liquid and the positive control that add 50 μ g/mL in positive controls are all prepared with not containing blood serum medium), 6 parallel holes established by each different components sample.For preventing microbiological contamination, sealing flat board with the sealed membrane after alcohol wipe and putting into 37 DEG C, 5% CO again
224 h are hatched in incubator.According to the detection method that test kit manufacturer provides, respectively by the secretory volume of NO, TNF-α, IL-6 in NO detection kit, mouse TNF-α ELISA kit, mouse IL-6 ELISA kit detection cell conditioned medium liquid.For Dictyophora rubrovalvata polysaccharide DP1 sample, in supernatant liquor, the content of NO, TNF-α, IL-6 is respectively as shown in Fig. 8, Figure 10, Figure 12.For Dictyophora rubrovalvata polysaccharide DP2 sample, in supernatant liquor, the content of NO, TNF-α, IL-6 is respectively as shown in Fig. 9, Figure 11, Figure 13.As seen from the figure, dictyophora fungus polysaccharide DP1 and DP2 is within the scope of 125 ~ 1000 μ g/mL, and in cell conditioned medium liquid, the content of NO, TNF-α, IL-6 is significantly higher than control group (Control), and presents dosage effect, along with the rising of polysaccharide concentration, the content of NO, TNF-α, IL-6 also raises.Therefore DP1 and DP2 all can significantly improve the content of NO, TNF-α, IL-6 in mouse macrophage RAW264.7 supernatant liquor, and the immunocompetence of DP2 component is better than DP1 component.
Claims (8)
1. one kind has the preparation method of immunocompetent Novel red holder dictyophora fungus polysaccharide, it is characterized in that, take Dictyophora rubrovalvata as raw material, prepare through following method: hot water extraction, Savege method removing protein, alcohol settling, obtain after then adopting ion-exchange chromatography, gel filtration chromatography, ultra-filtration and separation, the separation and purification of gel filtration chromatography four step separation.
2. preparation method according to claim 1, is characterized in that, described method comprises the following steps:
(1) by Dictyophora rubrovalvata sporophore in 40 ~ 70 DEG C of drying 0.5 ~ 4 h, then pulverize with powder beater;
(2) hot water extraction 1 ~ 4 h, solid-liquid ratio is 1: 20 ~ 1: 40 g/mL, and temperature is 60 ~ 100 DEG C, and extracting times is 1 ~ 3 time;
(3) adopt Sevage method to carry out deproteinated process 6 ~ 10 times to hot water extraction's thing, collect upper strata polysaccharide soln and then carry out concentrated and dry, obtain the Crude polysaccharides solution after deproteination;
(4) in the Crude polysaccharides solution after deproteination, add ethanolic soln, hold over night at 0 ~ 4 DEG C, centrifugal 10 ~ 20 min of 4000 ~ 8000 r/min, collecting precipitation thing, obtains Dictyophora rubrovalvata Crude polysaccharides after vacuum lyophilization;
(5) four step separations are adopted to carry out separation and purification: purification procedures is: first to adopt ion-exchange chromatography to carry out purifying to the Dictyophora rubrovalvata Crude polysaccharides that step (4) obtains, gradient elution is carried out by the NaCl solution of deionized water and 0.05 ~ 2 mol/L, collect elutriant, dialysis, concentrated postlyophilization; Then adopt gel filtration chromatography, make eluent with deionized water, dialysis, concentrated postlyophilization; Adopt Ultrafiltration Membrane to carry out purifying again, collect permeate and trapped fluid respectively, concentrated postlyophilization; Finally adopt gel filtration chromatography, make eluent with deionized water, dialysis, concentrated postlyophilization, obtain Dictyophora rubrovalvata polysaccharide.
3. preparation method according to claim 2, is characterized in that, in step (4), the volume fraction of used ethanol is 65% ~ 95% or uses dehydrated alcohol, and the add-on of ethanol is 1 ~ 5 times of Crude polysaccharides liquor capacity.
4. preparation method according to claim 2, it is characterized in that, in step (5), adopt ion-exchange chromatography, gel filtration chromatography, ultra-filtration and separation, gel filtration chromatography four step separation to carry out separation and purification, the embodiment of four step separations is as the one under type:
Mode one, adopts DEAE-52 or DEAE Sepharose FF anion exchange chromatography, Sephadex G-200 or Sephadex G-150 gel filtration chromatography, ultra-filtration and separation, Sephadex G-100 gel filtration chromatography;
Mode two, adopts DE 52 or QA 52 anion exchange chromatography, Sephadex G-200 or Sephadex G-150 gel filtration chromatography, ultra-filtration and separation, Sephadex G-100 gel filtration chromatography;
Mode three, DEAE-52 or Sepharose XL anion exchange chromatography, Sephadex G-200 or Sepharose Fast Flow gel filtration chromatography, ultra-filtration and separation, Superose 12 or Bio-Gel P-4 gel filtration chromatography;
Mode four, Sepharose XL or CM52 anion exchange chromatography, Sepharose 2B or Sepharose 4B gel filtration chromatography, ultra-filtration and separation, Sepharose 6B or Sephacryl S-400 HR gel filtration chromatography.
5. preparation method according to claim 2, is characterized in that, in the ultra-filtration and separation of step (5), the relative molecular mass that retains of the ultra-filtration membrane adopted is 20 ~ 1000 kDa.
6. prepare one by preparation method described in any one of claim 1-5 and there is immunocompetent Novel red holder dictyophora fungus polysaccharide.
7. Novel red holder dictyophora fungus polysaccharide according to claim 6 is applied to the medicine preparation of immune deficiency class disease and tumour.
8. Novel red holder dictyophora fungus polysaccharide according to claim 6 is applied to food or the protective foods that preparation improves immunologic function.
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| CN108546304A (en) * | 2018-03-26 | 2018-09-18 | 中国科学院华南植物园 | A method of preparing poly- arabogalactan aldehydic acid using dried orange peel |
| CN108948221A (en) * | 2018-08-03 | 2018-12-07 | 广东药科大学 | A kind of Cultivation of Dictyophora polysaccharide and its preparation and application with neuroprotective efficacy |
| CN108948223A (en) * | 2018-08-28 | 2018-12-07 | 华南师范大学 | Hill gooseberry's polysaccharide P1 and its separation method and preparing the application in blood lipid-lowering medicine |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106879831A (en) * | 2016-12-30 | 2017-06-23 | 新昌县拜特夫农业科技有限公司 | The preparation technology of sheep Chinese herbal composite feed additive |
| CN108546304A (en) * | 2018-03-26 | 2018-09-18 | 中国科学院华南植物园 | A method of preparing poly- arabogalactan aldehydic acid using dried orange peel |
| CN108948221A (en) * | 2018-08-03 | 2018-12-07 | 广东药科大学 | A kind of Cultivation of Dictyophora polysaccharide and its preparation and application with neuroprotective efficacy |
| CN108948223A (en) * | 2018-08-28 | 2018-12-07 | 华南师范大学 | Hill gooseberry's polysaccharide P1 and its separation method and preparing the application in blood lipid-lowering medicine |
| CN108948223B (en) * | 2018-08-28 | 2021-06-01 | 华南师范大学 | Myrtle polysaccharide P1, its separation method and application in preparing hypolipidemic medicine |
| US11369627B2 (en) | 2018-08-28 | 2022-06-28 | South China Normal University School of Life Sciences | Myrtle polysaccharide P1, the separation method thereof and the use in preparing hypolipidemic drugs therefor |
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