CN114106211A - Yunnan chanterelle polysaccharide and separation and purification method thereof - Google Patents
Yunnan chanterelle polysaccharide and separation and purification method thereof Download PDFInfo
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- CN114106211A CN114106211A CN202111336779.0A CN202111336779A CN114106211A CN 114106211 A CN114106211 A CN 114106211A CN 202111336779 A CN202111336779 A CN 202111336779A CN 114106211 A CN114106211 A CN 114106211A
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- chanterelle
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- 150000004676 glycans Chemical class 0.000 title claims abstract description 117
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 117
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 117
- 235000015722 Cantharellus cibarius Nutrition 0.000 title claims abstract description 100
- 244000191482 Cantharellus cibarius Species 0.000 title claims abstract description 100
- 238000000926 separation method Methods 0.000 title claims abstract description 22
- 238000000034 method Methods 0.000 title claims abstract description 21
- 238000000746 purification Methods 0.000 title claims abstract description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 39
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- WQZGKKKJIJFFOK-PHYPRBDBSA-N alpha-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 abstract description 8
- PYMYPHUHKUWMLA-WDCZJNDASA-N arabinose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-WDCZJNDASA-N 0.000 abstract description 8
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- 150000003214 pyranose derivatives Chemical class 0.000 description 4
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- PNNNRSAQSRJVSB-SLPGGIOYSA-N Fucose Natural products C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C=O PNNNRSAQSRJVSB-SLPGGIOYSA-N 0.000 description 3
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 3
- YVNQAIFQFWTPLQ-UHFFFAOYSA-O [4-[[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfophenyl)methyl]amino]-2-methylphenyl]methylidene]-3-methylcyclohexa-2,5-dien-1-ylidene]-ethyl-[(3-sulfophenyl)methyl]azanium Chemical compound C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S(O)(=O)=O)C)C=C1 YVNQAIFQFWTPLQ-UHFFFAOYSA-O 0.000 description 3
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 description 3
- 229910052799 carbon Inorganic materials 0.000 description 3
- 125000000524 functional group Chemical group 0.000 description 3
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- CVBNMWXECPZOLM-UHFFFAOYSA-N Rhamnetin Natural products COc1cc(O)c2C(=O)C(=C(Oc2c1)c3ccc(O)c(O)c3O)O CVBNMWXECPZOLM-UHFFFAOYSA-N 0.000 description 2
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- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 2
- 125000001434 methanylylidene group Chemical group [H]C#[*] 0.000 description 2
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 2
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- RYVMUASDIZQXAA-UHFFFAOYSA-N pyranoside Natural products O1C2(OCC(C)C(OC3C(C(O)C(O)C(CO)O3)O)C2)C(C)C(C2(CCC3C4(C)CC5O)C)C1CC2C3CC=C4CC5OC(C(C1O)O)OC(CO)C1OC(C1OC2C(C(OC3C(C(O)C(O)C(CO)O3)O)C(O)C(CO)O2)O)OC(CO)C(O)C1OC1OCC(O)C(O)C1O RYVMUASDIZQXAA-UHFFFAOYSA-N 0.000 description 2
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- AEMOLEFTQBMNLQ-BZINKQHNSA-N D-Guluronic Acid Chemical compound OC1O[C@H](C(O)=O)[C@H](O)[C@@H](O)[C@H]1O AEMOLEFTQBMNLQ-BZINKQHNSA-N 0.000 description 1
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- AEMOLEFTQBMNLQ-VANFPWTGSA-N D-mannopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@@H]1O AEMOLEFTQBMNLQ-VANFPWTGSA-N 0.000 description 1
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
- C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
- C07D249/04—1,2,3-Triazoles; Hydrogenated 1,2,3-triazoles
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Abstract
The invention provides a Yunnan chanterelle polysaccharide and a separation and purification method thereof, wherein the Yunnan chanterelle polysaccharide is prepared by pretreatment, water leaching, alcohol precipitation, decoloration treatment, separation and purification, the chanterelle polysaccharide extracted by the invention does not contain protein, polypeptide and nucleic acid, the extraction rate of the chanterelle crude polysaccharide is 9.38%, and the polysaccharide content in the chanterelle crude polysaccharide is 30.2%. The polysaccharide mainly contains 8 monosaccharides such as mannose, rhamnose, glucuronic acid, glucose, galactose, xylose, arabinose and L-fucose.
Description
Technical Field
The invention relates to the field of polysaccharide separation and purification, and particularly relates to a Yunnan chanterelle polysaccharide and a separation and purification method thereof.
Background
Chanterelle is classified in the family of the order gallinaceae. The chanterelle usually coexists with roots of broad-leaved trees such as pine, oak, fir, etc., has small and golden fruiting body, unique flavor, good crispness and tenderness, and rich nutrition, and is one of six famous mycorrhizal edible fungi in the world. About 70 species of the genus gallinacea are currently known globally, but the exact species and number need to be further examined. Chanterelle is widely distributed in temperate, subtropical, and tropical regions of europe, asia, and north america, and also in africa and oceania. At present, 17 kinds of chanterelle are reported in China. It is presumed that the southwest mountain, subalpine and tropical regions of China are a diversity distribution center of the genus gallinacea.
The chanterelle polysaccharide is mainly obtained from both fruiting bodies and mycelia, for example, CN201310480649.3, a method for extracting chanterelle mycelia polysaccharide, which is used for extracting polysaccharide from chanterelle mycelia. "the technological requirements for separation and extraction of mycelium polysaccharides and extracellular polysaccharides of chanterelle" was published in Liu Cheng Rong et al, the school newspaper of Chifeng academy of academic records, No. 8 of Chifeng, published in 2008, and discloses that the factors influencing the extraction of mycelium polysaccharides of chanterelle are extraction temperature, ethanol concentration and extraction time. None of the above patents mention how to separate and purify polysaccharides from chanterelle sporocarp.
Disclosure of Invention
In view of the above, the invention provides a separation and purification method of chanterelle Yunnanensis polysaccharide, and the polysaccharide prepared by the separation and purification method does not contain protein, polypeptide and nucleic acid and has high extraction rate.
The technical scheme of the invention is realized as follows:
(1) pretreatment: taking a Yunnan chanterelle fruiting body sample, crushing and sieving to prepare Yunnan chanterelle powder, wherein the material-liquid ratio is 1 g: 10-30 mL of ethanol solution, soaking for 12-16 h after ultrasonic extraction, centrifuging the soaked Yunnan chanterelle powder at the rotating speed of 3500-4500 r/min for 8-12 min, and removing supernatant to obtain filter residue.
(2) Water leaching: taking filter residues, and mixing the filter residues according to the material-liquid ratio of 1 g: adding 15-25 mL of distilled water, leaching in a water bath at 70-90 ℃ for 1.5-3 h, filtering, repeatedly extracting for 2-4 times, combining filtrates, and performing rotary evaporation and concentration to obtain a leaching solution.
(3) Alcohol precipitation: putting the leaching solution into a conical flask, and putting the conical flask on a magnetic stirrer according to the material-liquid ratio of 1 mL: adding 3-5 mL of ethanol solution, placing the mixture in a refrigerator with the temperature controlled to be 2-6 ℃ for refrigerating for 20-26 h, centrifuging, and collecting precipitates.
(4) And (3) decoloring treatment: adding the precipitate prepared in the step (3) into a mixture with a feed-liquid ratio of 1 g: dissolving 15-25 mL of distilled water to obtain an aqueous solution of a precipitate, mixing the aqueous solution of the precipitate with a Sevag reagent, extracting to obtain a first supernatant, adding the first supernatant into macroporous resin, decoloring to obtain a second supernatant, adding the second supernatant into an ethanol solution, placing the mixture into a refrigerator, standing for 12-16 h, centrifuging, collecting a precipitate, freezing the precipitate at-20 ℃ for 24h to obtain crude Yunnan chanterelle polysaccharide, and freeze-drying the crude Yunnan chanterelle polysaccharide to obtain the crude Yunnan chanterelle polysaccharide.
(5) Separation: dissolving the Yunnan chanterelle crude polysaccharide prepared in the step (4) in water to prepare a Yunnan chanterelle crude polysaccharide solution, wherein the material-liquid ratio is 1 g: 20-30 mL, separating the crude polysaccharide solution of the Yunnan chanterelle by using column chromatography, collecting CY-1 and CY-2 elution components as an eluent, and concentrating to obtain CY-1 and CY-2 elution component concentrated solutions.
(6) And (3) purification: and (3) putting the CY-1 and CY-2 elution component concentrated solutions separated in the step (5) into a dialysis bag, dialyzing with tap water and distilled water respectively, and freeze-drying for 24 hours after dialysis to obtain the Yunnan chanterelle polysaccharide. Further, in the step (1), the ultrasonic extraction is ultrasonic at 30-50 ℃ for 20-40 min, and the ultrasonic power is 50-150 Hz.
Further, the stirring speed of the magnetic stirrer is 50-150 r/min, and the mass fraction of the ethanol solution is 95%.
Further, in the step (4), the mass ratio of the precipitate to the Sevag reagent is 1:1, and the Sevag reagent is prepared from chloroform and n-butanol in a volume ratio of 4: 1.
Further, in the step (4), the extraction step includes mixing and oscillating the precipitate and the Sevag reagent for 20min, standing for 30min, collecting the upper layer of extract, and continuously extracting the upper layer of extract for 3 times to obtain a first supernatant.
Further, in the step (5), the concentration of the sodium chloride solution is 0.1-0.2 mol/L. Further, in the step (5), DEAE-52 cellulose (diethylaminoethyl cellulose 52) is used for the column chromatography.
Further, in the step (6), the tap water is dialyzed for 24 hours, the tap water is changed every 6 hours, then the distilled water is dialyzed for 12 hours, and the distilled water is changed every 4 hours, so that the concentrated solution is prepared.
Further, in the step (4), the model of the macroporous resin is SP 825.
Further, in the step (4), the macroporous resin adsorption step is as follows: and (3) putting the macroporous resin and the first supernatant into a triangular flask with a plug, and decoloring for 5-7 hours in a constant-temperature shaking table.
Further, in the step (4), the temperature of the constant temperature shaking table is 37 ℃, and the frequency is 150 r/min.
Further, in the step (5), the elution speed is 0.1-1 mL/min.
Further, the freeze drying temperature is-75 to-85 ℃, and the time is 23 to 25 hours.
Compared with the prior art, the invention has the beneficial effects that:
according to the method for separating and purifying the chanterelle polysaccharide, the extracted chanterelle polysaccharide does not contain protein, polypeptide and nucleic acid, the extraction rate of the chanterelle crude polysaccharide is 9.38%, and the polysaccharide content in the chanterelle crude polysaccharide is 30.2%. The polysaccharide mainly contains 8 monosaccharides such as mannose, rhamnose, glucuronic acid, glucose, galactose, xylose, arabinose and L-fucose.
Drawings
FIG. 1 glucose Standard Curve
FIG. 2 Standard Curve of proteins
FIG. 3 DEAE-52 cellulose chromatogram elution curve of Yunnan chanterelle polysaccharide
FIG. 4 Standard Curve for molecular weight determination
FIG. 5 GPC measurement chart of chanterelle CY-1 polysaccharide
FIG. 6 GPC measurement chart of chanterelle CY-2 polysaccharide
FIG. 7 chromatogram of derivatization products of monosaccharide standards
In the figure, the chromatographic peaks from left to right are respectively: guluronic acid, mannuronic acid, mannose, ribose, rhamnose, glucuronic acid, galactosamine, glucose, galacturonic acid, galactose, xylose, arabinose and L-fucose.
FIG. 8 chromatogram of derivatization product of polysaccharide hydrolysis sample of chanterelle Yunnan CY-1
In the figure, the chromatographic peaks from left to right are respectively: mannose, rhamnose, glucuronic acid, glucose, galactose, xylose, arabinose, and L-fucose.
FIG. 9 chromatogram of derivative of Yunnan chanterelle CY-2 polysaccharide hydrolysis sample
In the figure, the chromatographic peaks from left to right are respectively: mannose, rhamnose, glucuronic acid, glucose, galactose, xylose, arabinose, and L-fucose.
FIG. 10 ultraviolet spectrophotometric wavelength scanning chart of chanterelle CY-1 polysaccharide
FIG. 11 ultraviolet spectrophotometric wavelength scan of chanterelle CY-2 polysaccharide
FIG. 12 Infrared spectrum of chanterelle CY-1 polysaccharide
FIG. 13 Infrared spectrum of chanterelle CY-2 polysaccharide
FIG. 14 of chanterelle CY-1 polysaccharide from Yunnan1H-NMR
FIG. 15 preparation of chanterelle CY-1 polysaccharide from Yunnan13C-NMR
FIG. 16 of chanterelle CY-2 polysaccharide from Yunnan1H-NMR
FIG. 17 of chanterelle CY-2 polysaccharide from Yunnan13C-NMR
FIG. 18 electron microscope scanning image of polysaccharide of chanterelle CY-1 and CY-2
Detailed Description
In order to better understand the technical content of the invention, specific examples are provided below to further illustrate the invention.
The experimental methods used in the examples of the present invention are all conventional methods unless otherwise specified.
The materials, reagents and the like used in the examples of the present invention can be obtained commercially without specific description.
TABLE 1 apparatus
TABLE 2 reagents
Example 1 extraction method of crude polysaccharide of chanterelle Yunnanensis
(1) Pretreatment: taking a Yunnan chanterelle fruiting body sample, crushing and sieving to obtain Yunnan chanterelle powder, adding 95% ethanol solution by mass, wherein the material-liquid ratio is 1 g: 20mL, soaking for 14h after ultrasonic extraction, centrifuging the soaked Yunnan chanterelle powder at the rotating speed of 4000r/min for 8-12 min, and removing supernatant to obtain filter residue.
(2) Water leaching: taking filter residues, and mixing the filter residues according to the material-liquid ratio of 1 g: adding 20mL of distilled water, leaching in a water bath at 80 deg.C for 2h, filtering, extracting repeatedly for 3 times, mixing filtrates, rotary evaporating and concentrating to obtain leaching solution.
(3) Alcohol precipitation: putting the leaching solution into a conical flask, and putting the conical flask on a magnetic stirrer according to the material-liquid ratio of 1 mL: adding 95% ethanol solution into 4mL, refrigerating in a refrigerator at 4 deg.C for 24h, centrifuging at 4000r/min for 10min, and collecting precipitate.
(4) And (3) decoloring treatment: adding the precipitate prepared in the step (3) into a mixture with a feed-liquid ratio of 1 g: dissolving 20mL of distilled water to obtain an aqueous solution of a precipitate, mixing the aqueous solution of the precipitate with a Sevag reagent, wherein the Sevag reagent is prepared from chloroform and n-butyl alcohol in a volume ratio of 4:1, placing the precipitate and the Sevag reagent into a separating funnel, mixing and oscillating for 20min, standing for 30min, collecting an upper layer extract, continuously extracting the upper layer extract for 3 times to obtain a first supernatant, placing the first supernatant and SP825 macroporous resin into a triangular flask with a stopper, decoloring for 6h in a constant temperature shaking table, keeping the temperature of the constant temperature shaking table at 37 ℃, controlling the frequency at 150r/min to obtain a second supernatant, adding the second supernatant into an ethanol solution with the mass fraction of 95%, standing for 14h in a refrigerator, centrifuging, placing the precipitate at-20 ℃ for freezing for 24h to obtain crude Yunnan chanterelle crude polysaccharide, freeze-drying the Yunnan chanelle crude polysaccharide at the temperature of-80 ℃, the time is 24h, and the Yunnan chanterelle crude polysaccharide is prepared.
Example 2 separation method of crude polysaccharide of chanterelle Yunnanensis
Dissolving the Yunnan chanterelle crude polysaccharide prepared in example 1 in water to prepare a Yunnan chanterelle crude polysaccharide solution, wherein the material-liquid ratio is 1 g: 25mL, separating the crude polysaccharide solution of the Yunnan chanterelle by using a DEAE-52 column chromatography, eluting with sodium chloride solution and distilled water with the concentrations of 0.1mol/L, 0.2mol/L, 0.4mol/L, 0.8mol/L and 1.0mol/L respectively, collecting elution components of CY-0, CY-1, CY-2, CY-4, CY-8 and CY-10, and concentrating to obtain concentrated solutions of the elution components of CY-0, CY-1, CY-2, CY-4, CY-8 and CY-10.
Example 3 purification method of chanterelle Yunnanensis polysaccharide
And (2) putting the CY-1 and CY-2 elution component concentrated solutions obtained by separation in the embodiment 2 into a dialysis bag, dialyzing with tap water for 24 hours, changing the tap water every 6 hours, dialyzing with distilled water for 12 hours, changing the distilled water every 4 hours, and freeze-drying at the temperature of-80 ℃ for 24 hours after dialysis to obtain the Yunnan chanterelle polysaccharide.
Comparative example 1
In addition to example 1, the decolorization test was performed by replacing the SP825 macroporous resin with HP 2MGL, HP 20, and SP 850, respectively.
Test example 1 determination of polysaccharide content in chanterelle Yunnanensis
(1) Detection method
1. Method for determining total sugar of sample
1.1, precisely weighing 1.0012g of glucose standard substance, drying at 105 ℃ to constant weight, cooling for 0.5h in a dryer, precisely weighing 0.5003g, adding a small amount of distilled water to dissolve the glucose standard substance, fixing the volume in a 50mL volumetric flask to prepare a glucose standard solution with the concentration of 10mg/mL, precisely sucking 1mL of stock solution, fixing the stock solution in a 100mL volumetric flask, adding distilled water to dissolve and dilute the stock solution to a scale, and preparing the glucose standard solution with the concentration of 0.1mg/mL for later use.
1.2 creation of Standard Curve
0mL, 0.4mL, 0.8mL, 1.2mL, 1.6mL, 2.0mL of the glucose standard solution was removed and placed in 7 tubes, and distilled water was added to each tube to make up to 2 mL. Then adding 1mL of 5% phenol solution into each sample, shaking up, rapidly adding 5mL of concentrated sulfuric acid, shaking, mixing, standing for 10min, heating in 100 ℃ water bath for 15min, and rapidly cooling to room temperature. The absorbance was measured at 490 nm. Distilled water 2mL was similarly treated as a blank and run in triplicate to reduce error. The absorbance A was measured at 490 nm. And drawing a standard curve by taking the mass concentration of the glucose as an abscissa and the absorbance value A as an ordinate.
1.3 experimental results, see fig. 1, regression equation: 8.1621x-0.0024 for Y20.9994, indicating a good linear relationship.
2. Protein content determination
2.1 principle of measurement
The Coomassie brilliant blue is blue when the Coomassie brilliant blue is not combined with protein, the color of the Coomassie brilliant blue is changed into cyan when the Coomassie brilliant blue is combined with protein molecules, the absorbance value of the Coomassie brilliant blue is linearly changed with the protein content when the protein concentration is in the range of 0-1000 mu g/mL, and the Coomassie brilliant blue has maximum light absorption at the wavelength of 595nm, so the Coomassie brilliant blue can be used for quantitative determination of the protein.
2.2 preparation of reagents
Preparing a Coomassie brilliant blue G-250 solution: 0.0201G of Coomassie brilliant blue G-250 was weighed out accurately, dissolved in 10.0mL of 95% ethanol and then added 20.0mL of 85% concentrated H3PO4And adding purified water to a constant volume of 200mL, filtering by double-layer filter paper, and storing in dark place.
Preparing bovine serum albumin standard solution: accurately weighing 0.0204g of standard bovine serum albumin, pouring the standard bovine serum albumin into a beaker, adding deionized water to dissolve the standard bovine serum albumin, and carrying out constant volume to 200.0mL to obtain a bovine serum albumin standard substance with the concentration of 0.1020 mg/mL.
2.3 drawing of Standard Curve and determination of sample
Drawing a standard curve: respectively and accurately measuring standard bovine serum albumin solution 0.0mL, 0.2mL, 0.4mL, 0.6mL, 0.8mL and 1.0mL in 6 clean test tubes, and supplementing water until the volume of the solution in each test tube is 1.0 mL. Accurately transferring 5.0mL of Coomassie brilliant blue G-250 solution with a pipette gun, adding into 6 test tubes with the same specification, shaking uniformly, and measuring the absorbance at 595nm after 5 min. A standard curve was plotted with the concentration (mg/mL) of the standard bovine serum albumin solution as the abscissa and the absorbance (A595) as the ordinate.
2.4 experimental results, see fig. 2, regression equation: 6.5314x-0.0532, R20.9947, indicating a good linear relationship.
3. Content of polysaccharide
The polysaccharide content was determined using the phenol-sulfuric acid method.
(2) Results of the experiment
2.1 example 1 Experimental results
| Item | Crude polysaccharide extraction (%) | Polysaccharide content (%) |
| Example 1 | 9.38 | 30.2 |
2.2 separation results of crude polysaccharide of chanterelle Yunnanensis by sodium chloride solution with different concentrations
| Name (R) | CY–0 | CY–1 | CY–2 | CY–4 | CY–8 | CY–10 |
| Quality (g) | 0.0055 | 0.0548 | 0.1077 | 0.0157 | 0.0281 | 0.0072 |
| Polysaccharide content (%) | 98.33 | 80.8 | 68.18 | 33.20 | 30.10 | 28.00 |
Referring to FIG. 3, the test results show that 6 distinct elution peaks, different in content, were obtained after DEAE-52 cellulose column chromatography. The method is named as CY-0 (distilled water elution), CY-1 (0.1mol/L sodium chloride solution elution), CY-2 (0.2mol/L sodium chloride solution elution), CY-4 (0.4mol/L sodium chloride solution elution), CY-8 (0.8mol/L sodium chloride solution elution) and CY-10 (1.0mol/L sodium chloride solution elution), 6 elution peaks are subjected to freeze drying, weight calculation and polysaccharide content calculation, wherein the polysaccharide content of the distilled water component is the highest, the yield is lower than that of the CY-1 and CY-2 components, the separation quality and the polysaccharide content of the polysaccharide are comprehensively considered, and the sodium chloride solution with the concentration of 0.1-0.2 mol/L is selected for elution.
2.3 comparative example 1 test results
And (3) obtaining a comprehensive score M of 0.4a +0.4b +0.2c by respectively setting the weight coefficients of the decolorization rate (a), the polysaccharide retention rate (b) and the protein removal rate (c) to be 0.4, 0.4 and 0.2, and carrying out primary screening on the resin models.
Experimental results show that the purification effect of the macroporous adsorption resin SP825 selected in the invention is the best.
Test example 2 structural characterization of chanterelle polysaccharide
(1) Analytical method
1.1, carrying out HPLC analysis on a series of standard dextrans with the molecular weights of 3300Da, 8100Da, 18300Da and 100000Da respectively, carrying out linear regression equation analysis on the elution volume and the logarithm value of the standard molecular weight, preparing the Yunnan chanterelle polysaccharide into a solution with the concentration of 7mg/mL, wherein the sample injection amount is 10 mu L, the logarithm value of the known standard molecular weight is used as a vertical coordinate, and the elution volume is used as a horizontal coordinate, thus obtaining a standard curve.
1.2 results of the experiment see fig. 4, the regression equation is Log Mol Wt ═ 0.9295V +11.97, R2The linear relationship is good at 0.9988.
1.3 the molecular weight of the polysaccharide was determined using GPC gel chromatography.
1.4 identifying monosaccharide composition of chanterelle polysaccharide by high performance liquid chromatography analysis.
1.4.1 polysaccharide hydrolysis
Weighing 10mg polysaccharide sample, putting into a jaw bottle of 20mL, adding 5mL 2mol/L trifluoroacetic acid, filling nitrogen, sealing the tube (10L/min, 1min), and hydrolyzing in an oven at 100 ℃ for 2 h; after cooling, the lid was opened, 1mL of methanol was added to 1mL of the solution, and the mixture was heated in a water bath at 70 ℃ with N2Blowing, adding methanol repeatedly and blowing for 2 times by using nitrogen to remove TFA; adding 1mL0.3mol/LNaOH solution to dissolve the residue completely to obtain polysaccharide hydrolysate, diluting to a certain extent, and performing derivatization determination.
1.4.2 monosaccharide derivatization
Respectively putting 400 mu L of mixed monosaccharide standard solution or polysaccharide hydrolysate into a 5mL test tube with a plug, adding 400 mu L of LPMP methanol solution, and mixing uniformly in a vortex; reacting for 2 hours in a water bath at 70 ℃; taking out, standing and cooling to room temperature; adding 400 mu L0.3mol/L HCl to adjust the pH value to 6-7; adding water 1200 μ L, adding equal volume of chloroform, vortex mixing, shaking, standing, discarding chloroform phase, and extracting for 2 times. The aqueous phase was filtered through a 0.45 μm microporous membrane (aq) and analyzed by HPLC injection.
1.4.3 HPLC detection conditions
The instrument model is as follows: agilent1100, with DAD detector
Chromatographic conditions are as follows: chromatography column C18Column, 250mm × 4.6mm, 5 μm; mobile phase a was 90mmol/L sodium phosphate buffer (pH 7.8); the mobile phase B is acetonitrile; the detection wavelength is 250 nm; the column temperature is 30 ℃; the flow rate is 1 mL/min; the amount of sample was 10. mu.L.
Gradient elution schedule
| t/min | A | B％ | |
| 0 | 86 | 14 | |
| 9 | 83 | 17 | |
| 28 | 78 | 22 | |
| 29 | 50 | 50 | |
| 31 | 50 | 50 | |
| 32 | 86 | 14 | |
| 36 | 86 | 14 |
1.5 detecting heteroproteins, polypeptides and nucleic acids in chanterelle polysaccharide by using ultraviolet absorption spectroscopy.
The Yunnan chanterelle polysaccharide is prepared into 0.136mg/mL aqueous solution, and the ultraviolet absorption spectrum is scanned within the range of 200-400 nm by taking distilled water as blank.
1.6 the chemical structure of the chanterelle polysaccharide is determined using infrared spectroscopy and nuclear magnetic resonance.
1.6.1 Infrared Spectroscopy
Weighing 2mg of Yunnan chanterelle polysaccharide, mixing with KBr, tabletting, and keeping the mixture at 4000-400 cm-1The measured infrared absorption spectrum of (1).
1.6.2 nuclear magnetic resonance
Weighing 16mg of Yunnan chanterelle polysaccharide, dissolving in 0.7mL of heavy water (D)2O), mixing uniformly, adding into a nuclear magnetic tube, and sealing. On a nuclear magnetic resonance spectrometer1H-NMR and13C-NMR detection.
1.7 Electron microscopy
A small amount of vacuum dried polysaccharide sample is fixed on a sample holder, the sample is in a conductive state by vacuum gold plating, and the microscopic morphology of the sample is observed and recorded by a Sigma300 scanning electron microscope under the high vacuum condition of 500 times, 1000 times and 2000 times of magnification respectively under the acceleration voltage of 10 kV.
(2) Results of the experiment
2.1 molecular weights of polysaccharides of Yunnan chanterelle CY-1 and CY-2 components
| Peak number | Mn | Mw | MP | Polydispersity |
| CY-1 | 15036 | 30594 | 27696 | 2.03 |
| CY-2 | 15097 | 30495 | 26932 | 2.02 |
Mn is the number average molecular weight, Mw is the weight average relative molecular weight, MP is the peak molecular weight, Polydistorsity is the molecular weight distribution coefficient, and the breadth used to measure the molecular weight distribution of a polymer is the Mw/Mn ratio.
Referring to fig. 5 and 6, the aqueous phase GPC elution curves of the polysaccharides CY-1 and CY-2 of the chanterelle Yunnanensis have only one peak, and the experimental results show that the polysaccharide components are uniform.
2.2 chromatogram of derivative of polysaccharide hydrolysis sample of chanterelle CY-1 and CY-2
Referring to fig. 7, 8 and 9, analysis was performed to compare the 13 monosaccharide mixture controls, and the chanterelle yunnanensis CY-1 polysaccharide was composed of 8 monosaccharides, which were mannose Man, rhamnose Rham, glucuronic acid GlcUA, glucose Glc, galactose Gal, xylose Xyl, arabinose Ara, L-fucose Fuc, in a mass ratio of 2.253: 0.034: 0.026: 1.032: 0.068: 0.186: 0.087: 0.422: 4.109, the mol percentages are 54.83%: 0.83%: 0.64%: 25.11%: 1.64%: 4.53%: 2.12%: 10.28 percent. The chanterelle Yunnanensis CY-2 polysaccharide is composed of 8 monosaccharides, namely mannose Man, rhamnose Rham, glucuronic acid GlcUA, glucose Glc, galactose Gal, xylose Xyl, arabinose Ara and L-fucose Fuc, and the mass ratio of the substances is 1.653: 0.012: 0.052: 0.251: 0.023: 0.037: 0.040: 0.336, which comprises 68.82 percent of the following molar percentages: 0.48%: 2.16%: 10.44%: 0.95%: 1.52%: 1.66%: 13.97 percent. The result shows that the Yunnan chanterelle polysaccharide is composed of 8 monosaccharides, namely mannose, rhamnose, glucuronic acid, glucose, galactose, xylose, arabinose and L-fucose.
2.3 ultraviolet spectrophotometer wavelength scan pattern of polysaccharide of chanterelle CY-1 and CY-2
Referring to fig. 10 and fig. 11, no obvious absorption peak exists at 260nm and 280nm, and the result shows that the extracted Yunnan chanterelle polysaccharide does not contain heteroproteins, polypeptides and nucleic acids.
2.4 Infrared analysis results of polysaccharides CY-1 and CY-2 of chanterelle Yunnanensis
See FIG. 12, at 3500-3200 cm-1The interval has a wide and blunt strong absorption peak, the interval is mainly the characteristic peak of free hydroxyl or amino functional group, but the pattern is 1150-1050 cm-1A strong absorption peak, i.e. 1075cm, also appears-1Collateral peaks, mainly by stretching vibration (V) with C-OH bondC-OH) Caused, thus proving 3385cm-1The absorption peak is O-H stretching vibration absorption peak, belongs to hydroxyl compounds and is 3400-3200 cm-1The occurrence of strong and broad absorption peaks is a result of-OH stretching vibrations associated with intra-or intermolecular oxygen bonds of the polysaccharide molecules, which appear as-OH multimers.
2924cm-1The absorption peak is caused by the stretching vibration of C-H bond in the molecule, and the functional group is generally 1500-1300 cm-1Strong absorption peak appears in the interval as a side evidence peak which is just equal to 1416cm in the spectrogram-1Coincidental, and 1416cm-1Is the C-H bending vibration peak; 1651cm-1The absorption peak of hydrate of sugar or the asymmetric stretching vibration peak of C ═ O; 849cm-1The absorption peak at (b) belongs to 844 +/-4 cm-1Is the epimeric C-H vibration of the alpha end group of the D-pyran ring, and the C-H is an equatorial bond; 808cm-1At-800 cm-1Mannose is indicated nearby.
See FIG. 13, at 3500-3200 cm-1The interval has a wide and blunt strengthAn absorption peak, wherein the absorption peak is mainly a characteristic peak of a free hydroxyl group or an amino functional group in the interval, but the spectrum is 1150-1050 cm-1A strong absorption peak, i.e. 1078cm, also appears-1Collateral peaks, mainly by stretching vibration (V) with C-OH bondC-OH) Caused, thus demonstrating 3404cm-1The absorption peak is O-H stretching vibration absorption peak, belongs to hydroxyl compounds and is 3400-3200 cm-1The occurrence of strong and broad absorption peaks is a result of-OH stretching vibrations associated with intra-or intermolecular oxygen bonds of the polysaccharide molecules, which appear as-OH multimers.
2921cm-1The absorption peak is caused by the stretching vibration of C-H bonds in the molecule; 1647cm-1The absorption peak of hydrate of sugar or the asymmetric stretching vibration peak of C ═ O; 1418cm-1、1385cm-1The absorption peak is the bending vibration of C-H; 858cm-1Is an absorption peak of an alpha-type glycosidic bond; 985cm-1And 817cm-1Is a characteristic absorption peak of the pyran ring.
2.5 Nuclear magnetic resonance analysis results of polysaccharides CY-1 and CY-2 of chanterelle Yunnanensis
See FIG. 14, at CY-11In the H-NMR chart, the shift of polysaccharide is mostly concentrated between delta 3.5ppm and delta 4.4 ppm. In the anomeric proton region delta 04.4 ppm-delta 15.8ppm, there are 6 signals of delta 25.37ppm, delta 35.25ppm, delta 55.16ppm, delta 65.01ppm, delta 84.87ppm and delta 94.76 ppm. Wherein δ 5.37ppm, δ 05.25ppm, δ 5.16ppm and δ 5.01ppm are proton signals of the δ 4-type pyranose; delta 4.87ppm and delta 4.76ppm are proton signals of the delta 7 pyranose, and the absorption peak at delta 4.87ppm is stronger. Indicating that there is a repeating unit consisting of 6 sugar residues in the CY-1 sugar chain; 3.29ppm, 3.43ppm and the like, and in the region of 3.0-4.0 ppm, methine and methylene resonance regions of non-anomeric protons in the sugar residues, namely, structural information resonance signals. Wherein, the delta 3.43ppm is the signal of the galactose 6-position methyl, and the delta 1.18ppm is the signal of the fucose methyl.
See FIG. 15, at CY-113In a C-NMR spectrum, the chemical shift of anomeric carbon is between delta 90ppm and delta 110ppm, and delta 103.12ppm is a signal of beta-type pyranose anomeric C1, so that the signal is strong. Delta 101.00ppm and delta 100.65ppm, delta 99.46ppm and delta 98.44ppm are signals of alpha anomeric C1, and the content is higher;no signal in the region of delta 82ppm to delta 84ppm, indicating that all sugar residues are pyranoside; δ 078.70ppm, δ 178.60ppm are signals for C4 of the (1 → 4) glycosidic bond undergoing oxygen substitution, δ 276.04ppm, δ 375.68ppm, δ 75.02ppm, δ 73.33ppm, δ 73.17ppm and δ 71.96ppm are signals for C5, C4, C3 and C2, respectively; a large amount of signal was accumulated between δ 67ppm to δ 70ppm, indicating that C-6 was mostly substituted in the sugar chain of CY-1; δ 61.15ppm and δ 60.83ppm are free C6 signals, slightly weaker. Further illustrating that C-6 is more substituted; 15.47ppm CH for fucose3Resonance signal of C above, i.e. fucose in polysaccharide has CH3And (5) structure. From the results of IR and NMR, it was presumed that CY-1 may have a linkage of two glycosidic bonds of α (1 → 4) and β (1 → 6) and contains fucose.
See FIG. 16, at CY-21In the H-NMR chart, the polysaccharide shift is mostly concentrated on signals of delta 24.82ppm, delta 34.72ppm, delta 44.45ppm and delta 54.43ppm at delta 3.5ppm to delta 4.4ppm in an anomeric carbon region delta 04.4ppm to delta 15.8 ppm. Wherein, δ 4.82ppm is the proton signal of β type pyranose, δ 4.72 is the residual hydrogen peak of solvent heavy water, indicating that there is a repeating unit consisting of 3 sugar residues in CY-2 sugar chain. The chemical shifts of 3.61ppm, 3.71ppm and the like are methine and methylene resonance regions of non-anomeric protons in the sugar residues in the region of delta 3.0 to delta 4.0ppm, namely, structural information resonance signals. δ 3.41ppm is a signal for the methyl group at the 6-position of galactose; δ 1.15ppm and δ 1.13ppm are fucomethyl doublets.
See FIG. 17, at CY-213In the C-NMR spectrum, the delta 102.99ppm and the delta 99.41ppm are signals of delta 1 anomeric C1, the content is higher, and no signals exist in the delta 082 ppm-delta 84ppm area, which indicates that all sugar residues are pyranoside. In the non-anomeric carbon region delta 70-75 ppm, delta 75.57ppm, delta 73.10ppm and delta 72.91ppm are signals of C5, C4 and C3 respectively, and a large amount of signals are accumulated near delta 70ppm, which indicates that most of C-6 in the sugar chain of CY-2 is substituted; delta 60.77ppm is the free C6 signal, weaker, and further indicates more C-6 substitution. From the results of IR and NMR, it was speculated that CY-2 may have an α (1 → 6) glycosidic linkage.
2.6 Electron microscopy analysis results of polysaccharides of chanterelle CY-1 and CY-2
Referring to fig. 18, under the scanning electron microscope, CY-1 is observed to be in a mesh shape under the electron microscope, and is connected with each other to form an integral multi-layer branched structure, the molecules are crossed, the edges of the mesh structure are smooth and flat, and the integral structure is loose. The CY-2 has a rough lower surface on the electron microscope, has a spongy small cavity and is compact as a whole.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (10)
1. A separation and purification method of Yunnan chanterelle polysaccharide is characterized by comprising the following steps:
(1) pretreatment: taking a Yunnan chanterelle fruiting body sample, crushing and sieving to prepare Yunnan chanterelle powder, wherein the material-liquid ratio is 1 g: 10-30 mL of ethanol solution, soaking for 12-16 h after ultrasonic extraction, centrifuging the soaked Yunnan chanterelle powder at the rotating speed of 3500-4500 r/min for 8-12 min, and removing supernatant to obtain filter residue;
(2) water leaching: taking filter residues, and mixing the filter residues according to the material-liquid ratio of 1 g: adding 15-25 mL of distilled water, leaching in a water bath at 70-90 ℃ for 1.5-3 h, filtering, repeatedly extracting for 2-4 times, combining filtrates, and performing rotary evaporation concentration to obtain a leaching solution;
(3) alcohol precipitation: 1mL of the leaching solution according to the material-liquid ratio: adding 3-5 mL of ethanol solution, placing the mixture in a refrigerator with the temperature controlled to be 2-6 ℃ for 20-26 h, centrifuging at the rotating speed of 3500-4500 r/min for 8-12 min, and collecting precipitates;
(4) and (3) decoloring treatment: adding the precipitate prepared in the step (3) into a mixture with a feed-liquid ratio of 1 g: dissolving 15-25 mL of distilled water to obtain an aqueous solution of a precipitate, mixing the aqueous solution of the precipitate with a Sevag reagent, extracting to obtain a first supernatant, adding the first supernatant into macroporous resin, decoloring to obtain a second supernatant, adding the second supernatant into an ethanol solution, putting the second supernatant into a refrigerator, standing for 12-16 h, centrifuging, collecting a precipitate, freezing for 24h at-20 ℃ in the refrigerator to obtain crude Yunnan chanterelle polysaccharide, and freeze-drying the crude Yunnan chanterelle polysaccharide to obtain crude Yunnan chanterelle polysaccharide;
(5) separation: dissolving the Yunnan chanterelle crude polysaccharide prepared in the step (4) in water to prepare a Yunnan chanterelle crude polysaccharide solution, wherein the material-liquid ratio is 1 g: 20-30 mL, separating the crude polysaccharide solution of the Yunnan chanterelle by using column chromatography, collecting target elution components and concentrating to prepare a target elution component concentrated solution, wherein the eluent is a sodium chloride solution;
(6) and (3) purification: and (3) filling the target elution component concentrated solution obtained by separation in the step (5) into a dialysis bag, dialyzing by using tap water and distilled water respectively, and freeze-drying after dialysis to obtain the Yunnan chanterelle purified polysaccharide.
2. The separation and purification method of the Yunnan chanterelle polysaccharide as claimed in claim 1, characterized in that in the step (1), the ultrasonic extraction is ultrasonic at 30-50 ℃ for 20-40 min, and the ultrasonic power is 50-150 Hz.
3. The Yunnan chanterelle polysaccharide of claim 1, wherein in the step (1), the mass fraction of the ethanol solution is 95%.
4. The separation and purification method of Yunnan chanterelle polysaccharide as claimed in claim 1, wherein in the step (4), the mass ratio of the precipitate to the Sevag reagent is 1:1, and the Sevag reagent is prepared from chloroform and n-butanol with the volume ratio of 4: 1.
5. The separation and purification method of Yunnan chanterelle polysaccharide as claimed in claim 1, wherein in the step (4), the extraction operation is to mix and shake the precipitate and Sevag reagent for 20min, stand for 30min, collect the upper layer of extract, and extract the upper layer of extract for 3 times to obtain the first supernatant.
6. The method for separating and purifying the Yunnan chanterelle polysaccharide as claimed in claim 1, wherein in the step (5), the concentration of the sodium chloride solution is 0.1-0.2 mol/L, and the filler of the column chromatography is DEAE-52 cellulose.
7. The method for separating and purifying the Yunnan chanterelle polysaccharide as claimed in claim 1, wherein in the step (6), the concentrated solution is prepared by dialyzing with tap water for 24 hours, changing the tap water every 6 hours, dialyzing with distilled water for 12 hours and changing the distilled water every 4 hours.
8. The separation and purification method of the Yunnan chanterelle polysaccharide as claimed in claim 1, characterized in that, in the step (4), the model of the macroporous resin is SP825, and the macroporous resin adsorption step is as follows: and (3) putting the macroporous resin and the first supernatant into a triangular flask with a plug, and decoloring for 5-7 hours in a constant-temperature shaking table.
9. The method for separating and purifying the Yunnan chanterelle polysaccharide as claimed in claim 1, wherein the freeze-drying temperature is-75 to-85 ℃ and the time is 23-25 h.
10. A yunnan chanterelle polysaccharide characterized by being prepared by the separation and purification method according to any one of claims 1 to 9.
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| US20140357871A1 (en) * | 2012-01-10 | 2014-12-04 | Lupin Limited | Process for preparation of rufinamide |
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| WO2014013511A2 (en) * | 2012-07-20 | 2014-01-23 | Hetero Research Foundation | Rufinamide solid dispersion |
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