CN110724207A

CN110724207A - Method for extracting and separating polysaccharide from codium spinulosum

Info

Publication number: CN110724207A
Application number: CN201910978370.5A
Authority: CN
Inventors: 陈荫; 王腾; 董喆; 孙坤来; 赵玉勤; 王斌
Original assignee: Zhejiang Ocean University ZJOU
Current assignee: Zhejiang Ocean University ZJOU
Priority date: 2019-10-15
Filing date: 2019-10-15
Publication date: 2020-01-24

Abstract

The invention relates to the technical field of extraction of polysaccharides. In view of the problems of low extraction rate and difficult separation of polysaccharides extracted from Pinus spinosa, a method for extracting and separating polysaccharides from Pinus spinosa is provided. Including the pretreatment of degreasing; extracting the pine algae with hot water, then ethanol precipitation, standing overnight, suction filtration, adding ethanol for washing, dehydration, and drying to obtain the pine algae hot water to extract the crude polysaccharide; finally, use a strong anion exchange chromatographic column Separation and extraction of crude polysaccharide from Pine algae with hot water: gradient elution with NaCl solution, partial collector collection, concentration of each component eluate under reduced pressure, dialysis desalination, and freeze-drying to obtain product polysaccharide. The water of the invention greatly promotes the rupture of the cell wall and the release of polysaccharides through the fatty acid ether sodium sulfate and polysorbate-80 active polypeptide in advance, reduces the extraction time, improves the extraction rate of polysaccharides, and is washed in a strong anion exchange chromatography column through a suitable gradient. De-prepared polysaccharides with anti-HPV activity.

Description

Method for extracting and separating polysaccharide from codium spinulosum

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.

Sulfate content 20%, other results are similar to example 1.

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

1. the method for extracting and separating polysaccharide from Pinus spinosa, is characterized in that, comprises the following steps:

1) Dry the pine algae after pretreatment including degreasing;

2) The dried spiny algae was extracted with water at 80-100 °C for 1-4 h, centrifuged, and the algal residue was repeatedly extracted for several times. The combined supernatant was concentrated and then alcohol-precipitated, left standing at 3-9 °C overnight, suction filtered, Add ethanol for washing, dehydration, and drying to obtain the crude polysaccharide from Pine algae with hot water;

3) Use a strong anion exchange chromatographic column to separate the crude polysaccharide from Pine algae with hot water: gradient elution with NaCl solution, partial collector collection, concentration of each component eluent under reduced pressure, dialysis desalination, and freeze-drying to obtain the product polysaccharide.

2. The method for extracting and separating polysaccharides from Pinus spinosa according to claim 1, wherein the pretreatment in the step 1) further comprises destroying the cell wall, and the reagents used are fatty acid ether sodium sulfate and polysorbate- 80% of the ethanol solution, wherein the added mass of fatty acid ether sodium sulfate is 0.5-2% of the pine algae, and the added quality of polysorbate-80 is 3-5% of the pine algae.

3 . The method for extracting and separating polysaccharides from Pinus spinosa according to claim 2 , wherein the process of destroying the cell wall is: dissolving Pinus spinosa in a minimum amount of water at 50-70° C., and adding ethanol. 4 . The volume of the ethanol solution is 5-20% of that of water, vigorously stirred for 2-3 h, centrifuged to obtain the aqueous phase, and filtered to obtain the filter cake.

4. The method for extracting and separating polysaccharides from Pinus spinosa according to claim 1, wherein the quality of the water for extraction in the step 2) is 10-30 times that of Pinus spinosa, and the ethanol for washing is 2- 5 times the volume of 95% ethanol, dehydrated with absolute ethanol.

5. The method for extracting and separating polysaccharides from Pinus spinosa according to claim 1, wherein the concentration of NaCl solution gradient elution in the step 3) is 0, 0.1M, 0.25M, 0.5M, 0.75M, 1M and 2M.

6. the method for extracting and separating polysaccharides from Pinus spinosa according to claim 1 or 5, is characterized in that, described step 3) middle column volume 60 mL; Loading volume 60 mg; Flow velocity 0.5 mL/min wash-out Elute at 2 mL/min after 2 column volumes.

7. The method for extracting and separating polysaccharides from Pinus spinosa according to claim 1 or 2 or 3, wherein the monosaccharide in the polysaccharide is composed of galactose and arabinose, and the sulfate radical content is 16-25 %.