CN103130907A - Chinese cordyceps sinensis protein polysaccharide and preparation and application thereof - Google Patents
Chinese cordyceps sinensis protein polysaccharide and preparation and application thereof Download PDFInfo
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Abstract
The invention discloses a Chinese cordyceps sinensis protein polysaccharide and preparation and application thereof. The Chinese cordyceps sinensis protein polysaccharide is composed of polysaccharide with 55%-60% weight percentage and protein with 40%-45% weight percentage; and the ingredients of the polysaccharide are mannose, ribose, glucose, galactose, xylose and arabinose; the ratio of amount of substances among the mannose, the ribose, the glucose, the galactose, the xylose and the arabinose is 6.42-6.51: 2.20-2.32: 65.85-65.95: 2.10-2.18: 4.20-4.30. The Chinese cordyceps sinensis protein polysaccharide preparation method comprises the following steps: extracting the Chinese cordyceps sinensis protein polysaccharide by aqueous extract and alcohol precipitation, removing the protein, dialyzing, exchanging chromatography by anion and purifying gel filter layer chromatography through an enzyme-Sevage combination method, and freezing, drying and separating purification components in vacuum, and the Chinese cordyceps sinensis protein polysaccharide is acquired. According to the preparation and the application thereof, proximate analysis, structural identification and immunologic function study are carried out on the components of polysaccharide after purification, and the facts that the Chinese cordyceps sinensis protein polysaccharide has strong immunoregulation activity and is a potential immunological enhancement substance are discovered.
Description
Technical field
The present invention relates to fungi exocellular polysaccharide field, be specifically related to a kind of China pilose spore protein-polysaccharide and preparation and purposes, belong to polymer and biology field.
Background technology
The Cordyceps sinensis of classifying one of China's three large tonics together with ginseng, pilose antler as is the traditional famous and precious tonic Chinese medicine material of China, it colonizes in the Hepialus larva in alpine meadow soil by the Cordyceps fungus of Hypocreales Clavicipitaceae Cordyceps, larva is ossify, under optimum conditions, go out long bar-shaped stroma by bombys batryticatus head end pumping and form summer.China pilose spore (Hirsutella sinensis Liu, Guo, Yu, Zeng) be the Conidial Stage of Cordyceps sinensis, once with life Hirsutella hepiali Chen et Shen (Hirsutella hepialid Chen, Zhen) (Liu Xijin etc., 1989, the separation in Cordyceps sinensis imperfect stage and evaluation, the fungi journal, 8(1): 35-40).
Wild cordyceps is scarcity of resources due to the impact that is subject to special ecological environment, and people adopt the method for solid or liquid submerged fermentation to obtain the substitute of Cordyceps sinensis mostly now.Chinese patent ZL200710156844.5(Granted publication CN101200748B) in, Chinese caterpillar fungus extracellular polysaccharide High-efficient Production and separation purification method are disclosed, a kind of method of cultural method and film dialysis fast separating and purifying exocellular polysaccharide of Cordyceps militaris spawn extracellular polysaccharide produced by submerged fermentation is provided, comprise following standing cultivation Cordyceps militaris (L.) Link. step: access Cordyceps militaris spawn in substratum, inoculum size 5%-10%, under 22-27 ℃ of condition, standing cultivation 5-9 days, wherein substratum used comprises in weight percentage: yeast extract powder 0.5-1.5, brown sugar 1-3%, male Bombycis mori oil 3-5%, KNO
30.8-0.95%, MgSO
40.1-0.2% and KH
2pO
40.1-0.2%.A kind of extracting method of Cordyceps mycelium polysaccharide is disclosed Chinese patent ZL200810116219.2(Granted publication CN101307112B), mainly comprise that Cordyceps mycelium that particle diameter is less than to 830 μ m obtains the Cordyceps mycelium polysaccharide through steps such as degreasing, water extraction, concentrated, alcohol precipitation, deproteinated and ion column chromatographies, total sugar content can reach 98.26%.Chinese patent ZL200710142960.1(Granted publication CN101104006B) disclose a kind of preparation method and application of Chinese caterpillar fungus extracting concentrated liquid in, extracting solution merging after alcohol extract, aqueous extract and enzymolysis is homogenized and obtains concentrated solution.
Above-mentioned report is to extract and the method for separating about the Cordyceps sinensis exocellular polysaccharide mostly, does not relate to the evaluation of active polysaccharide or higher structure.For separation and the structural analysis of China pilose spore fermentation exocellular polysaccharide, such as its monose composition, glycosidic link mode of connection etc. are had no to report; And increasing research shows that the performance of polysaccharide critical function is determined by its constitutional features, its higher structure (secondary and tertiary structure) is more close, the biological activity of polysaccharide and its molecular weight, molecular chain conformation (Conformation) are closely related, and the conformation of understanding this glycan molecule more contributes to illustrate its biological action mechanism.So find new polysaccharide fraction and activity, to the research and development new drug, be to there is very important scientific meaning.
Summary of the invention
The purpose of this invention is to provide a kind of China pilose spore protein-polysaccharide of determining chemical structure characteristic that separates from China pilose spore submerged fermentation liquid.
Another object of the present invention is to provide preparation and the purposes of described China pilose spore protein-polysaccharide, the preparation method of this China pilose spore protein-polysaccharide, have advantages of simple to operate, be easy to control.
The technical solution adopted for the present invention to solve the technical problems is:
A kind of China pilose spore protein-polysaccharide, the albumen that the polysaccharide that is 55 ℅-60% by weight percentage and weight percentage are 40 ℅-45% forms; Described polysaccharide consist of seminose, ribose, glucose, semi-lactosi, wood sugar and pectinose, the structural unit of polysaccharide is that to take 1 → 3 α-ribose (Rib) connected, 1 → 4 α-wood sugar (Xyl) connected and 1 → 4 β-glucose (Glc) connected be main chain, and by beta galactose (Gal) and β-seminose (Man) replacement, α-pectinose (Ara), is terminal group respectively on the C6 position of β-glucose; The ratio of the amount of substance of described seminose, ribose, glucose, semi-lactosi, wood sugar and pectinose is 6.42-6.51:2.20-2.32:65.85-65.95:2.10-2.18:4.20-4.30.
Described α-ribose is preferably α-D-ribose, described α-wood sugar is preferably alpha-D-xylose, described β-glucose is preferably β-D-Glucose, described beta galactose is preferably β-D-semi-lactosi, described β-seminose is preferably β-D-MANNOSE, and described α-pectinose is preferably α-L-arabinose.
The side chain of described protein-polysaccharide has multiple different variations combination, as there is the structural unit as shown in the structural formula I:
The molecular-weight average of described China pilose spore protein-polysaccharide is 40KDa-50KDa, and KDa is kilodalton.
Described China pilose spore protein-polysaccharide is separated and is obtained by hirsutella sinensis fungal liquid submerged fermentation, extraction.Concrete technical scheme is as follows:
A kind of preparation method of China pilose spore protein-polysaccharide comprises step:
(1) submerged fermentation: access China pilose spore bacterial classification in substratum, inoculum size 5%-10%(weight percent),, under 15-22 ℃ of condition, standing cultivation 30-50 days, obtain the China pilose spore fermented liquid; Wherein substratum used comprises (weight percent): glucose 15%-25%, peptone 3%-7%, cicada pupa powder 6%-12%, KH
2pO
40.6%-1.2%, MgSO
4.7H
2o 0.1%-1.0%;
(2) alcohol precipitation: the China pilose spore fermented liquid in step (1) is concentrated, obtain concentrated solution, then add aqueous ethanolic solution, stirring and evenly mixing, precipitation is spent the night, centrifugal, gets centrifugal gained precipitation and obtains the China pilose spore Crude polysaccharides one time;
(3) Deproteinization: by the aqueous solution protease hydrolyzed of a China pilose spore Crude polysaccharides of step (2) gained, enzyme centrifugal metaprotein and the enzyme removed go out, centrifugal gained supernatant liquor is centrifugal except sub-cloud organic phase and middle egg white layer with organic solvent again, repeat by the centrifugal step of organic solvent until, without the white precipitate generation, obtain extracting solution;
(4) dialysis: the dialysis tubing that the extracting solution of step (3) gained is 6000Da-10000Da with aperture is dialysed in deionized water, collects the extracting solution after dialysing, and vacuum lyophilization obtains secondary China pilose spore Crude polysaccharides;
(5) purifying: the secondary China pilose spore Crude polysaccharides that step (4) is obtained obtains the secondary China pilose spore Crude polysaccharides aqueous solution with deionized water dissolving, through diethylaminoethyl cellulose-52(DEAE Mierocrystalline cellulose-52) the ion exchange column chromatography, the elutriant of collecting detects polysaccharide and detects albumen by the lowry method by the phenolsulfuric acid method, collect the elutriant of the second elution peak through gel permeation chromatography, the collected elutriant of gel permeation chromatography detects polysaccharide and protein peak by phenolsulfuric acid method and lowry method, collect the elutriant of common proteoglycan, through concentrated, dialysis and freeze-drying obtain the cotton-shaped China pilose spore protein-polysaccharide of white loose, called after HS002-II.
In order to reach better invention effect, preferably:
In step (2), the consumption of described aqueous ethanolic solution is preferably 4 times-5 times of concentrated solution volume; The concentration expressed in percentage by volume of described aqueous ethanolic solution is preferably 90 ℅-96%; The temperature that described precipitation is spent the night is preferably 2 ℃-5 ℃.
In step (3), described proteolytic enzyme is trypsinase; The 1%-2% that the weight of described proteolytic enzyme is a China pilose spore Crude polysaccharides weight;
Described organic solvent is chloroform and propyl carbinol, and wherein the volume ratio of chloroform and propyl carbinol is 4:1.
In step (4), the time of dialysing in deionized water is preferably 80h-100h.
In step (5), the concentration of the described secondary China pilose spore Crude polysaccharides aqueous solution is 3mg/mL-25mg/mL, and flow velocity is 1ml/min-1.5ml/min.
In step (5), the condition of described diethylaminoethyl cellulose 52 ion exchange column chromatographies is: adopt gradient elution, the NaCl aqueous solution that elutriant is 0.1mol/L-1.0mol/L, flow velocity 1ml/min-1.5ml/min.
In step (5), the condition of described gel permeation chromatography is: flow velocity 0.5ml/min, and the NaCl aqueous solution that elutriant is 0.05mol/L phosphate buffered saline buffer and 0.15mol/L, wherein the volume ratio of phosphate buffered saline buffer and the NaCl aqueous solution is 2-3:1.
The method that the compound method of described phosphate buffered saline buffer is general according to this area, generally can be with reference to version " Chinese pharmacopoeia in 2005.
Described China pilose spore bacterial classification adopts commercially available prod.
Described China pilose spore protein-polysaccharide has the mouse macrophage strain RAW264.7NO of promotion and TNF-α discharges, and can reach immunoregulatory effect by activating I κ B-NF-κ B signal path, can be used for preparing immunomodulator.
Compared with prior art, the present invention has following advantage:
First passage of the present invention is to the submerged fermentation of China pilose spore bacteria liquid, extract to separate and obtain a kind of bioactive macromole China pilose spore protein-polysaccharide HS002-II that has, form and identify that this polysaccharide of discovery is comprised of seminose, ribose, glucose, semi-lactosi, wood sugar and pectinose through monose, the ratio of its amount of substance is 6.42-6.51:2.20-2.32:65.85-65.95:2.10-2.18:4.20-4.30, as 6.47:2.27:65.89:16.17:2.13:4.26; FTIR proof HS002-II is proteoglycan, contains α and beta comfiguration; It is that one-component and molecular weight are 40-50KDa for the agarose electrophoresis proof, and β-elimination reaction explanation albumen is connected with the N-ethanoyl with polysaccharide; Nuclear magnetic resonance map shows that it is α and beta comfiguration, contains N-ethanoyl structure, illustrates in this sample and contains albumen and polysaccharide.
The inventive method is easy and simple to handle, is easy to control, can obtain there is higher order, macromole that structure is clear and definite, provide researching value for furtheing investigate its higher structure and functional relationship.Utilize the present invention to prepare the China pilose spore protein-polysaccharide, do not affect its natural structure and activity, the method is lower to equipment requirements, cost is low, is beneficial on industrial production and promotes on a large scale, develops and use.
China pilose spore protein-polysaccharide of the present invention has the mouse macrophage strain RAW264.7NO of promotion and TNF-α discharges, and can reach immunoregulatory effect by activating I κ B-NF-κ B signal path, can be used for preparing immunomodulator, be conducive to this fungus resource of further Efficient Development.
The accompanying drawing explanation
The elutriant that Fig. 1 is the collection of China pilose spore HS002-II protein-polysaccharide DEAE Mierocrystalline cellulose-52 ion exchange chromatography is at the absorbancy curve of 490nm and 280nm; Tube number is the pipe number;
Fig. 2 is HS002-II agarose electrophoresis collection of illustrative plates; Lane is zone;
Fig. 3 is the detection collection of illustrative plates of HS002-II at automatic analyzer for amino acids; Left figure is the contrast collection of illustrative plates, and right figure is the sample collection of illustrative plates;
The HPLC collection of illustrative plates that Fig. 4 is HS002-II solution after 1-phenyl-3-methyl-5-pyrazolones ketone (PMP) derivatize; A is the contrast collection of illustrative plates, and B is the sample collection of illustrative plates;
The infrared spectra that Fig. 5 is HS002-II;
Fig. 6 is HS002-II's
1h-NMR collection of illustrative plates (A) with
13c-NMR collection of illustrative plates (B);
Fig. 7 is HS002-II atomic force microscope test pattern;
Fig. 8 is that HS002-II is to NO(A in mouse macrophage strain RAW264.7) and TNF-α (B) immunostimulant figure.
Embodiment
(1) submerged fermentation: access China pilose spore bacterial classification in substratum, inoculum size 6%(weight percent),, under 18 ℃ of conditions, standing cultivation 40 days, obtain the China pilose spore fermented liquid; Wherein substratum used comprises (weight percent): glucose 20%, peptone 5%, cicada pupa powder 10%, KH
2pO
41.0%, MgSO
4.7H
2o 0.5%, and surplus is sterilized water.
(2) alcohol precipitation: concentrated to the China pilose spore fermented liquid in step (1), obtain concentrated solution, add 4 times of concentrated solution volumes, concentration expressed in percentage by volume is 95% aqueous ethanolic solution, stirring and evenly mixing, being placed in 4 ℃ of refrigerator precipitations spends the night, in 4 ℃ of centrifugal 25min of 6000rpm, get centrifugal gained precipitation and obtain the China pilose spore Crude polysaccharides one time again;
(3) Deproteinization: a China pilose spore Crude polysaccharides of step (2) gained is water-soluble, obtain the aqueous solution of a China pilose spore Crude polysaccharides, trypsin solution is added in the aqueous solution of a China pilose spore Crude polysaccharides, tryptic weight is 1.5% of a China pilose spore Crude polysaccharides weight, then at 50 ℃ of water-bath 2h, the enzyme 15min that goes out under 100 ℃ again, be cooled to room temperature, and the centrifugal 30min of 10000rpm removes metaprotein and enzyme; The Sevage reagent (chloroform and the propyl carbinol that add 1/2nd supernatant liquor volumes in centrifugal gained supernatant liquor, wherein the volume ratio of chloroform and propyl carbinol is 4:1), violent jolting 15min, centrifugal except sub-cloud organic phase and middle egg white layer, repeat the step 3 centrifugal with Sevage reagent time and extremely produce without white precipitate, obtain extracting solution;
(4) dialysis: the dialysis tubing that the extracting solution of step (3) gained is 6000Da-10000Da with aperture is at the deionized water 96h that dialyses, this process can be removed oligosaccharides, pigment, organic solvent, inorganic salt etc., collect the extracting solution after dialysing, vacuum lyophilization obtains secondary China pilose spore Crude polysaccharides;
(5) purifying: the secondary China pilose spore Crude polysaccharides that step (4) is obtained obtains the secondary China pilose spore Crude polysaccharides aqueous solution of 3mg/mL with deionized water dissolving;
With deionized water balance diethylaminoethyl cellulose-52 ion exchange column (3cm * 26cm), by the secondary China pilose spore Crude polysaccharides aqueous solution through diethylaminoethyl cellulose 52 ion exchange column chromatographies, applied sample amount 5ml, flow velocity 1.5ml/min; Elutriant is the 0.1mol/L-1.0mol/L NaCl aqueous solution, flow velocity 1.5ml/min, and gradient elution, the elutriant of collection detects polysaccharide by the phenolsulfuric acid method and 280nm, 490nm detect protein (as Fig. 1), collects the elutriant of the second elution peak;
The elutriant of the second elution peak of collection is further purified through gel permeation chromatography (Sephadex G-200), the specification of chromatography column is 2.6cm * 100cm, applied sample amount 5ml, elutriant is 0.05mol/L phosphate buffered saline buffer (pH7.0)+0.15mol/LNaCl aqueous solution (wherein the volume ratio of phosphate buffered saline buffer and the NaCl aqueous solution is 2:1), flow velocity 0.5ml/min, the collected elutriant of gel permeation chromatography detects the polysaccharide peak by the phenolsulfuric acid method and 280nm detects protein peak, collect the elutriant of proteoglycan, through concentrated, the China pilose spore protein-polysaccharide that the dialysis tubing dialysis that aperture is 6000Da-10000Da and freeze-drying obtain the cotton-shaped homogeneous of white loose, called after HS002-II.
(1) submerged fermentation: access China pilose spore bacterial classification in substratum, inoculum size 5%(weight percent),, under 15 ℃ of conditions, standing cultivation 50 days, obtain the China pilose spore fermented liquid; Wherein substratum used comprises (weight percent): glucose 15%, peptone 3%, cicada pupa powder 6%, KH
2p
o4 0.6%, MgSO
4.7H
2o 0.1%, and surplus is sterilized water.
(2) alcohol precipitation: concentrated to the China pilose spore fermented liquid in step (1), obtain concentrated solution, add 5 times of concentrated solution volumes, concentration expressed in percentage by volume is 96% aqueous ethanolic solution, stirring and evenly mixing, being placed in 2 ℃ of refrigerator precipitations spends the night, in 2 ℃ of centrifugal 30min of 5000rpm, get centrifugal gained precipitation and obtain the China pilose spore Crude polysaccharides one time again;
(3) Deproteinization: a China pilose spore Crude polysaccharides of step (2) gained is water-soluble, obtain the aqueous solution of a China pilose spore Crude polysaccharides, trypsin solution is added in the aqueous solution of a China pilose spore Crude polysaccharides, tryptic weight is 1% of a China pilose spore Crude polysaccharides weight, then at 55 ℃ of water-bath 1.5h, the enzyme 15min that goes out under 100 ℃ again, be cooled to room temperature, and the centrifugal 30min of 10000rpm removes metaprotein and enzyme; The Sevage reagent (chloroform and the propyl carbinol that add 1/2nd supernatant liquor volumes in centrifugal gained supernatant liquor, wherein the volume ratio of chloroform and propyl carbinol is 4:1), violent jolting 15min, centrifugal except sub-cloud organic phase and middle egg white layer, repeat the step secondary centrifugal with Sevage reagent to producing without white precipitate, obtain extracting solution;
(4) dialysis: the dialysis tubing that the extracting solution of step (3) gained is 6000Da-10000Da with aperture is at the deionized water 80h that dialyses, this process can be removed oligosaccharides, pigment, organic solvent, inorganic salt etc., collect the extracting solution after dialysing, vacuum lyophilization obtains secondary China pilose spore Crude polysaccharides;
(5) purifying: the secondary China pilose spore Crude polysaccharides that step (4) is obtained obtains the secondary China pilose spore Crude polysaccharides aqueous solution of 25mg/mL with deionized water dissolving;
With deionized water balance diethylaminoethyl cellulose-52 ion exchange column (3cm * 26cm), by the secondary China pilose spore Crude polysaccharides aqueous solution through diethylaminoethyl cellulose 52 ion exchange column chromatographies, applied sample amount 5ml, flow velocity 1ml/min; Elutriant is the 0.1mol/L-1.0mol/L NaCl aqueous solution, flow velocity 1ml/min, and gradient elution, the elutriant of collection detects polysaccharide by the phenolsulfuric acid method and detects protein (result is consistent with Fig. 1) with 280nm, 490nm, collects the elutriant of the second elution peak;
The elutriant of the second elution peak of collection is further purified through gel permeation chromatography (Sephadex G-200), the specification of chromatography column is 2.6cm * 100cm, applied sample amount 5ml, elutriant is 0.05mol/L phosphate buffered saline buffer (pH7.0)+0.15mol/LNaCl aqueous solution (wherein the volume ratio of phosphate buffered saline buffer and the NaCl aqueous solution is 2:1), flow velocity 0.5ml/min, the collected elutriant of gel permeation chromatography detects the polysaccharide peak by the phenolsulfuric acid method and 280nm detects protein peak, collect the elutriant of proteoglycan, through concentrated, the China pilose spore protein-polysaccharide that the dialysis tubing dialysis that aperture is 6000Da-10000Da and freeze-drying obtain the cotton-shaped homogeneous of white loose, called after HS002-II.
(1) submerged fermentation: access China pilose spore bacterial classification in substratum, inoculum size 10%(weight percent),, under 22 ℃ of conditions, standing cultivation 30 days, obtain the China pilose spore fermented liquid; Wherein substratum used comprises (weight percent): glucose 25%, peptone 7%, cicada pupa powder 12%, KH
2pO
41.2%, MgSO
4.7H
2o 1%, and surplus is sterilized water.
(2) alcohol precipitation: concentrated to the China pilose spore fermented liquid in step (1), obtain concentrated solution, add 4 times of concentrated solution volumes, concentration expressed in percentage by volume is 90% aqueous ethanolic solution, stirring and evenly mixing, being placed in 5 ℃ of refrigerator precipitations spends the night, in 5 ℃ of centrifugal 30min of 4000rpm, get centrifugal gained precipitation and obtain the China pilose spore Crude polysaccharides one time again;
(3) Deproteinization: a China pilose spore Crude polysaccharides of step (2) gained is water-soluble, obtain the aqueous solution of a China pilose spore Crude polysaccharides, trypsin solution is added in the aqueous solution of a China pilose spore Crude polysaccharides, tryptic weight is 2% of a China pilose spore Crude polysaccharides weight, then at 45 ℃ of water-bath 2h, the enzyme 15min that goes out under 100 ℃ again, be cooled to room temperature, and the centrifugal 30min of 10000rpm removes metaprotein and enzyme; The Sevage reagent (chloroform and the propyl carbinol that add 1/2nd supernatant liquor volumes in centrifugal gained supernatant liquor, wherein the volume ratio of chloroform and propyl carbinol is 4:1), violent jolting 15min, centrifugal except sub-cloud organic phase and middle egg white layer, repeat the step 3 centrifugal with Sevage reagent time and extremely produce without white precipitate, obtain extracting solution;
(4) dialysis: the dialysis tubing that the extracting solution of step (3) gained is 6000Da-10000Da with aperture is at the deionized water 100h that dialyses, this process can be removed oligosaccharides, pigment, organic solvent, inorganic salt etc., collect the extracting solution after dialysing, vacuum lyophilization obtains secondary China pilose spore Crude polysaccharides;
(5) purifying: the secondary China pilose spore Crude polysaccharides that step (4) is obtained obtains the secondary China pilose spore Crude polysaccharides aqueous solution of 10mg/mL with deionized water dissolving;
With deionized water balance diethylaminoethyl cellulose-52 ion exchange column (3cm * 26cm), by the secondary China pilose spore Crude polysaccharides aqueous solution through diethylaminoethyl cellulose 52 ion exchange column chromatographies, applied sample amount 5ml, flow velocity 1.2ml/min; Elutriant is the 0.1mol/L-1.0mol/L NaCl aqueous solution, flow velocity 1ml/min, and gradient elution, the elutriant of collection detects polysaccharide by the phenolsulfuric acid method and detects protein (result is consistent with Fig. 1) with 280nm, 490nm, collects the elutriant of the second elution peak;
The elutriant of the second elution peak of collection is further purified through gel permeation chromatography (Sephadex G-200), the specification of chromatography column is 2.6cm * 100cm, applied sample amount 5ml, elutriant is 0.05mol/L phosphate buffered saline buffer (pH7.0)+0.15mol/LNaCl aqueous solution (wherein the volume ratio of phosphate buffered saline buffer and the NaCl aqueous solution is 2:1), flow velocity 0.5ml/min, the collected elutriant of gel permeation chromatography detects the polysaccharide peak by the phenolsulfuric acid method and 280nm detects protein peak, collect the elutriant of proteoglycan, through concentrated, the China pilose spore protein-polysaccharide that the dialysis tubing dialysis that aperture is 6000Da-10000Da and freeze-drying obtain the cotton-shaped homogeneous of white loose, called after HS002-II.
Below the embodiment to HS002-II Structural Identification or performance analysis:
Embodiment 4: physico-chemical property component and molecular weight detection
The China pilose spore protein-polysaccharide HS002-II that embodiment 1 is made, detecting total polysaccharides content by the phenolsulfuric acid method is 57.9 ℅, detecting protein content with coomassie brilliant blue (Bradford method) is 42.1%.With β-elimination reaction detect this protein-polysaccharide at the 240nm place without uv-absorbing, prove that its albumen is connected with N-acetyl cardohydrata-peptide linkage with polysaccharide.Utilize agarose electrophoresis to be judged its purity and molecular weight, concrete operations: first with the about 30min of 60V electrophoresis, to sample concentration behind 5% concentrated glue bottom, change the 120V constant voltage power supply into, when tetrabromophenol sulfonphthalein arrives 12% separation gel bottom (about 60min), stop electrophoresis, coomassie brilliant blue staining.Simultaneously with standard protein Marker (20,27,36,50,90,118KDa) compare, tetrabromophenol sulfonphthalein is made indicator, the molecular-weight average that obtains HS002-II is that 44KDa(is shown in Fig. 2).Utilize automatic analyzer for amino acids to obtain protein part and be comprised of 17 seed amino acids, 17 seed amino acids are specially: aspartic acid (Asp), Threonine (Thr), Serine (Ser), L-glutamic acid (Glu), glycine (Gly), L-Ala (Ala), Gelucystine (Cys), α-amino-isovaleric acid (Val), methionine(Met) (Met), Isoleucine (Ile), leucine (Leu), tyrosine (Tyr), phenylalanine (Phe), Methionin (Lys), Histidine (His), tryptophane (Trp) and arginine (Arg); Wherein aspartic acid (Asp), L-glutamic acid (Glu), glycine (Gly), L-Ala (Ala) content are maximum, the minimum (see figure 3) of Gelucystine (Cys) content.
The China pilose spore protein-polysaccharide HS002-II that embodiment 2 is made, detecting total polysaccharides content by the phenolsulfuric acid method is 55 ℅, detecting protein content with coomassie brilliant blue (Bradford method) is 45%.With β-elimination reaction detect this protein-polysaccharide at the 240nm place without uv-absorbing, prove that its albumen is connected with N-acetyl cardohydrata-peptide linkage with polysaccharide.Utilize agarose electrophoresis to be judged its purity and molecular weight, concrete operations: first with the about 30min of 60V electrophoresis, to sample concentration behind 5% concentrated glue bottom, change the 120V constant voltage power supply into, when tetrabromophenol sulfonphthalein arrives 12% separation gel bottom (about 60min), stop electrophoresis, coomassie brilliant blue staining.Simultaneously with standard protein Marker (20,27,36,50,90,118KDa) compare, tetrabromophenol sulfonphthalein is made indicator, the molecular-weight average that obtains HS002-II is 50KDa.Utilize automatic analyzer for amino acids to obtain protein part and be comprised of 17 seed amino acids, 17 seed amino acids are specially: aspartic acid (Asp), Threonine (Thr), Serine (Ser), L-glutamic acid (Glu), glycine (Gly), L-Ala (Ala), Gelucystine (Cys), α-amino-isovaleric acid (Val), methionine(Met) (Met), Isoleucine (Ile), leucine (Leu), tyrosine (Tyr), phenylalanine (Phe), Methionin (Lys), Histidine (His), tryptophane (Trp) and arginine (Arg); Wherein aspartic acid (Asp), L-glutamic acid (Glu), glycine (Gly), L-Ala (Ala) content are maximum, and Gelucystine (Cys) content is minimum.
The China pilose spore protein-polysaccharide HS002-II that embodiment 3 is made, detecting total polysaccharides content by the phenolsulfuric acid method is 60 ℅, detecting protein content with coomassie brilliant blue (Bradford method) is 40%.With β-elimination reaction detect this protein-polysaccharide at the 240nm place without uv-absorbing, prove that its albumen is connected with N-acetyl cardohydrata-peptide linkage with polysaccharide.Utilize agarose electrophoresis to be judged its purity and molecular weight, concrete operations: first with the about 30min of 60V electrophoresis, to sample concentration behind 5% concentrated glue bottom, change the 120V constant voltage power supply into, when tetrabromophenol sulfonphthalein arrives 12% separation gel bottom (about 60min), stop electrophoresis, coomassie brilliant blue staining.Simultaneously with standard protein Marker (20,27,36,50,90,118KDa) compare, tetrabromophenol sulfonphthalein is made indicator, the molecular-weight average that obtains HS002-II is 40KDa.Utilize automatic analyzer for amino acids to obtain protein part and be comprised of 17 seed amino acids, 17 seed amino acids are specially: aspartic acid (Asp), Threonine (Thr), Serine (Ser), L-glutamic acid (Glu), glycine (Gly), L-Ala (Ala), Gelucystine (Cys), α-amino-isovaleric acid (Val), methionine(Met) (Met), Isoleucine (Ile), leucine (Leu), tyrosine (Tyr), phenylalanine (Phe), Methionin (Lys), Histidine (His), tryptophane (Trp) and arginine (Arg); Wherein aspartic acid (Asp), L-glutamic acid (Glu), glycine (Gly), L-Ala (Ala) content are maximum, and Gelucystine (Cys) content is minimum.
Embodiment 5: monose forms
The China pilose spore protein-polysaccharide HS002-II5mg that embodiment 1, embodiment 2 or embodiment 3 make, add 2mol/L aqueous sulfuric acid 2ml, be placed in tool plug test tube, nitrogen sealing, 100 ℃ of hydrolysis 12h, be cooled to room temperature, with the barium sulfate neutralization, centrifugal, the supernatant liquor freeze-drying obtains lyophilized powder (being the HS002-II hydrolyzation sample), treats derivatize.Various monose and uronic acid standard substance are dissolved in to 0.3M(mol/L) in aqueous sodium hydroxide solution every kind of monose of preparation and uronic acid concentration be 5mmol/L(mM) monose and uronic acid standard solution, protein-polysaccharide HS002-II hydrolyzation sample is dissolved in to the HS002-II solution that in the 0.3M aqueous sodium hydroxide solution, preparation protein-polysaccharide HS002-II hydrolyzation sample concentration is 5mmol/L, then get respectively 75 μ l monose and uronic acid standard solution, get 75 μ lHS002-II solution, respectively add 50 μ l0.5M PMP methanol solutions, mix, 70 ℃ of water-bath 100min, be cooled to room temperature, add 75 μ l, 0.3M HCl aqueous solution neutralization, the centrifugal 3min of 10000rpm, supernatant liquor is transferred to another clean centrifuge tube, add water to 1ml, add the equal-volume chloroform, fully concussion, collect water after stratification, in order to remove PMP, the impurity such as superfluous reaction reagent, the water of collecting, repeat " to add water to 1ml, add the equal-volume chloroform, fully concussion, stratification " step 3 time, cross 0.22 μ m film, obtain respectively after the PMP derivatize HS002-II solution after monose and uronic acid standard solution and PMP derivatize, treat that HPLC detects.
HPLC condition: pillar Inertsil-ODS-SP(5 μ m, 4.6 * 250mm), detect wavelength 245nm, flow velocity 1.0ml/min, column temperature: room temperature, inject volume: HS002-II solution after monose and uronic acid standard solution or 10 μ l PMP derivatizes after 20 μ l PMP derivatizes, mobile phase A (acetonitrile): Mobile phase B (0.05mol/L phosphate buffered saline buffer (PH6.9))=17:83(volume ratio).
As Fig. 4, corresponding monose and uronic acid standard substance, the monose of embodiment 1HS002-II polysaccharide part consists of by seminose, ribose, glucose, semi-lactosi, wood sugar and pectinose and forms, and the ratio of its amount of substance is 6.47:2.27:65.89:16.17:2.13:4.26; Illustrate that HS002-II take glucose as main chain, and the polysaccharide that contains branch.
Corresponding monose and uronic acid standard substance, the monose of embodiment 2HS002-II polysaccharide part consists of by seminose, ribose, glucose, semi-lactosi, wood sugar and pectinose and forms, and the ratio of its amount of substance is 6.42:2.20:65.95:16.10:2.10:4.30; Illustrate that HS002-II take glucose as main chain, and the polysaccharide that contains branch.
Corresponding monose and uronic acid standard substance, the monose of embodiment 3HS002-II polysaccharide part consists of by seminose, ribose, glucose, semi-lactosi, wood sugar and pectinose and forms, and the ratio of its amount of substance is 6.51:2.32:65.85:16.23:2.18:4.20; Illustrate that HS002-II take glucose as main chain, and the polysaccharide that contains branch.
Embodiment 6:FITR
Get the China pilose spore protein-polysaccharide HS002-II that 5mg embodiment 1, embodiment 2 or embodiment 3 make, use the KBr compressing tablet, U.S. Nicolet5700 infrared spectrometer 4000-600cm
-1infrared scan.
As Fig. 5,1021.8cm
-1the vibration of O-H angle, the absorption peak of C-H is more weak, at 3000-2800cm
-1between, so 2900.0cm
-1the C-H stretching vibration, 1252.45cm
-1-1070.58cm
-1be the vibration of C-H angle, this compound is carbohydrate.The O-H stretching vibration is at 3600-3200cm
-1one broad peak appears, so 3400.0cm
-1it is the O-H stretching vibration; 1650.1cm
-1be-the asymmetric stretching vibration of C=O in CONH 1418.39cm
-1it is the C-N symmetrical stretching vibration; At 1635.18cm
-1the sharper absorption peak in left and right is C=O (carbonyl) stretching vibration and the vibration of N-H angle; At 1373.45cm
-1the weak absorption peak in left and right is C-H angle absorption of vibrations; At 1021cm
-1absorption peak show that this molecule all contains pyranoid ring; 919.82cm
-1, 896.51cm
-1, 800.34cm
-1illustrate in sugar chain exist simultaneously α-, β-glycosidic link.
Embodiment 7: methylation analysis
Get the China pilose spore protein-polysaccharide HS002-II sample that 2mg embodiment 1, embodiment 2 or embodiment 3 make and be dissolved in 1mlDMSO, logical nitrogen-sealed, ultrasonic a moment hydrotropy; then according to Ciucanu; et al. method methylated preparation (Ciucanu, L. ， & Kerek, F..A simple and repid method for permethylation of carbohydrates.Carbohydrate Research, 131,209-217).
HS002-II is after cyclonite, then, through acid hydrolysis, reduction, acetylize is prepared into part methyl ALDI alcohol acetic ester derivative, carries out GC-MS analysis (in Table 1).As seen from table: (1) 1 → 4 is connected to main of bonding in HS002-II, and 1 → 6 and 1 → 3 of bonding takes second place, and non-reducing end has pectinose (Ara); (2) this polysaccharide PRELIMINARY RESULTS is inferred as glucose (Glc) with 1 → 4, and 6 connect, and semi-lactosi (Gal), seminose (Man) are with 1 → 6 key, and wood sugar (Xyl) connects with 1 → 4, and ribose (Rib) connects with 1 → 3 key, and Ara connects with end; (3) to add up be that 32.93, Gal residue summation is 7.1 to the Glc residue, and all kinds of residue molar ratios are Glcp:Galp:Manp:Arap:Ribp:Xylp=32.93:8.2:3.08:1.93:1.12:1 .05, consistent with the result of HPLC;
Table 1HS002-II methylation analysis
Embodiment 8: nucleus magnetic resonance
Get the China pilose spore protein-polysaccharide HS002-II that 50mg embodiment 1, embodiment 2 or embodiment 3 make and be dissolved in the 1ml deuterium-oxide, Switzerland Bruker-AVIII500M carries out 500MHz NMR scanning.
1h-NMR is mainly used in determining glycosidic link configuration in polysaccharide structures.
1in the H-NMR spectrogram, the signal great majority of polysaccharide concentrate in the narrow and small scope of δ 3.2-5.5ppm, and δ 3.5-4.5ppm is sugar ring proton signal, and δ 1.9ppm shows the hydrogen proton of residue of protein part N ethanoyl.The proton peak of δ 4.7-5.2ppm anomer hydrogen further confirm this polysaccharide contain simultaneously α-, β-glycosidic link, consistent with infrared spectra; And because the peak integrated value is very little, show that its content in sample is few, thus not the major constituent glucose signals of HS002-II, and then can infer that the glucose in this component exists with the β type.
13c-NMR can be by anomeric carbon the number at resonance region (δ 90-110) peak determine quantity and the relative content of saccharide residue.Usually, the chemical shift of α type glucosides anomeric carbon is in δ 95-103 scope, and the chemical shift of most β type glucosides anomeric carbon is positioned at δ 103-110.In addition, by
13the C-NMR characteristic signal can be determined some saccharide residue or functional group, as the carboxylic acid carbon signal of uronic acid or kharophen signal appear at low place δ 170-180; The methyl carbon signal of 6 desoxy sugars appears at high field region δ 15-20; The methyl carbon signal of ethanoyl appears at high field district δ 22-23.5.From the carbon spectrum, find out, δ 107.83 is residue (1 → 4, the anomeric carbon signal of 6)-linked β-D-pyrans glucosan, the anomeric carbon signal that δ 106.93 is residue (1 → 4)-linked β-D-pyrans glucosan, the anomeric carbon signal that δ 103.38 is side chain residue (1 → 6)-linked β-D-mannosans, the anomeric carbon signal that δ 102.94 is side chain residue (1 → 6)-linked β-D-Polygalactan, the anomeric carbon signal that δ 101.23 is residue (1 → 3)-linked α-D-core glycan, the anomeric carbon signal that δ 100.67 is residue (1 → 4)-linked α-D-xylan, the CH that δ 22.531 is the N-ethanoyl
3signal, δ 174.140 is N kharophen anomeric carbon signal, has confirmed that the HS002-II component is protein-polysaccharide.The concrete structure unit is as shown in the structural formula I.
Embodiment 9: the atomic force microscope test
The embodiment 1 that is 5%-10% by mass percentage concentration, the China pilose spore protein-polysaccharide HS002-II aqueous solution that embodiment 2 or embodiment 3 make spreads upon the mica on-chip testing, the atom that obtains this polysaccharide is tried hard to, as Fig. 7, wherein Fig. 7 a is 2 dimension atomic power shape appearance figures, Fig. 7 b is 3 dimension stereoscopic pattern figure, Fig. 7 c is each topography measurement value, the diameter that can find out sample particle from result is 40-70nm, be highly 1.2nm-2.8nm, thereby can judge that great majority are formed by a plurality of molecular aggregatess, further confirm that HS002-II is the stretching, extension stiff chain with multiple-limb.
The present invention has set up the method for China pilose spore protein-polysaccharide extraction purifying, obtains homogeneous polysaccharide, and its primary structure of preliminary study, and the discussion of further carrying out biological activity and structure activity relationship is had to important meaning.
Embodiment 10: immunoregulatory activity
The present invention detected China that embodiment 1, embodiment 2 or embodiment 3 make by hair spore HS002-II protein-polysaccharide the multiplication capacity to NO and tumor necrosis factor TNF-alpha.
Mouse monokaryon scavenger cell RAW264.7 cell with RPMI1640 (containing 20 μ g/ml PMB, 10% foetal calf serum, 100U/ml penicillin, 100U/ml Streptomycin sulphate) complete culture solution in 37 ℃, 5%CO
2in cell culture incubator, cultivate, 4th ~ 6 acute pyogenic infection of finger tip count vegetative period cell for the experiment.
In 96 orifice plates, every hole adds RAW264.7 cell suspension (3 * 10
5individual/ml, the trypan blue cell counting, determine that cell survival rate is more than 95%) 100 μ l, be placed in 37 ℃, 5%CO
2incubator is hatched 2h, discards nutrient solution in each hole, adds fresh RPMI1640 complete culture solution 100 μ l/ holes (positive control LPS stimulates hole to add the RPMI1640 nutrient solution that does not contain PMB), continues to cultivate 24h.Suck the substratum supernatant, then add respectively each 200 μ l of HS002-II diluent (12.5,25,50,100 μ g/ml) of RPMI1640 nutrient solution, LPS diluent (1 μ g/ml) or different concns, repeat 5 holes.37 ℃, 5%CO
224h in incubator, be transferred to 96 new porocyte culture plates by cell conditioned medium liquid 100 μ l, and every hole adds Griess A and Griess B mix reagent (1: 1, volume ratio) 100 μ l, dark place lucifuge reaction 20min, measure the OD value in the 540nm place, three groups of parallel tests are averaged.
In 24 porocyte culture plates, every hole adds RAW264.7 cell suspension (2.0 * 10
5individual/ml, the trypan blue cell counting, determine that cell survival rate is more than 95%) 500 μ l, be placed in 37 ℃, 5%CO
2cultivate 2h in incubator, discard nutrient solution, add fresh RPMI1640 complete culture solution 500 μ l (positive control LPS stimulates hole to add the RPMI1640 nutrient solution that does not contain PMB), continue to cultivate 24h.Suck the substratum supernatant, then add respectively each 500 μ l of HS002-II diluent (12.5,25,50,100,200 μ g/ml) of RPMI1640 nutrient solution, LPS diluent (1 μ g/ml) or different concns, be placed in 37 ℃, 5%CO
2cultivate 24h in incubator.The collecting cell supernatant, measure cytokine content according to the explanation of mouse cytokine TNF-α ELISA detection kit: get the enzyme plate of envelope antigen, add respectively standard substance (1000,500,250,125,62.5,31.25,0pg/ml) or sample 100 μ l/ holes, hatch 90min for 37 ℃; Get rid of liquid in enzyme plate, pat dry, every hole adds biotin labeled anti-mouse specific antibody working fluid 100 μ l, hatches 60min for 37 ℃; Get rid of liquid in enzyme plate, the 1min left and right is soaked in PBS300 μ l/ hole washing 3 times at every turn; Every hole adds avidin-superoxide enzyme complex (ABC) working fluid (37 ℃ of pre-equilibration 30min) 100 μ l, hatches 30min for 37 ℃; Get rid of liquid in enzyme plate, 1min is soaked in PBS300 μ l/ hole washing 5 times at every turn; Every hole adds at 1,37 ℃ of lucifuge 15 ~ 20min of the TMB of 37 ℃ of balance 30min nitrite ion, 90 μ, with several holes of reference liquid high density, occurs that the gradient blueness is as the criterion; Every hole adds TMB stop buffer 100 μ l, and blueness transfers yellow to.Measure the OD value at the 450nm place with microplate reader.According to typical curve, calculate each cytokine content, three groups of parallel tests are averaged.
As seen from Figure 8, with control group, compare, positive control LPS can significantly improve the ability that the RAW264.7 cell discharges NO by the utmost point.HS002-II can significantly strengthen the RAW264.7 cell and discharge the ability of NO and the ability that stimulates rear its TNF secretion-α in 12.5 ~ 100 μ g/ml concentration ranges, and presents the finite concentration dependency.HS002-II has potential immunoregulatory activity to scavenger cell.
Claims (9)
1. a China pilose spore protein-polysaccharide, is characterized in that, the albumen that the polysaccharide that is 55 ℅-60% by weight percentage and weight percentage are 40 ℅-45% forms; Described polysaccharide consist of seminose, ribose, glucose, semi-lactosi, wood sugar and pectinose, the structural unit of polysaccharide is that to take 1 → 3 α-ribose connected, 1 → 4 α-wood sugar connected and 1 → 4 β-glucose connected be main chain, and on the C6 position of β-glucose respectively by beta galactose and β-seminose replace, α-pectinose is terminal group; The ratio of the amount of substance of described seminose, ribose, glucose, semi-lactosi, wood sugar and pectinose is 6.42-6.51:2.20-2.32:65.85-65.95:2.10-2.18:4.20-4.30.
2. China pilose spore protein-polysaccharide according to claim 1, it is characterized in that, described α-ribose is α-D-ribose, α-wood sugar is alpha-D-xylose, β-glucose is β-D-Glucose, beta galactose is β-D-semi-lactosi, and β-seminose is β-D-MANNOSE, and α-pectinose is α-L-arabinose.
3. China pilose spore protein-polysaccharide according to claim 1 and 2, is characterized in that, the molecular-weight average of described China pilose spore protein-polysaccharide is 40KDa-50KDa.
4. according to the preparation method of the described China pilose spore protein-polysaccharide of claim 1-3 any one, it is characterized in that, comprise step:
(1) submerged fermentation: access China pilose spore bacterial classification in substratum, inoculum size 5%-10%, under 15-22 ℃ of condition, standing cultivation 30-50 days, obtain the China pilose spore fermented liquid; Wherein substratum used comprises: glucose 15%-25%, peptone 3%-7%, cicada pupa powder 6%-12%, KH
2pO
40.6%-1.2%, MgSO
4.7H
2o 0.1%-1.0%; % is weight percentage;
(2) alcohol precipitation: the China pilose spore fermented liquid in step (1) is concentrated, obtain concentrated solution, then add aqueous ethanolic solution, stirring and evenly mixing, precipitation is spent the night, centrifugal, gets centrifugal gained precipitation and obtains the China pilose spore Crude polysaccharides one time;
(3) Deproteinization: by the aqueous solution protease hydrolyzed of a China pilose spore Crude polysaccharides of step (2) gained, enzyme centrifugal metaprotein and the enzyme removed go out, centrifugal gained supernatant liquor is centrifugal except sub-cloud organic phase and middle egg white layer with organic solvent again, repeat by the centrifugal step of organic solvent until, without the white precipitate generation, obtain extracting solution;
(4) dialysis: the dialysis tubing that the extracting solution of step (3) gained is 6000Da-10000Da with aperture is dialysed in deionized water, collects the extracting solution after dialysing, and vacuum lyophilization obtains secondary China pilose spore Crude polysaccharides;
(5) purifying: the secondary China pilose spore Crude polysaccharides that step (4) is obtained obtains the secondary China pilose spore Crude polysaccharides aqueous solution with deionized water dissolving, through diethylaminoethyl cellulose-52(DEAE Mierocrystalline cellulose-52) the ion exchange column chromatography, the elutriant of collecting detects polysaccharide and detects albumen by the lowry method by the phenolsulfuric acid method, collect the elutriant of the second elution peak through gel permeation chromatography, the collected elutriant of gel permeation chromatography detects polysaccharide and protein peak by phenolsulfuric acid method and lowry method, collect the elutriant of common proteoglycan, through concentrated, dialysis and freeze-drying obtain the cotton-shaped China pilose spore protein-polysaccharide of white loose, called after HS002-II.
5. preparation method according to claim 4, is characterized in that, in step (2), the consumption of described aqueous ethanolic solution is 4 times-5 times of concentrated solution volume; The concentration expressed in percentage by volume of described aqueous ethanolic solution is 90 ℅-96%.
6. preparation method according to claim 4, is characterized in that, in step (3), described proteolytic enzyme is trypsinase; The 1%-2% that the weight of described proteolytic enzyme is a China pilose spore Crude polysaccharides weight.
7. preparation method according to claim 4, is characterized in that, in step (3), described organic solvent is chloroform and propyl carbinol, and wherein the volume ratio of chloroform and propyl carbinol is 4:1.
8. preparation method according to claim 4, is characterized in that, in step (5), the concentration of the described secondary China pilose spore Crude polysaccharides aqueous solution is 3mg/mL-25mg/mL.
9. according to the application of the described China pilose spore protein-polysaccharide of claim 1-3 any one, it is characterized in that the application of described China pilose spore protein-polysaccharide in preparing immunomodulator.
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