CN101385909A - Radial Chromatographic Purification of Natural Soluble Polysaccharides - Google Patents

Abstract

The invention relates to a method for radial chromatographic purification of natural soluble polysaccharides, wherein the crude polysaccharide solution enters the radial chromatographic column, flows from the periphery of the chromatographic column to the center, flows through the stationary phase filled in the chromatographic column, and uses the corresponding solvent as the flow The phase is eluted, and then flows out from the center outlet. The invention utilizes the characteristics that the radial chromatographic column has a large contact area between the sample and the filler at the beginning and converges at the liquid outlet to separate or purify the natural soluble polysaccharide.

Description

Radial Chromatographic Purification of Natural Soluble Polysaccharides

technical field

The invention belongs to the field of biotechnology, in particular to a method for radial chromatographic purification of natural soluble polysaccharides.

Background technique

As early as the 1950s, with the continuous research of chromatography technology, radial chromatography developed rapidly. This chromatographic technique is mainly evolved from the traditional axial chromatographic technique by Rhee et al. At present, this chromatographic technique has been widely used in molecular separation techniques.

In the relevant reports comparing radial chromatography with axial chromatography, radial chromatography has shown the advantages of large processing capacity and fast speed, and also has the advantages of large contact area between the sample and the filler at the beginning and convergence at the outlet. features. In the mid-1980s, radial chromatography technology began to be industrialized, mainly used in the separation and purification of proteins, such as the purification of monoclonal antibodies, the separation and purification of IGase enzymes, and the separation and purification of human urinary kallikrein. There are few reports on the application of chromatography to the separation and purification of polysaccharides.

Natural polysaccharides are mainly extracted and isolated from animals, plants, fungi and microorganisms or their secretions, and are polar macromolecular compounds. However, crude polysaccharides extracted from plant tissues or animal tissues usually contain many different polysaccharides and impurities. Therefore, to separate polysaccharides with different molecular weights or compositions or to remove non-sugar components, it is necessary to separate and purify the polysaccharide mixture. . Commonly used polysaccharide separation and purification methods are as follows: fractional precipitation method, quaternary ammonium salt precipitation method, salting out method, metal complex method and column chromatography, among which column chromatography includes ion exchange chromatography and coagulation Two methods of gel chromatography.

Ion exchange chromatography is based on the electrostatic combination between the cation or anion of the separated polysaccharide and the ion exchanger with the opposite charge, that is, according to the difference in the acidity, alkalinity and polarity of the substance, the adsorption and desorption between the ions will separate the The polysaccharide components of the separation solution are separated; after the sample is added, it is eluted with an electrolyte solution or a buffer solution, and various substances with different charges have different affinities for the ion exchanger. By changing the ionic strength and pH value of the eluent, This affinity is controlled so that the different components and impurities of these polysaccharides are eluted from the chromatographic column in sequence according to the order of affinity, so as to achieve the purpose of separation and purification.

Gel chromatography is to slowly flow the crude polysaccharide solution through the gel chromatographic column, and each molecule undergoes two different movements in the column at the same time: moving along the direction of the mobile phase and non-directional diffusion movement in the gel stationary phase . Due to the large diameter, macromolecular substances are not easy to enter the micropores of the gel particles, but can only be distributed between the particles, so they move forward faster during elution. In addition to diffusing in the gaps of gel particles, small molecular substances can also enter the micropores of gel particles, that is, enter the gel phase. During the elution process of the mobile phase, after diffusing from a gel to the particle gap Then enter another gel particle, and so continuously enter and diffuse, the moving speed of small molecular substances lags behind that of large molecular substances, so that the large molecules in the sample flow out of the chromatographic column first, the medium molecules flow out last, and the smallest molecules flow out last , so as to separate polysaccharides of different molecular weights or to separate polysaccharides from small molecular non-sugar impurities.

Contents of the invention

The invention provides a method for radial chromatographic purification of natural soluble polysaccharides, which uses the characteristics of radial chromatographic samples and mobile phases to converge from the outer circumference of the column to the center of the column, and has the characteristics of large processing capacity and fast speed to separate and purify the natural soluble polysaccharides.

To achieve the above object, the present invention adopts the following technical solutions:

A method for radial chromatographic purification of natural soluble polysaccharides, characterized in that: after the crude polysaccharide solution enters the radial chromatographic column, it flows from the periphery of the chromatographic column to the center, flows through the stationary phase filled with the chromatographic column, and uses the corresponding solvent as the flow The phase is eluted, and then flows out from the center outlet.

Moreover, the radius R of the stationary phase of the chromatographic column should be greater than or equal to 1 cm.

Moreover, the crude polysaccharides are obtained from animals, plants, fungi and microorganisms or their secretions, including chitosan, ginseng polysaccharide, hair vegetable polysaccharide, spirulina polysaccharide, angelica polysaccharide, grifola frondosa polysaccharide, polyporus polysaccharide, Poria cocos polysaccharide Polysaccharides, ganoderma polysaccharides, aloe polysaccharides, seaweed polysaccharides, lotus seed polysaccharides, tea polysaccharides, tremella polysaccharides, enteromorpha polysaccharides, lentinan, xanthan gum, gellan gum, heparin, chondroitin sulfate, Bupleurum polysaccharides, pullulans , Cordyceps polysaccharide, wolfberry polysaccharide, dendrobium polysaccharide, pullulan polysaccharide astragalus polysaccharide, tremella polysaccharide, angelica polysaccharide, fungus polysaccharide, bitter gourd polysaccharide, Gastrodia elata polysaccharide, pullulan, dextran and sclerotin, etc., its molecular weight is in In the range of 5,000 to 5,000,000, and contain more than one polysaccharide component, or the total polysaccharide content is less than 97%, and the solubility in aqueous solution is greater than 0.2g/L at normal temperature.

Moreover, the crude polysaccharide solution is extracted by water or alcohol to obtain a crude polysaccharide extract, and ethanol and water are mixed in various proportions during alcohol extraction.

Moreover, the radial chromatographic column stationary phase is a cation exchange resin or anion exchange resin of ion exchange resin or gel resin, such as styrene type ion exchange resin, acrylic ion exchange resin, agarose ion exchange resin, cross-linked glucose Polysaccharide ion exchange resin and cellulose ion exchange resin.

Moreover, the stationary phase of the radial chromatographic column can be a gel resin, including cross-linked dextran gel, agarose gel, polyacrylamide gel and polystyrene gel, and its gel type is G-10 , G-15, G-25, G-50, G-75, G-100, G-150, G-200, Sephadex LH-20, Sepharose and Styrogel.

Moreover, the eluting mobile phase refers to respectively, the eluent in the ion exchange chromatography is a KCl solution with a concentration of 0 to 4.7mol/L, a NaCl solution with a concentration of 0 to 6.2mol/L, and a concentration of 0 to 6.2mol/L. 9.4mol/L NH ₄ Cl solution and a buffer solution composed of hydrogen phosphate such as sodium salt, potassium salt or ammonium salt and dihydrogen phosphate such as sodium salt, potassium salt or ammonium salt, or trihydroxymethylaminomethane ( Tris)-NaCl buffer, Tris-KCl buffer; wherein the eluent in the gel chromatography is deionized water or distilled water.

Advantage and positive effect of the present invention are:

When performing chromatographic separation in the present invention, the mobile phase flows from the periphery of the chromatographic column to the center, carrying polysaccharides and flowing through the stationary phase in the radial direction, so that different substances in the polysaccharides are separated due to their different moving speeds in the stationary phase, achieving separation and purification The purpose of different constituents of polysaccharides. The radial chromatographic column of the present invention has the characteristics of a large contact area between the sample and the filler at the beginning and convergence at the liquid outlet, and can separate and purify natural soluble polysaccharides.

Description of drawings

Fig. 1 is a schematic structural diagram of a radial chromatographic column of the present invention;

Fig. 2 is ion-exchange chromatography elution curve of the present invention;

Fig. 3 is the gel chromatography elution curve of the present invention.

Detailed ways

Below in conjunction with embodiment, the present invention is further described; Following embodiment is illustrative, not limiting, can not limit protection scope of the present invention with following embodiment.

In the present invention, the radial chromatographic column 1 is composed of a coaxial porous ring composed of two filter screens 4, as shown in Fig. 1, the space between the two filter screens is used to fill the stationary phase filler. The method of radial chromatographic purification of natural soluble polysaccharides is as follows: the filler enters from the filler port 2 and is discharged from the discharge port 5; the mobile phase and sample enter from the liquid inlet 3, and radially enter the filler from the periphery of the cylinder through the outer porous filter The stationary phase migrates to the center, and finally flows out from the liquid outlet 6 through the inner porous filter, and the different components in the sample are separated from each other due to the different migration speeds in the stationary phase.

The advanced nature of the present invention is further proved by specific examples below.

Example 1:

(1). Choose DEAE-650M anion exchange resin to fill the radial chromatographic column (see Figure 1 for the size), weigh 15 mg of Fat Cai crude polysaccharide and dissolve it in deionized water to make a polysaccharide solution with a concentration of 0.5 mg/mL, and remove it by centrifugation After precipitation, ion exchange chromatography was performed.

(2). Carry out linear gradient elution from 01.0mol/L with 1000mL NaCl solution, the elution rate is 1mL/min, and collect the eluate with a partial collector.

(3). Use the phenol-sulfuric acid method to detect the absorbance A ₄₉₀ of each tube. Taking the elution volume as the abscissa, the absorbance value and the concentration gradient change of the NaCl solution as the ordinate, the ion exchange chromatography elution curve of the polysaccharide , see Figure 2. As can be seen from the figure, the elution curve presents three independent peaks, that is, the polysaccharides of Nostocypris chinensis presents three different components, and the resolution of the first peak and the second peak is R ₁₂ and the second peak and the second peak The resolution R ₂₃ of the three peaks reaches 1.191 and 1.001 respectively after passing through the radial chromatographic column (resolution, also known as resolution, resolution, is a measure of the separation degree of two adjacent peaks in the chromatogram).

(4). The eluents from the three different peaks were combined, dialyzed, and freeze-dried to obtain three different single-component polysaccharides with a NaCl content of about 1.5%, 4.2mg, 4.5mg and 3.9mg.

Example 2:

(1). Choose Gel G-100 to fill a self-made radial chromatographic column (see Figure 1 for size), weigh 3 mg of Facai polysaccharide (the first component of Facai polysaccharide obtained in Example 1), dissolve it in deionized Water was made into a polysaccharide solution with a concentration of 0.5 mg/mL, and after centrifugation to remove the precipitate, gel chromatography was performed to remove most of the electrolyte NaCl contained therein.

(2). Use 200mL deionized water for elution, the elution rate is 0.3mL/min, and use a partial collector to collect the eluate.

(3). The absorbance A ₄₉₀ of each tube is detected by the phenol-sulfuric acid method, and the elution volume is taken as the abscissa, and the absorbance value and the change in the conductivity of NaCl are drawn as the ordinate, and the gel chromatography elution curve of the polysaccharide ( See Figure 3).

(4). The eluents before the intersection of the two curves were combined, concentrated under reduced pressure, and freeze-dried to obtain 2.4 mg of NaCl content of 0.06% Fatsai polysaccharide.

Claims (7)

1. the method for a radial chromatography purifying natural soluble polysaccharide, it is characterized in that: after thick polysaccharide solution enters radial chromatographic column, by around the chromatographic column to center flow, the fixedly phase that the chromatographic column of flowing through is filled, carry out wash-out mutually with the corresponding solvent work is mobile, flow out by the center liquid outlet then.

2. the method for radial chromatography purifying natural soluble polysaccharide according to claim 1 is characterized in that: described chromatographic column fixedly the phase radius R more than or equal to 1cm.

3. the method for radial chromatography purifying natural soluble polysaccharide according to claim 1, it is characterized in that: described thick polysaccharide is from animal, plant, obtain in fungi and microorganism or its secretion, comprise shitosan, the panaxan, the polysaccharide of delivering vegetables, spirulina polysaccharide, Radix Angelicae Sinensis polysaccharide, grifolan, grifola polysaccharide, pachymaran, GL-B, aloe polysaccharide, laver amylose, the lotus nut polysaccharide, tea polysaccharide, tremella polysaccharides, sea grass polysaccharide, lentinan, xanthans, gellan gum, heparin, chondroitin sulfate, the radix bupleuri polysaccharide, the mould polysaccharide of short stalk, Chinese caterpillar fungus polysaccharide, LBP-X, dendrobium polysaccharide, the pulullan polysaccharide astragalus polyose, tremella polysaccharides, Radix Angelicae Sinensis polysaccharide, Auricularia polysaccharide, bitter melon polysaccharide, gastrodia elata polysaccharide, the mould polysaccharide of short stalk, dextran and pyrenomycetes dextran etc., its molecular weight is 5,000～5,000, in 000 scope, and contain more than one polysaccharide components, or polysaccharide total content＜97%, solubility is at normal temperatures greater than 0.2g/L in the aqueous solution.

4. the method for radial chromatography purifying natural soluble polysaccharide according to claim 1 is characterized in that: described thick polysaccharide solution is carried by water or alcohol extracting gets the polysaccharide crude extract, and ethanol and water are mixed with various ratios during alcohol extracting.

5. the method for radial chromatography purifying natural soluble polysaccharide according to claim 1, it is characterized in that: described radial chromatographic column is fixing to be the cationic ion-exchange resin or the anion exchange resin of ion exchange resin or gel resin mutually, as benzethylene type ion exchange resin, acrylic acid series ion exchange resin, agarose ion exchange resin, cross-link dextran ion exchange resin and cellulose ion exchanger resin.

6. the method for radial chromatography purifying natural soluble polysaccharide according to claim 1 or 5, it is characterized in that: described radial chromatographic column is fixing can be gel resin mutually, comprise sephadex, Ago-Gel, polyacrylamide gel and Aquapak A-440, its gel type is G-10, G-15, G-25, G-50, G-75, G-100, G-150, G-200, Sephadex LH-20, Sepharose and Styrogel.

7. the method for radial chromatography purifying natural soluble polysaccharide according to claim 1, it is characterized in that: described wash-out flows and is meant respectively that mutually the eluent in the ion-exchange chromatography is that concentration is that the KCl solution of 0～4.7mol/L, NaCl solution, the concentration that concentration is 0～6.2mol/L are the NH of 0～9.4mol/L ₄The cushioning liquid that Cl solution and hydrophosphate such as sodium salt, sylvite or ammonium salt and dihydric phosphate such as sodium salt, sylvite or ammonium salt are formed, or trishydroxymethylaminomethane (Tris)-NaCl buffer solution, Tris-KCl buffer solution; Wherein the eluent in the gel chromatography is deionized water or distilled water.

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