阅读说明：本技术 一种裸藻多糖含量的定量测定方法 (Quantitative determination method for euglena polysaccharide content ) 是由 张道敬 鞠海军 于 2020-04-24 设计创作，主要内容包括：本发明提供一种裸藻多糖含量的定量测定方法,以刚果红染色的样品溶液作为待测溶液,在最大吸收波长540nm下测定所述待测溶液的吸光度,并根据标准方程计算出样品溶液中裸藻多糖的含量。(The invention provides a quantitative determination method of euglena polysaccharide content, which takes Congo red dyed sample solution as solution to be determined, determines the absorbance of the solution to be determined under the maximum absorption wavelength of 540nm, and calculates the content of the euglena polysaccharide in the sample solution according to a standard equation.)

1. The quantitative determination method is characterized in that a Congo red dyed sample solution is used as a solution to be determined, the absorbance of the solution to be determined is determined at the maximum absorption wavelength of 540nm, and the content of the euglena polysaccharide in the sample solution is calculated according to a standard equation.

2. The quantitative determination method according to claim 1, wherein the sample solution is dyed with a congo red solution, and the solution to be measured is formed by mixing the sample solution and the congo red solution at a volume ratio of 1: 2.

3. The quantitative determination method according to claim 2, wherein the solvent of the congo red solution is a phosphate buffer solution having a pH of 8.

4. The quantitative determination method according to claim 3, wherein the concentration of the phosphate buffer is 0.1 mol/L.

5. The quantitative determination method of claim 3, wherein the concentration of the Congo red solution is 150 μ g/mL.

6. The quantitative determination method of claim 1, wherein the solvent of the sample solution is a dimethyl sulfoxide solution.

7. The quantitative determination method of claim 2, wherein the dimethyl sulfoxide solution has a volume concentration of 10%.

8. A quantitative determination method for euglena polysaccharide content is characterized by comprising the following steps:

measuring the absorbance of the standard solution under the maximum absorption wavelength of 540nm, drawing a standard curve and obtaining a standard equation;

taking a Congo red dyed sample solution as a solution to be detected, and determining the absorbance of the solution to be detected under the maximum absorption wavelength of 540 nm; and the number of the first and second groups,

calculating and obtaining the content of the euglena polysaccharide in the sample solution according to the standard equation; wherein the content of the first and second substances,

the standard solution is a mixed solution of a euglena polysaccharide standard solution and a Congo red solution according to the volume ratio of 1:2,

the solvent of the standard solution of the euglena polysaccharide is dimethyl sulfoxide solution, and the mass concentration of the euglena polysaccharide in the standard solution of the euglena polysaccharide is 5-100 mug/mL;

the concentration of the Congo red solution is 150 mug/mL, and the solvent is a phosphate buffer solution with the pH value of 8;

dyeing the sample solution by using the Congo red solution, wherein the solution to be detected is formed by mixing the sample solution and the Congo red solution according to the volume ratio of 1: 2; and the number of the first and second electrodes,

the solvent of the sample solution is dimethyl sulfoxide solution.

9. The quantitative determination method according to claim 8, wherein the concentration of the phosphate buffer is 0.1 mol/L.

10. The quantitative determination method of claim 8, wherein the dimethyl sulfoxide solution has a volume concentration of 10%.

Technical Field

The invention relates to the technical field of analytical chemistry, in particular to a quantitative determination method for euglena polysaccharide content.

Background

The euglena polysaccharide is a specific linear polysaccharide with a beta-1, 3-D-glucan structure of the euglena, is not easy to digest like dietary fiber, and numerous small holes are found in a spirally wound complex structure after the euglena polysaccharide is degraded by microwave treatment. The interior is a porous structure, so that the adsorption performance is excellent, and the redundant substances in the body such as cholesterol, neutral fat, heavy metal, alcohol and the like can be adsorbed and discharged out of the body, and meanwhile, the activity of beneficial enzymes in the body is promoted, and the intestines and the stomach are adjusted. The Euglena polysaccharide has antioxidant and free radical scavenging effects. Has obvious effects of reducing cholesterol and reducing blood sugar.

The euglena polysaccharide has potential properties as an immunostimulant and immunopotentiator, and sulfated derivatives thereof also exhibit anti-HIV activity. In addition, the euglena polysaccharide and the modifier thereof can stimulate the immune system, especially macrophages, enhance the immune activity, and have extremely strong anticancer, antibacterial and antiviral activities. These immunomodulatory and anti-tumor activities represent a potential application of euglena gracilis in the biomedical field. The euglena polysaccharide has a relieving effect on acute hepatotoxicity of mice and has important potential to become a liver-protecting medicine.

In some reported patents and documents, the content of polysaccharide in euglena is determined by conventional methods for determining the content of polysaccharide, such as phenol-sulfuric acid method and anthrone method, but the conventional methods usually result in inaccurate measurement results due to other monosaccharides and different kinds of polysaccharide. In view of the structure of the euglena polysaccharide, the structure is highly concentrated and the euglena polysaccharide is a polysaccharide body consisting of linear beta-1, 3-glucan only, so that a specific measuring method of the beta-glucan can be considered to improve the accuracy of measuring the content of the euglena polysaccharide. The accurate determination of the content of the beta-glucan is always one of the main difficulties in research and development.

At present, the quantitative determination method of β -glucan is mainly an enzymatic method and a Flow Injection (FIA) fluorescence method, the former is often inaccurate due to the low purity of the enzyme, and the high purity specific β -glucan hydrolase is very expensive, which greatly limits the use of this method. The FIA method has the advantages of high efficiency and high accuracy, but the instrument is expensive. The binding capacity of the optical brightener calcium calcofour to beta-glucan is influenced by many factors, such as the luminescence of calcofour, experimental conditions, and the like. It is also difficult to apply this method widely.

The related patents are disclosed in part for the quantitative determination of polysaccharide content.

Chinese patent CN109682893A discloses a method for determining the content of lentinan in a lentinan composition, comprising: and (3) eluting the lentinan composition sample test solution by adopting a high performance liquid chromatography, and then measuring the content of lentinan. The method can effectively eliminate the influence of auxiliary materials such as dextran and the like on the content determination of the lentinan, improves the accuracy of the content determination, and is suitable for the content determination of various lentinan preparations.

Chinese patent CN103969384B discloses a method for measuring the content of arca subcrenata polysaccharide, firstly, determining that the polysaccharide in a sample to be measured is arca subcrenata polysaccharide by using a gel chromatography column high performance liquid chromatography, and then, measuring the content by using a spectrophotometry.

Chinese patent CN1220876C discloses a method for measuring the starch content in grifolan, which comprises the steps of removing fat-soluble components, removing soluble carbohydrate interference substances, decomposing starch by an enzyme method, detecting starch enzymolysis, processing starch enzymolysis liquid, measuring the reducing sugar content by a Fehling method and the like. The method has the advantages of strong operability, stable and reliable measurement result, small variation coefficient of the measurement result, small error, high reproducibility and the like.

Chinese patent CN100567956C discloses a method for measuring litchi polysaccharide content, the invention provides a method for solving protein interference when measuring litchi polysaccharide by using phenol-sulfuric acid method, the main content comprises: (1) preparing crude polysaccharide; (2) preparing refined litchi polysaccharide; (3) preparing and measuring a sample solution; (4) determining the net polysaccharide content in the crude litchi polysaccharide; (5) establishing a functional relation between the absorbance and the content of the protein contained in the polysaccharide when the polysaccharide is measured by a phenol-sulfuric acid method; (6) calculating the absorbance generated by the protein in the sample liquid; (7) and calculating the polysaccharide content of the sample.

Chinese patent CN102252978B discloses a method for measuring cordyceps mycelium mispolysaccharide, which comprises the following steps: (1) preparing a test solution; (2) preparing a standard curve; (3) determining the absorption coefficient of crude polysaccharide in the cordyceps sinensis mycelia; (4) repeating the steps (1) - (3) for n times to obtain absorption coefficients E2, E3 and E4 … … En of a plurality of test sample solutions to be tested, and calculating the average value E of the E1, the E2, the E3 and the E4 … … En, namely the absorption coefficient of the crude polysaccharide in the cordyceps mycelia; (5) calculating the content of crude polysaccharide in Cordyceps mycelium according to the following formula by using the determined absorption coefficient E.

However, the above-mentioned methods for measuring various polysaccharides are only suitable for each specific polysaccharide, and experiments show that these methods cannot accurately measure the content of euglena polysaccharide, and the measurement results are inaccurate.

Therefore, a new method for quantitative determination of polysaccharide content in Euglena is needed to solve the above technical problems.

Disclosure of Invention

The invention aims to provide a method for measuring the content of euglena polysaccharide, which has good repeatability and high accuracy.

In order to achieve the above object, according to an aspect of the present invention, there is provided a method for quantitatively determining a content of a euglena polysaccharide, the method comprising using a sample solution dyed with congo red as a solution to be measured, measuring an absorbance of the solution to be measured at a maximum absorption wavelength of 540nm, and calculating the content of the euglena polysaccharide in the sample solution according to a standard equation.

In some embodiments, the sample solution is dyed by a congo red solution, and the solution to be tested is formed by mixing the sample solution and the congo red solution according to a volume ratio of 1: 2.

In some embodiments, the solvent of the congo red solution is phosphate buffer at pH 8.

In some embodiments, the concentration of the phosphate buffer is 0.1 mol/L.

In some embodiments, the concentration of the Congo red solution is 150 μ g/mL. Namely, 150. mu.g of commercially available Congo red powder was contained in 1mL of the phosphate buffer.

In some embodiments, the solvent of the sample solution is dimethyl sulfoxide solution (DMSO solution).

In some embodiments, the dimethyl sulfoxide solution has a concentration of 10% by volume. Namely, the dimethyl sulfoxide solution is prepared by the volume ratio of the commercial pure dimethyl sulfoxide to water being 1: 9.

In a preferred embodiment of the present application, there is provided a method for quantitative determination of polysaccharide content in euglena, comprising the steps of: measuring the absorbance of the standard solution under the maximum absorption wavelength of 540nm, drawing a standard curve and obtaining a standard equation; taking a Congo red dyed sample solution as a solution to be detected, and determining the absorbance of the solution to be detected under the maximum absorption wavelength of 540 nm; and calculating to obtain the content of the euglena polysaccharide in the sample solution according to the standard equation; wherein the content of the first and second substances,

the standard solution is a mixed solution formed by mixing a euglena polysaccharide standard solution and a Congo red solution according to a volume ratio of 1: 2; the solvent of the standard solution of the euglena polysaccharide is dimethyl sulfoxide solution, and the mass concentration of the euglena polysaccharide in the standard solution of the euglena polysaccharide is 5-100 mug/mL; the concentration of the Congo red solution is 150 mug/mL, and the solvent is a phosphate buffer solution with the pH value of 8; dyeing the sample solution by using the Congo red solution, wherein the solution to be detected is formed by mixing the sample solution and the Congo red solution according to the volume ratio of 1: 2; and the solvent of the sample solution is dimethyl sulfoxide solution.

According to the method for quantitatively determining the content of the euglena polysaccharide, the Congo red is used for dyeing the euglena polysaccharide in the sample, so that the influence of impurities such as protein, monosaccharide and the like in the sample on quantitative determination is greatly reduced, and the accuracy of determining the content of the euglena polysaccharide is improved. The verification shows that the method for quantitatively determining the content of the euglena polysaccharide can accurately determine the content of the euglena polysaccharide in a sample, has high accuracy, and the calculated relative standard deviation is only about 3 percent. And, when various impurities are contained in the sample, the impurities do not influence the determination of the euglena polysaccharide content.

Drawings

FIG. 1: determination of the maximum absorption wavelength;

FIG. 2: according to the standard curve in the quantitative determination method of the euglena polysaccharide content, disclosed by the embodiment of the invention.

Detailed Description

Hereinafter, the technique of the present invention will be described in detail with reference to specific embodiments. It should be understood that the following detailed description is only for the purpose of assisting those skilled in the art in understanding the present invention, and is not intended to limit the present invention.

Example 1 determination of the wavelength of maximum absorption

In this example, the maximum absorption wavelength in the quantitative determination method of euglena polysaccharide content according to the present invention was first determined by a full wavelength scanning (190-800nm) method with an ultraviolet-visible spectrophotometer.

A quantity of highly purified euglena polysaccharide (cat #89862, Sigma Aldrich Corporation, St. Louis, MO, USA) was dissolved in 10% DMSO to make up a euglena polysaccharide stock solution. 0.015g of Congo red powder was dissolved in 0.1mol/L, pH 8.0.0 of phosphate buffer, and the volume was 100 mL. And (3) performing full-waveband absorption spectrum scanning on the mixed solution of 1mL of euglena polysaccharide mother solution and 2mL of Congo red solution by using an ultraviolet-visible spectrophotometer, and taking the mixed solution of 1mL of 10% DMSO solution and 2mL of Congo red solution as a blank control to obtain the detection result shown in figure 1. Thus, the most suitable maximum absorption wavelength (measurement wavelength) for determining the quantitative determination method of the polysaccharide content of Euglena according to the present invention is 540 nm.

Example 2 quantitative determination of polysaccharide content in Euglena

In this embodiment, a method for quantitatively determining the content of euglena polysaccharide is provided, which comprises the steps of: measuring the absorbance of the standard solution under the maximum absorption wavelength of 540nm, drawing a standard curve and obtaining a standard equation; taking a Congo red dyed sample solution as a solution to be detected, and determining the absorbance of the solution to be detected under the maximum absorption wavelength of 540 nm; and calculating to obtain the content of the euglena polysaccharide in the sample solution according to the standard equation; wherein the content of the first and second substances,

the standard solution is a mixed solution formed by mixing a euglena polysaccharide standard solution and a Congo red solution according to a volume ratio of 1: 2; the solvent of the standard solution of the euglena polysaccharide is dimethyl sulfoxide solution, and the mass concentration of the euglena polysaccharide in the standard solution of the euglena polysaccharide is 5-100 mug/mL; the concentration of the Congo red solution is 150 mug/mL, and the solvent is a phosphate buffer solution with the pH value of 8; dyeing the sample solution by using the Congo red solution, wherein the solution to be detected is formed by mixing the sample solution and the Congo red solution according to the volume ratio of 1: 2; and the solvent of the sample solution is dimethyl sulfoxide solution.

Specifically, in this embodiment, the method for quantitatively determining the content of euglena polysaccharide includes the following steps:

preparation of (I) Standard solution

Purified euglena polysaccharide (cat #89862, Sigma Aldrich Corporation, st. louis, MO, USA) was taken, dissolved in a small amount of 10% DMSO solvent with ultrasound-assisted to prepare a euglena polysaccharide mother liquor. 0.015g of Congo red powder was dissolved in 0.1mol/L, pH 8.0.0 of phosphate buffer, and the volume was 100 mL.

10 sets of 10mL tubes were taken, the first containing only one tube and the other containing 3 parallel tubes per set. The euglena polysaccharide mother liquor is added into each group by 0, 5, 10, 20, 50, 100, 150, 200, 500 and 1000 mu g respectively, and then is supplemented to 1mL by 10% DMSO so as to obtain euglena polysaccharide standard liquor with the concentration of 0.5 mu g/mL, 1.0 mu g/mL, 2.0 mu g/mL, 5.0 mu g/mL, 10 mu g/mL, 15 mu g/mL, 20 mu g/mL, 50 mu g/mL and 100 mu g/mL.

2mL of Congo red solution was added to the above test tube, and shaken well to allow sufficient reaction to obtain a standard solution.

(II) drawing a standard curve

Measuring the photometric absorption value (OD value) at 540nm of each standard solution, plotting the euglena polysaccharide concentration as abscissa (X) and the absorbance value as ordinate (Y) to obtain the standard curve shown in FIG. 2, and obtaining regression equation of Y ═ 0.0021X +0.0251 and correlation coefficient (R value)²＝0.9975)。

In this step, it is concluded that: the method provided by the invention conforms to the beer law within the mass concentration range of the euglena polysaccharide concentration of 5-100 mug/mL, and the regression effect is obvious.

(III) measurement of sample

A sample solution was prepared by dissolving 0.050g of the sample in 10% DMSO. And (3) taking 1mL of sample solution, adding 2mL of Congo red solution, and uniformly mixing. And (3) after the reaction is completed, determining the OD value at 540nm, and calculating the content of the euglena polysaccharide in the sample according to the regression equation in the step (II) and the standard curve shown in the figure 2.

Example 3 verification of examples

In this example, the quantitative determination of the euglena polysaccharide content of example 2 was verified. The verification step comprises:

preparing purified euglena polysaccharide: weighing 1g of dry euglena powder, suspending the powder in 20mL of SDS solution of 8.5g/L, performing ultrasonic treatment for 18.5min (ultrasonic treatment for 3s, interval for 4s), centrifuging the suspension (4000rpm,5min) to remove supernatant, adding 40mL of 1g/L SDS solution into the precipitate, placing the precipitate in 95 ℃ water bath for 1h, centrifuging at 4000rpm for 5min to remove supernatant, adding 0.1g/L SDS solution into the precipitate, performing 50 ℃ water bath for 0.5 h, centrifuging, and washing twice with distilled water. Freeze drying to obtain euglena polysaccharide, suspending and centrifuging (4000rpm,5min), removing supernatant, adding 1 g/L40 mL SDS solution into the precipitate, placing in 95 deg.C water bath for 1h, centrifuging at 4000rpm to remove supernatant, washing with distilled water twice, and freeze drying to obtain purified euglena polysaccharide.

Dissolving 0.050g of the purified euglena polysaccharide in 10% DMSO to prepare a sample solution, taking 1mL of the sample solution, adding 2mL of Congo red solution, and uniformly mixing until the reaction is complete. The content of euglena polysaccharide in the purified euglena polysaccharide was 0.9569g/g as determined by the quantitative determination method described in example 2, measuring the OD at 540nm, and calculating according to the regression equation of step (II) in example 2 and the standard curve shown in FIG. 2.

The result of the quantitative determination method is accurate.

Example 4 influence of impurities on the assay results

In this example, the effect of reaction impurities on the quantitative determination method of the content of euglena polysaccharide according to the present invention is considered.

The results of the method in which 1mg and 10mg of glucose, soluble starch and casein were added to 1mL of the measurement system, respectively, and the effect of these impurities on the method were examined are shown in Table I.

TABLE I absorbance of Euglena polysaccharide standards and impurities-containing Euglena polysaccharide standards

Wherein, the sample I is a euglena polysaccharide standard sample (mu g/mL), the sample II is a standard sample plus glucose (mu g/mL), the sample III is a standard sample plus soluble starch (mu g/mL), and the sample IV is a standard sample plus protein (mu g/mL).

As can be seen from the results of the Table, in the case of a content much higher than that of β -glucan, the measurement results were not greatly affected by other impurities except for 10mg of soluble starch. In the experimental process, it was found that adding 10mg of soluble starch to 1mL of the reaction system did not completely dissolve, which resulted in turbidity of the reaction system and an influence on OD. This indicates that the combination of Euglena polysaccharide and Congo red has good specificity.

Example 5 precision test

Relative standard deviation RSD refers to: the ratio of the standard deviation to the arithmetic mean of the measurements. The precision of the results can be analyzed in the test work. Therefore, in this example, the same sample was tested 5 times, and in this example, the precision of the quantitative determination method of the content of euglena polysaccharide of the present invention was considered, and the results shown in table two were obtained.

TABLE II, relative Standard deviations of standards

As can be seen from the table two, the RSD value of the content of the euglena polysaccharide measured by the quantitative determination method of the content of the euglena polysaccharide is only about 3%, which shows that the quantitative determination method of the invention has better repeatability and high precision.

The present invention has been described in relation to the above embodiments, which are only exemplary of the implementation of the present invention. It must be noted that the disclosed embodiments do not limit the scope of the invention. Rather, modifications and equivalent arrangements included within the spirit and scope of the claims are included within the scope of the invention.