阅读说明：本技术 磷酸化扁藻胞外多糖及其制备方法 (Phosphorylated Platymonas extracellular polysaccharide and preparation method thereof ) 是由 吴斌华 易志恒 翁正芬 梁少华 于 2020-12-30 设计创作，主要内容包括：本发明公开了一种磷酸化扁藻胞外多糖及其制备方法,其中磷酸化扁藻胞外多糖的制备方法包括以下步骤：将扁藻胞外多糖与磷酸化试剂和硫酸钠在一定pH条件下,加热反应一段时间,进行磷酸化分子修饰,以产物的磷酸根含量为指标,以试剂配比、反应温度、反应pH值、反应时间为因素,用正交试验优选出扁藻胞外多糖磷酸化修饰的最佳条件。本发明技术方案能够实现工业化生产,且能够提高产率。(The invention discloses a phosphorylated tetraselmis extracellular polysaccharide and a preparation method thereof, wherein the preparation method of the phosphorylated tetraselmis extracellular polysaccharide comprises the following steps: heating and reacting the tetraselmis extracellular polysaccharide with a phosphorylation reagent and sodium sulfate for a period of time under a certain pH condition, carrying out phosphorylation molecule modification, and preferably selecting the optimal condition for the phosphorylation modification of the tetraselmis extracellular polysaccharide by an orthogonal test by taking the phosphate radical content of a product as an index and taking reagent proportion, reaction temperature, reaction pH value and reaction time as factors. The technical scheme of the invention can realize industrial production and improve the yield.)

1. A preparation method of phosphorylated tetraselmis extracellular polysaccharide is characterized by comprising the following steps:

dissolving sodium tripolyphosphate, sodium trimetaphosphate and water according to a preset proportion to prepare a phosphorylation reagent;

adding the tetraselmis extracellular polysaccharide PEPs into the phosphorylation reagent to prepare a tetraselmis extracellular polysaccharide solution;

adding a catalyst into the Platymonas vernaliella exopolysaccharide solution, heating for phosphorylation reaction to prepare a mixed solution, and adjusting the pH value of the mixed solution until the pH value reaches a target pH value to prepare a semi-finished product solution;

and (3) dialyzing and freeze-drying the semi-finished product solution in sequence to obtain the phosphorylated Platymonas extracellular polysaccharide pPEPs.

2. The method for preparing phosphorylated tetraselmis extracellular polysaccharide according to claim 1, wherein the mass ratio of sodium tripolyphosphate to sodium trimetaphosphate in the phosphorylating reagent is 4: 3, 5: 2 or 6: 1; the catalyst is sodium sulfate.

3. The method for preparing phosphorylated tetraselmis exopolysaccharide according to claim 1 or 2, wherein the target PH of the mixed solution is 8 to 10.

4. The method for preparing phosphorylated tetraselmis extracellular polysaccharide according to claim 3, wherein the phosphorylation reaction is carried out for 4 to 8 hours at a temperature of 80 to 100 ℃.

5. The method of claim 1, further comprising, prior to the step of drying the intermediate solution by dialysis:

adding 2-3 times of 95% ethanol solution into the semi-finished product solution;

and centrifugally mixing the semi-finished product solution and the ethanol solution, and then placing the mixture in an environment of 45 +/-3 ℃ for 2-3 hours to remove residual ethanol to obtain the alcohol-precipitated polysaccharide.

6. The method of claim 5, wherein the step of dialyzing the intermediate solution comprises:

freeze drying the alcohol precipitated polysaccharide, adding ultrapure water, re-dissolving in water bath at 50 ℃, placing in a dialysis bag with the molecular weight cutoff of 800-1200, dialyzing for more than 24h, dialyzing for more than 1 time, and collecting the trapped fluid to obtain the phosphorylated Platymonas extracellular polysaccharide solution.

7. The method of claim 6, wherein the step of lyophilizing the intermediate solution comprises: and (3) carrying out vacuum freeze drying on the phosphorylated tetraselmis extracellular polysaccharide solution to obtain the phosphorylated tetraselmis extracellular polysaccharide.

8. The method for preparing phosphorylated tetraselmis extracellular polysaccharide according to claim 1, wherein the phosphorylating reagent is prepared by mixing sodium tripolyphosphate and sodium trimetaphosphate in a mass ratio of 6: 1 with water, the pH of the mixed solution is 8, the reaction temperature is 100 ℃, and the reaction time is 6 hours.

9. A phosphorylated tetraselmis extracellular polysaccharide, which is obtainable by the process according to any one of claims 1 to 8.

Technical Field

The invention belongs to the technical field of biological pharmacy, and relates to phosphorylated Platymonas sobria extracellular polysaccharide and a preparation method thereof.

Background

According to modern scientific research, polysaccharides are important macromolecular substances in organisms, and have various biological activities, such as pharmacological activities of oxidation resistance, virus resistance, bacteria resistance, tumor resistance and the like. The main component of the pure flat algae culture solution is polysaccharide, and the flat algae has strong adaptability and rapid growth and propagation, and is one of the algae species which are easy to culture. Research has proved that the polysaccharide of algae has the function of regulating the activity of immunocyte and the growth of tumor cell. However, the extracted natural polysaccharide has generally low bioactivity and poor water solubility, and cannot be easily subjected to deep pharmacological research and clinical preparation application research.

Disclosure of Invention

The invention mainly aims to provide a preparation method of phosphorylated tetraselmis extracellular polysaccharide, which aims to produce phosphorylated tetraselmis extracellular polysaccharide by taking tetraselmis as a raw material and improve the yield by a novel phosphorylation modification technology.

In order to achieve the technical purpose, the technical scheme of the invention is as follows:

a preparation method of phosphorylated tetraselmis extracellular polysaccharide comprises the following steps:

dissolving sodium tripolyphosphate, sodium trimetaphosphate and water according to a preset proportion to prepare a phosphorylation reagent;

adding the tetraselmis extracellular polysaccharide PEPs into the phosphorylation reagent to prepare a tetraselmis extracellular polysaccharide solution;

adding a catalyst into the Platymonas vernaliella exopolysaccharide solution, heating for phosphorylation reaction to prepare a mixed solution, and adjusting the pH value of the mixed solution until the pH value reaches a target pH value to prepare a semi-finished product solution;

and (3) dialyzing and freeze-drying the semi-finished product solution in sequence to obtain the phosphorylated Platymonas extracellular polysaccharide pPEPs.

Preferably, the mass ratio of the sodium tripolyphosphate to the sodium trimetaphosphate in the phosphorylation reagent is 4: 3, 5: 2 or 6: 1; the catalyst is sodium sulfate.

Preferably, the target PH of the mixed solution is 8 to 10.

Preferably, the phosphorylation reaction is carried out for a reaction time of 4 to 8 hours and at a reaction temperature of 80 to 100 ℃.

Preferably, before the dialysis drying of the semi-finished solution, the method further comprises:

adding 2-3 times of 95% ethanol solution into the semi-finished product solution;

and centrifugally mixing the semi-finished product solution and the ethanol solution, and then placing the mixture in an environment of 45 +/-3 ℃ for 2-3 hours to remove residual ethanol to obtain the alcohol-precipitated polysaccharide.

Preferably, the step of subjecting the semi-finished solution to dialysis comprises:

freeze drying the alcohol precipitated polysaccharide, adding ultrapure water, re-dissolving in water bath at 50 ℃, placing in a dialysis bag with the molecular weight cutoff of 800-1200, dialyzing for more than 24h, dialyzing for more than 1 time, and collecting the trapped fluid to obtain the phosphorylated Platymonas extracellular polysaccharide solution.

Preferably, the step of lyophilizing the intermediate solution comprises: and (3) carrying out vacuum freeze drying on the phosphorylated tetraselmis extracellular polysaccharide solution to obtain the phosphorylated tetraselmis extracellular polysaccharide.

Preferably, the phosphorylation reagent is prepared by mixing sodium tripolyphosphate and sodium trimetaphosphate with water according to the mass ratio of 6: 1, the pH of the mixed solution is 8, the reaction temperature is 100 ℃, and the reaction time is 6 hours.

The invention also provides phosphorylated tetraselmis extracellular polysaccharide which is prepared by the preparation method.

The invention has the beneficial effects that: the invention adopts the tetraselmis extracellular polysaccharide and the phosphorylation reagent as reaction raw materials, uses sodium sulfate as a catalyst, and is prepared by adjusting the reaction time, the PH, the proportion of the phosphorylation reagent and the reaction temperature. The pure phosphorylated tetraselmis extracellular polysaccharide product is obtained by dialysis separation and freeze-drying, and the method can effectively phosphorylate polysaccharide, retain the polysaccharide structure to the maximum extent and improve the biological activity of the tetraselmis extracellular polysaccharide. The tetraselmis extracellular polysaccharide PEPs and the phosphorylation derivative pPEPs thereof obtained by the invention have certain inhibition effect on the proliferation of Raw264.7 cells; compared with the tetraselmis galbana exopolysaccharide, the phosphorylation derivative pPEPs has more obvious inhibition effect on the proliferation of Raw264.7 cells, and has statistical significance.

Drawings

FIG. 1 is a schematic flow chart of a method according to an embodiment of the present invention;

FIG. 2 is a standard curve diagram of phosphate radical content of phosphorylated Platymonas extracellular polysaccharide;

FIG. 3 is a graph of the results of infrared scanning of the extracellular polysaccharide of Pantoea alga;

FIG. 4 is a graph of the results of infrared scanning of phosphorylated tetraselmis extracellular polysaccharide;

FIG. 5 shows the effect of PEPs and pPEPs on Raw264.7 cells.

Detailed Description

In order to make the objects, features and advantages of the present invention more apparent, the present invention will be described in further detail with reference to the following embodiments. These examples are intended to illustrate the invention and are not intended to limit the invention.

The invention provides a preparation method of phosphorylated Platymonas sobria extracellular polysaccharide. In one embodiment, the method comprises the steps of:

step S10, dissolving sodium tripolyphosphate and sodium trimetaphosphate in water according to a preset ratio to prepare a phosphorylation reagent;

step S20, adding the tetraselmis extracellular polysaccharide PEPs into the phosphorylation reagent to prepare a tetraselmis extracellular polysaccharide solution;

step S30, adding a catalyst into the Platymonas vernalis extracellular polysaccharide solution, heating for phosphorylation reaction to prepare a mixed solution, and adjusting the pH value of the mixed solution until the pH value reaches a target pH value to prepare a semi-finished product solution;

and step S40, sequentially dialyzing and freeze-drying the semi-finished product solution to obtain the phosphorylated Platymonas vernalis exopolysaccharide pPEPs.

In the embodiment of the invention, the sodium tripolyphosphate and the sodium trimetaphosphate can be in a mass ratio of 4: 3, 5: 2 or 6: 1. In the embodiment of the invention, the catalyst can be selected from sodium sulfate, and the target pH value of the mixed solution is 8-10.

In the embodiment of the invention, the reaction time of the phosphorylation reaction is 4 to 8 hours, and the reaction temperature is 80 to 100 ℃.

In a preferred embodiment, before the dialysis drying of the semi-finished solution, the method further comprises:

adding 2-3 times volume of 95% ethanol solution into the semi-finished product solution;

and centrifugally mixing the semi-finished product solution and the ethanol solution, and then placing the mixture in an environment of 45 +/-3 ℃ for 2-3 hours to remove residual ethanol to obtain the alcohol-precipitated polysaccharide.

In a preferred embodiment, the step of dialyzing the semi-finished solution comprises:

freeze drying the alcohol precipitated polysaccharide, adding ultrapure water, re-dissolving in water bath at 50 ℃, placing in a dialysis bag with the molecular weight cutoff of 800-1200, dialyzing for more than 24h, dialyzing for more than 1 time, and collecting the trapped fluid to obtain the phosphorylated Platymonas extracellular polysaccharide solution.

In one embodiment, the step of lyophilizing the intermediate solution comprises: and (3) carrying out vacuum freeze drying on the phosphorylated tetraselmis extracellular polysaccharide solution to obtain the phosphorylated tetraselmis extracellular polysaccharide.

In a preferred embodiment, the phosphorylation reagent is prepared by mixing sodium tripolyphosphate and sodium trimetaphosphate in a mass ratio of 6: 1 with water, the pH of the mixed solution is 8, the reaction temperature is 100 ℃, and the reaction time is 6 hours.

Example 1: preparation of phosphorylated Platymonas extracellular polysaccharide.

Dissolving 7g of phosphorylation reagent (6g of sodium tripolyphosphate +1g of sodium trimetaphosphate) and 5g of anhydrous sodium sulfate by 100ml of ultrapure water, adding 1g of refined tetraselmis algae extracellular polysaccharide PEPs after dissolution, adjusting the pH of the solution to 9 by NaHCO3, reacting for 6h at 80 ℃, adding 95% ethanol solution with 3 times of volume into the obtained semi-finished product solution, standing and precipitating for 24h, centrifuging, collecting precipitate, placing in an environment of 45 ℃ for 2h, removing residual ethanol, freeze-drying, re-dissolving in a water bath at 50 ℃, placing in a dialysis bag with the molecular weight cutoff of 800-1200, dialyzing for more than 24h, dialyzing for more than 1 time, collecting retentate, and freeze-drying to obtain the phosphorylated tetraselmis algae extracellular polysaccharide pPEPs.

Example 2: detection of degree of modification of extracellular polysaccharide of phosphorylated Platymonas

Phosphate radical standard curve determination (molybdenum blue colorimetry):

preparing a Tris buffer solution: 3.6g of Tris-hydroxymethyl aminomethane and 120mg of MgCl were weighed₂·6H₂Dissolving O in ultrapure water, and diluting with ultrapure waterReleasing to 300ml, and adjusting the pH to 7 by using 1mol/L HCl to obtain a Tris buffer solution.

Preparing a phosphorus determination reagent: respectively taking VC aqueous solution with the mass fraction of 20 percent and H with the mass fraction of 3mol/L₂SO₄And uniformly mixing the solution and the ammonium molybdate aqueous solution with the mass fraction of 3% in an equal volume manner to obtain the phosphorus determination reagent.

Preparation of a phosphate standard solution: precisely weighing 1g of potassium dihydrogen phosphate, dissolving in ultrapure water, diluting to a constant volume to a scale by using a 100mL volumetric flask, and diluting by 100 times to obtain a 0.1mg/mL phosphate radical standard solution.

Drawing a standard curve: accurately sucking 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5 and 5mL of phosphate radical standard solution, respectively transferring into 20mL test tubes, adding ultrapure water into each tube to supplement to 5mL, adding 3mL of Tris buffer solution, shaking uniformly, adding 3mL of phosphorus determination reagent, heating in a 45 ℃ constant temperature water bath kettle in a water bath for 30min, taking out, measuring absorbance at 580nm, and drawing a standard curve by taking the phosphate radical concentration as an abscissa (x) and the corresponding absorbance as an ordinate (y).

The obtained standard curve y is 0.0963x +0.0214, the correlation coefficient is 0.9876, see fig. 2, and the correlation is good.

And (3) measuring phosphate radicals:

adopting molybdenum blue colorimetry, taking 0.1g of sample, adding 1mL of concentrated sulfuric acid and 1mL of concentrated nitric acid into a beaker, heating until smoke is generated, cooling, and adding 1mL of 30% H₂O₂And slowly heating the solution, and repeating the steps until the beaker does not smoke any more, so that the solution is colorless and transparent or light yellow. After cooling, 1mL of hydrochloric acid of 6mol/L was added, and the mixture was heated to completely decompose the acid and transferred to a 50mL volumetric flask to a constant volume. 5mL of the solution is taken, the absorbance is measured according to a standard curve operation method, and the phosphate content is calculated according to the following formula.

Mass fraction of phosphate radical w (PO)₄ ^3-) Calculating the formula:

in the formula: m is measured at 580nm for the sampleThe mass of phosphate radical corresponding to absorbance is mg; m is₀Is the mass of the sample in g.

Infrared spectrum determination: respectively grinding dried Platymonas sobria extracellular polysaccharide and phosphorylated Platymonas polysaccharide with dried potassium bromide particles uniformly, tabletting a little mixture, and setting infrared wavelength measurement range of 4000-^-1And carrying out infrared spectrum analysis on the tabletted sample.

The infrared scanning results of the tetraselmis algae exopolysaccharide and its phosphorylated derivatives are shown in fig. 3 and fig. 4, respectively.

Referring to FIG. 3, the infrared absorbance of the Tetraselmis extracellular polysaccharide was detected to find that the result was 3414.00cm^-1An absorption peak exists, and is a characteristic-OH absorption peak; 1620.21cm^-1An absorption peak exists, the characteristic absorption peak is C ═ O, and hydrogen bonds exist; at 1134.14cm^-1An absorption peak exists, and is a C-O characteristic absorption peak; at 999.13cm^-1The relevant absorption peak at (A) is the characteristic absorption peak of C-O-C on the pyran ring, so that it can be concluded that the polysaccharide is a pyran-type polysaccharide.

Referring to FIG. 4, the chemical bonds in the exopolysaccharides of group 9 phosphorylated Platymonas were detected. It can be seen that 3456.44cm^-1The absorption peak is a characteristic-OH absorption peak, has weaker intensity than the non-phosphorylated polysaccharide absorption peak, and is presumed to be caused by the reduction of the content of-OH in polysaccharide with the same quality after phosphorylation; 1654.92cm^-1An absorption peak exists, the absorption peak is a characteristic absorption peak of C ═ O, the intensity of the absorption peak is weaker than that of the absorption peak of unphosphorylated polysaccharide, and the change of-OH concentration of phosphorylated polysaccharide is presumed to be caused; at 1099.43cm^-1An absorption peak exists, and is a C-O characteristic absorption peak. Compared with the non-phosphorylated tetraselmis galbana exopolysaccharide, the phosphorylated tetraselmis algaes exopolysaccharide still contains the characteristic absorption peak of the tetraselmis algaes polysaccharide, which shows that the main structure of the polysaccharide is not obviously changed. In addition, the phosphorylation was at 1265.30cm^-1The characteristic absorption peak is a P ═ O stretching vibration absorption peak, 891.11cm^-1The absorption peak should be the absorption peak of P-O-C bond, so it can be concluded that the Tetraselmis verticillata exopolysaccharide is successfully phosphorylated and the pyran type polysaccharide structure is still remained. The sodium tripolyphosphate and the sodium trimetaphosphate are in the range of 480-550cm^-1Within the range of its characteristicsSexual absorption Peak [ PO₃]Comparison of FIGS. 1 and 2 shows that the IR spectrum of phosphorylated tetraselmis algae exopolysaccharide is 505cm^-1The polysaccharide has obvious absorption peaks, but does not appear in the infrared spectrum of unphosphorylated polysaccharide, which indicates that phosphate groups are combined on polysaccharide molecules after phosphorylation.

Example 3: orthogonal assay of phosphorylated tetraselmis extracellular polysaccharide.

According to the preparation method, an orthogonal experiment design method is adopted, the proportion (A) of a phosphorylation reagent, the temperature (B), the time (C) and the pH (D) in a phosphorylation experiment are selected as investigation factors, each factor is selected from three levels (shown in table 1), and L is selected₉(3⁴) Orthogonal tables orthogonal experiments were performed. The orthogonal experimental design and results are shown in table 2:

TABLE 1 orthogonal experimental factors and levels

TABLE 2 Quadrature experiments and results

In the orthogonal test, the yield and the phosphate content are used as evaluation indexes, and four conditions of phosphorylation reagent proportion, reaction temperature, reaction time and pH value are screened. The worst analysis results using yield as an indicator show that the influencing factors B > A > D > C, i.e. the reaction temperature has the greatest influence on the yield, followed by the ratio of the phosphorylating reagent, followed by the pH value and finally the reaction time. The range analysis results using phosphate content as an index show that the influence factors B > D > A > C, i.e., the reaction temperature has the greatest influence on the phosphate content, the pH value, the ratio of the phosphorylation reagent, and the reaction time. By combining the influence of the two indexes, the optimal reaction condition is determined when the ratio of the phosphorylation reagents is 6: 1, the reaction temperature is 100 ℃, the reaction time is 6h, and the reaction pH value is 8.

Example 4: MTT method for detecting cell proliferation activity

Mouse macrophage cells Raw264.7 in logarithmic growth phase and in good condition were digested with pancreatin and then resuspended. Inoculating into 96-well culture plate to make cell density per well 1 × 10⁴Placing in 5% CO₂Culturing for 16h in an incubator at 37 ℃ to allow the cells to adhere to the walls, adding equivalent cells and culture solution to a normal cell control group for normal culture, adding equivalent culture medium to a blank control group without adding cells, adding tetraselmis galbana exopolysaccharide and phosphorylated tetraselmis exopolysaccharide (the mass concentration is 50, 100, 150, 200 and 250 mu g/ml respectively) to an administration group, setting 5 multiple holes for each concentration, allowing the medicine to act on the cells for 24h, adding 20 mu L of MTT solution (5mg/ml) to each hole, and continuously putting the holes into the incubator for culture for 4 h; after the culture is finished, the supernatant is discarded, 150 mu L of DMSO solution is added into each compound hole, the compound hole is wrapped by tinfoil and protected from light, and the compound hole is shaken on a shaking table for 10min to completely dissolve crystals. The absorbance (OD) of each well at 570nm was measured using a microplate reader. And calculating the cell viability using the formula: cell bioavailability/% (OD)_{Sample (I)}-OD_{Blank space})/(OD_Control-OD_{Blank space}) X 100. The experimental results were data processed and statistically analyzed using Graphpad Prism8 software.

PEPs and pPEPs with different concentration gradients act on the cells respectively, and the influence on the proliferation capacity of Raw264.7 cells is shown in FIG. 5. Compared with a blank control group, the PEPs group has a remarkable inhibiting effect (p is less than 0.05) on the proliferation of Raw264.7 cells at 100 mu g/mL. The pPEPs group has obvious inhibition effect on the proliferation of Raw264.7 cells at 50 mu g/mL (p is less than 0.05); compared with the PEPs group, the two groups have very significant difference (p < 0.01) in the inhibition effect on Raw264.7 proliferation at the drug concentration of 150 mu g/mL, and have significant difference (p < 0.05) at 200 and 250 mu g/mL.

The method is used for preparing the phosphorylated polysaccharide from the extraction and purification of the tetraselmis galbana exopolysaccharide, and has the advantages of quick and efficient operation, high product purity, less usage amount of a phosphorylation agent, shorter phosphorylation reaction time, high stability of the phosphorylated polysaccharide and high biological value.

The invention also provides a phosphorylated tetraselmis extracellular polysaccharide which is prepared by the preparation method of the phosphorylated tetraselmis extracellular polysaccharide, and the phosphorylated tetraselmis extracellular polysaccharide has high stability, has the effects of regulating the activity of immune cells and inhibiting the growth of tumor cells, and has higher medicinal value.

It should be noted that the technical solutions of the embodiments of the present invention can be combined with each other, but must be based on the realization of the technical solutions by those skilled in the art, and when the technical solutions are contradictory or can not be realized, the combination of the technical solutions should be considered to be absent and not to be within the protection scope of the present invention.

The above description is only a preferred embodiment of the present invention, and not intended to limit the scope of the present invention, and it will be apparent to those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.