CN110724207A - Method for extracting and separating polysaccharide from codium spinulosum - Google Patents
Method for extracting and separating polysaccharide from codium spinulosum Download PDFInfo
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- CN110724207A CN110724207A CN201910978370.5A CN201910978370A CN110724207A CN 110724207 A CN110724207 A CN 110724207A CN 201910978370 A CN201910978370 A CN 201910978370A CN 110724207 A CN110724207 A CN 110724207A
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- codium
- spinulosum
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
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- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
Abstract
The invention relates to the technical field of extraction of polysaccharide, and provides a method for extracting and separating polysaccharide from codium spinulosum, aiming at the problems of low extraction rate and difficult separation of the polysaccharide from the codium spinulosum, which comprises the following steps: firstly, carrying out pretreatment including degreasing on the codium spinulosum; extracting Codium spinulosum with hot water, precipitating with ethanol, standing overnight, filtering, washing with ethanol, dehydrating, and oven drying to obtain Codium spinulosum hot water extract crude polysaccharide; and finally, separating the crude polysaccharide of the hot water extraction of the codium spinulosum by using a strong anion exchange chromatographic column: gradient eluting with NaCl solution, collecting partial eluate with collector, concentrating the eluate under reduced pressure, dialyzing for desalting, and freeze drying to obtain polysaccharide product. Before water extraction, the method greatly promotes the rupture of cell walls and the release of polysaccharide through the fatty acid ether sodium sulfate and the polysorbate-80 active polypeptide, reduces the extraction time, improves the extraction rate of the polysaccharide, and prepares the polysaccharide with the anti-HPV activity in a strong anion exchange chromatographic column through proper gradient elution.
Description
Technical Field
The invention relates to the technical field of extraction of polysaccharide, in particular to a method for extracting and separating polysaccharide from codium spinulosum.
Background
The Codium spinulosum belongs to Chlorophyta and is a perennial green alga. The algae is in a branch shape, mostly is antler-shaped branches and cylindrical branches, has a few flat shapes, has a sponge-shaped structure inside, is formed by interweaving tubular filaments, and has stick-shaped capsules formed by closely arranged filaments on the surface. The extraction of macromolecules containing sugars from codium spiniferum has been studied for a long time, for example love J et al demonstrate that aqueous extracts of this alga contain glucans, mannans, highly sulfated and pyruvylated galactans and sulfated arabinans. However, the isolation and purification of the extracted polysaccharides remains a problem. In addition, the cell wall of this seaweed is a highly integrated structure comprising a molecular chain of 31% (w/w) mannose units linked together, 9% of pyruvylated arabinogalactan sulphate and a small amount of hydroxyproline glycoprotein epitopes, the mannose and pyruvylated arabinogalactan sulphate being in the middle of the cell wall and the hydroxyproline glycoprotein epitopes being in the border region of the cell wall, especially in the apical region of the oocysts. This structure leads to low extraction of polysaccharides from the codium spinulosum and troublesome treatment for destroying cell wall structure, so an ideal method is needed to solve this problem.
Disclosure of Invention
The invention aims to overcome the problems of low extraction rate and difficult separation of polysaccharide extracted from codium spinulosum, and provides a method for extracting and separating polysaccharide from codium spinulosum, wherein before water extraction, the rupture of cell walls and the release of polysaccharide are greatly promoted by using fatty acid ether sodium sulfate and polysorbate-80 active polypeptide, the extraction time is reduced, the polysaccharide extraction rate is improved, and the polysaccharide with anti-HPV activity is prepared in a strong anion exchange chromatographic column by proper gradient elution.
In order to achieve the purpose, the invention adopts the following technical scheme:
the method for extracting and separating polysaccharide from codium spinulosum comprises the following steps:
1) performing pretreatment including degreasing on the codium spinulosum, and drying for later use;
2) extracting dried Codium spinulosum with water at 80-100 deg.C for 1-4 hr, centrifuging, repeatedly extracting algae residue for several times, mixing the supernatants, concentrating, precipitating with ethanol, standing at 3-9 deg.C overnight, filtering, washing with ethanol, dehydrating, and oven drying to obtain Codium spinulosum hot water extract crude polysaccharide;
3) separating the crude polysaccharide of the codium spinulosum hot water extraction by using a strong anion exchange chromatographic column: gradient eluting with NaCl solution, collecting partial eluate with collector, concentrating the eluate under reduced pressure, dialyzing for desalting, and freeze drying to obtain polysaccharide product.
The invention prepares the coarse polysaccharide of the spiny pine algae by hot water extraction and alcohol precipitation after the spiny pine algae is pretreated by degreasing and the like. The ion exchange chromatographic column has an open support skeleton, allows polysaccharide macromolecules to freely enter and rapidly diffuse, so that the adsorption capacity is large, and the ion exchange chromatographic column has the advantages of porosity, large surface area, large exchange capacity and high recovery rate; the anion exchange chromatographic column has hydrophilicity, is not firm in adsorption of macromolecules, can be eluted under mild conditions, and cannot cause denaturation.
Preferably, the pretreatment in the step 1) further comprises cell wall breaking, and the used reagents are ethanol solution of sodium fatty acid ether sulfate and polysorbate-80, wherein the added mass of the sodium fatty acid ether sulfate is 0.5-2% of that of the codium spinulosum, and the added mass of the polysorbate-80 is 3-5% of that of the codium spinulosum. Polysorbate-80 has a strong affinity for hydrophobic substances, and can bind and solubilize lipids in the cell wall, helping to increase the permeability of the cell wall. The fatty acid ether sodium sulfate can react with hydroxyproline glycoprotein antigenic determinant to destroy the outer layer of cell wall, so that polysaccharide is easy to exude and be extracted; in addition, the sodium fatty acid ether sulfate can also dissolve protein, thereby playing a role in removing protein. The ethanol can be used as solvent, and can also dissolve phospholipid layer in cell wall to promote cell wall breaking. The fatty acid ether sodium sulfate and the polysorbate-80 ethanol solution are used for increasing the permeability of cell walls, and compared with a physical cell breaking valve, the cell breaking valve can keep complete cell appearance, has few fragments and low slurry viscosity, and is easy for subsequent filtration and separation.
Preferably, the process of disrupting cell walls is: dissolving Codium spinulosum in a minimum amount of water of 50-70 deg.C, adding ethanol solution with volume of 5-20% of water, stirring vigorously for 2-3 hr, centrifuging to obtain water phase, and filtering to obtain filter cake.
Preferably, the mass of the water for extraction in the step 2) is 10-30 times of that of the spiny algae, the volume of the ethanol for washing is 2-5 times of that of the 95% ethanol, and the absolute ethanol for dehydration is used.
Preferably, the NaCl solution gradient elution concentration in the step 3) is 0, 0.1M, 0.25M, 0.5M, 0.75M, 1M and 2M in sequence. On the basis of a large number of tests, the elution effect under the condition is optimal.
Preferably, the column volume in the step 3) is 60 mL; the loading amount is 60 mg; elution was performed at a flow rate of 0.5 mL/min for 2 column volumes and then at 2 mL/min.
Preferably, the monosaccharide composition in the polysaccharide is galactose and arabinose, and the sulfate content is 16-25%. The prepared polysaccharide is sulfated galactaraban, has high purity and good quality, has high in-vitro antiviral activity, has equivalent anti-HPV activity to acyclovir, has high sulfate group content and strong anticoagulation activity, and has good development and utilization prospects.
Therefore, the invention has the following beneficial effects: (1) before water extraction, the method greatly promotes the rupture of cell walls and the release of polysaccharide through the fatty acid ether sodium sulfate and the polysorbate-80 active polypeptide, reduces the extraction time, improves the extraction rate of the polysaccharide of the codium spinulosum, can keep the complete appearance of cells compared with a physical broken cell valve, and obtains slurry with low viscosity which is easy for subsequent filtration and separation; (2) the ion exchange chromatographic column has an open support skeleton, allows polysaccharide macromolecules to freely enter and rapidly diffuse, so that the adsorption capacity is large, and the ion exchange chromatographic column has the advantages of porosity, large surface area, large exchange capacity and high recovery rate; the anion exchange chromatographic column has hydrophilicity, is not firm in adsorption of macromolecules, can be eluted under mild conditions, and cannot cause denaturation; (3) the prepared polysaccharide is sulfated galactaraban, has high purity and good quality, has equivalent anti-HPV activity to acyclovir due to in vitro antiviral activity, and has good development and utilization prospects.
Drawings
FIG. 1 is a Q-Sepharose FF chromatographic separation chart of the polysaccharide of example 1.
FIG. 2 is a monosaccharide analysis spectrum of the polysaccharide of example 1.
FIG. 3 is a graph showing the results of molecular weight measurements of the polysaccharide of example 1.
Detailed Description
The technical solution of the present invention is further illustrated by the following specific examples.
In the present invention, unless otherwise specified, all the raw materials and equipment used are commercially available or commonly used in the art, and the methods in the examples are conventional in the art unless otherwise specified.
Example 1
1) Drying the codium spiniferum at 40 ℃, crushing and sieving by a 60-mesh sieve for later use. Taking the acanthopanax spinosus algae powder, carrying out reflux degreasing for 2 hours by 85% ethanol, repeating the reflux degreasing for 2 times, and drying to obtain the degreased acanthopanax spinosus algae powder. Then cell wall breaking treatment is carried out: heating a small amount of water to 50 ℃, adding 100g of degreased acanthopanax spinosus powder, stirring, adding water in batches until the degreased acanthopanax spinosus powder is just dissolved, dissolving 0.5 g of fatty acid ether sodium sulfate and 5 g of polysorbate-80 in ethanol with the volume of 5% of the water volume, adding the ethanol solution into the degreased acanthopanax spinosus powder aqueous solution, stirring vigorously for 2 hours, centrifuging to obtain a water phase, filtering to obtain a filter cake, and drying for later use;
2) adding water with the volume about 20 times that of the dried acanthomonas spinosa, stirring and extracting for 2 hours in a water bath at 100 ℃ by an electronic stirrer, centrifuging, repeatedly extracting algae residues for 2 times, combining supernate, concentrating, precipitating with ethanol, standing in a refrigerator at 4 ℃ overnight, filtering, adding 95% ethanol with the volume 4 times that of the algae, washing, dehydrating by absolute ethanol, and drying to obtain crude polysaccharide extracted by the acanthomonas spinosa through hot water;
3) separating and purifying by AKTA FPLC liquid chromatograph and Q-Sepharose Fast Flow (QFF) strong anion exchange chromatographic column, eluting by 0, 0.1M, 0.25M, 0.5M, 0.75M, 1M and 2M NaCl solution for 2 column volumes respectively, and detecting by sulfuric acid-phenol method. Chromatographic conditions are as follows: the column volume is 60 mL; the loading amount is 60 mg; elution was performed at a flow rate of 0.5 mL/min for 2 column volumes and then at 2 mL/min. Collecting eluate of each component by a partial collector, concentrating under reduced pressure, dialyzing, desalting, and freeze-drying to obtain polysaccharide product.
The separation results are shown in FIG. 1. As can be seen in FIG. 1, 6 peaks were obtained after the sample was eluted with a gradient of 0, 0.1M, 0.25M, 0.5M, 0.75M, 1M and 2M NaCl.
And (3) adopting a PMP derivatization method to derivatize monosaccharide compositions of the monosaccharide mixed standard substance and the low molecular weight polysaccharide, and then carrying out high performance liquid chromatography analysis. A sample of 1mg was taken, 2ml of 2 mol/L TFA was added, the tube was sealed and then hydrolyzed in an oven at 105 ℃ for 10 hours, methanol was added to remove TFA by repeated rotary evaporation, and then the hydrolyzate was subjected to PMP derivatization. Chromatographic conditions are as follows: a chromatographic column: agilent ZORBAX Eclipse XDB-C18 column (5 μm, 4.6 x 150 mm); column temperature: 35 ℃; mobile phase: 0.1M PBS CH3CN =83:17 (V: V), flow rate: 1.0 mL/min; a detector: DAD (254 nm). The monosaccharide composition analysis spectrogram of each purified component is shown in FIG. 2, and the PMP pre-column derivatization chromatogram determines that the main monosaccharide compositions are galactose and arabinose, the ratio is 1:4, the sulfate radical content is 25%, so the target polysaccharide is sulfated galactan.
As shown in FIG. 3, the molecular weight of the polysaccharide was measured to be about 63 ten thousand.
In addition, in vitro antiviral activity showed that the product polysaccharide had comparable anti-HPV activity to acyclovir.
Example 2
1) The defatted acanthopanax japonicus powder of example 1 was subjected to cell wall disruption: heating a small amount of water to 70 ℃, adding 100g of degreased acanthopanax spinosus powder, stirring, adding water in batches until the degreased acanthopanax spinosus powder is just dissolved, dissolving 2 g of fatty acid ether sodium sulfate and 3 g of polysorbate-80 in ethanol with the volume of 20% of the water volume, adding the ethanol solution into the degreased acanthopanax spinosus powder aqueous solution, stirring vigorously for 3 hours, centrifuging to obtain a water phase, filtering to obtain a filter cake, and drying for later use;
2) adding water with the volume about 30 times that of the dried codium spinulosum into the dried codium spinulosum, stirring and extracting for 4 h in a water bath at 80 ℃ by an electronic stirrer, centrifuging, repeatedly extracting algae residues for 2 times, combining supernate, concentrating, precipitating with ethanol, standing in a refrigerator at 9 ℃ overnight, performing suction filtration, adding 95% ethanol with the volume 2 times that of the algae, washing, dehydrating by absolute ethanol, and drying to obtain the crude polysaccharide extracted by the hot water of the codium spinulosum;
3) the other conditions were the same as 3) in example 1, and the gradient elution was changed to elution with 0, 0.2M and 0.4M NaCl solutions, respectively.
Example 3
The defatted acanthopanax japonicus powder of example 1 was subjected to cell wall disruption: heating a small amount of water to 60 ℃, adding 100g of degreased acanthopanax spinosus powder, stirring, adding water in batches until the degreased acanthopanax spinosus powder is just dissolved, dissolving 1 g of fatty acid ether sodium sulfate and 4 g of polysorbate-80 in ethanol with the volume of 10% of the water volume, adding the ethanol solution into the degreased acanthopanax spinosus powder aqueous solution, stirring vigorously for 3 hours, centrifuging to obtain a water phase, filtering to obtain a filter cake, and drying for later use;
2) adding water with the volume about 20 times that of the dried acanthomonas spinosa, stirring and extracting for 1 h in a water bath at 90 ℃ by an electronic stirrer, centrifuging, repeatedly extracting algae residues for 2 times, combining the supernatant, concentrating, precipitating with ethanol, standing in a refrigerator at 3 ℃ overnight, filtering, adding 95% ethanol with the volume 5 times that of the supernatant, washing, dehydrating with absolute ethanol, and drying to obtain crude polysaccharide extracted by the acanthomonas spinosa with hot water;
3) the other conditions were the same as 3) in example 1, and the gradient elution was changed to elution with 0, 0.5M, 0.8M, 1.0M, 1.2M, and 1.4M NaCl solutions, respectively.
Sulfate content 16%, other results are similar to example 1.
Although the present invention has been described with reference to a preferred embodiment, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (7)
1. The method for extracting and separating polysaccharide from codium spinulosum is characterized by comprising the following steps:
1) performing pretreatment including degreasing on the codium spinulosum, and drying for later use;
2) extracting dried Codium spinulosum with water at 80-100 deg.C for 1-4 hr, centrifuging, repeatedly extracting algae residue for several times, mixing the supernatants, concentrating, precipitating with ethanol, standing at 3-9 deg.C overnight, filtering, washing with ethanol, dehydrating, and oven drying to obtain Codium spinulosum hot water extract crude polysaccharide;
3) separating the crude polysaccharide of the codium spinulosum hot water extraction by using a strong anion exchange chromatographic column: gradient eluting with NaCl solution, collecting partial eluate with collector, concentrating the eluate under reduced pressure, dialyzing for desalting, and freeze drying to obtain polysaccharide product.
2. The method for extracting and separating polysaccharide from codium spinulosum according to claim 1, wherein the pretreatment in step 1) further comprises cell wall disruption, and the reagents are ethanol solution of sodium fatty acid ether sulfate and polysorbate-80, wherein the sodium fatty acid ether sulfate is added by 0.5-2% of the codium spinulosum, and the polysorbate-80 is added by 3-5% of the codium spinulosum.
3. The method for extracting and separating polysaccharide from codium spinulosum according to claim 2, wherein the process for breaking cell wall is as follows: dissolving Codium spinulosum in a minimum amount of water of 50-70 deg.C, adding ethanol solution with volume of 5-20% of water, stirring vigorously for 2-3 hr, centrifuging to obtain water phase, and filtering to obtain filter cake.
4. The method as claimed in claim 1, wherein the extraction water used in step 2) is 10-30 times the mass of the echinacea, the washing ethanol is 2-5 times the volume of 95% ethanol, and the dehydrated ethanol is anhydrous ethanol.
5. The method for extracting and separating polysaccharide from codium spinulosum according to claim 1, wherein the concentration of NaCl solution gradient elution in step 3) is 0, 0.1M, 0.25M, 0.5M, 0.75M, 1M and 2M in sequence.
6. The method for extracting and separating polysaccharide from codium spinulosum according to claim 1 or 5, wherein the column volume in step 3) is 60 mL; the loading amount is 60 mg; elution was performed at a flow rate of 0.5 mL/min for 2 column volumes and then at 2 mL/min.
7. The method as claimed in claim 1, 2 or 3, wherein the monosaccharide composition of the polysaccharide is galactose and arabinose, and the sulfate content is 16-25%.
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| CN112094357A (en) * | 2020-09-15 | 2020-12-18 | 淄博职业学院 | Purification method of crude polysaccharide |
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| CN112094357A (en) * | 2020-09-15 | 2020-12-18 | 淄博职业学院 | Purification method of crude polysaccharide |
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