Preparation method and application of elaeagnus angustifolia gum homogeneous polysaccharide

Abstract

The invention discloses a preparation method and application of elaeagnus angustifolia gum homogeneous polysaccharide. The method comprises the following steps: removing impurities from elaeagnus angustifolia gum, crushing, and extracting with pure water; precipitating the extracting solution with ethanol to obtain crude elaeagnus angustifolia gum polysaccharide; loading the crude polysaccharide on an ion exchange chromatography column for elution, and collecting the polysaccharide at the elution part with water; freeze-drying to obtain polysaccharide at the neutral part of elaeagnus angustifolia gum; and loading the neutral part polysaccharide sample on a gel chromatographic column for purification, eluting with water, collecting the peak top end, and freeze-drying to obtain the elaeagnus angustifolia gum homogeneous polysaccharide. The obtained elaeagnus angustifolia gum homogeneous polysaccharide is a white flaky flocculate, the molecular weight is 158.8 * 10 < 4 > Dalton, the purity is high, monosaccharide is composed of rhamnose, arabinose, xylose, mannose and galactose, and detection shows that the elaeagnus angustifolia gum homogeneous polysaccharide has remarkable immunoregulation and antioxidant activity, and can be used for preparing the immunopotentiator. The compound can be used as an immunomodulatory component or / and an antioxidant active component for preparing health care products and food, and has wide application prospects and market value.

Description

Technical Field

[0001] The invention belongs to a preparation method and application of an angustifolia gum uniform polysaccharide, and relates to the technical development and application field of traditional Chinese medicine. Background Art

[0002] Natural plant gums come from the stems and branches of trees that are attacked by sandstorms, artificial mechanical damage, insect bites and infections, and other external invasions. The stems and branches secrete exudative gum juice, which covers the injured area to prevent infection of cracked or injured tissue surfaces, inhibit water loss, and the invasion and spread of microorganisms to the stems and branches. The production of these exudative gums can also be increased by making artificial cuts and incisions on the bark of trees or shrubs. The production of natural gums is high every summer, which means that dry and hot environments are conducive to the production of exudative gums. Plant gums can be divided into exudative and non-exudative types according to their release form. The exudative release process occurs in epithelial cells as a defense mechanism against environmental stress (high temperature) and physical damage or microbial attack on the cortex, while non-exudative polysaccharide reserves are mainly found in seeds and plant mucus. Exudative natural gums are amorphous in nature. After drying, they form glassy hard secretions, such as white (gum arabic), translucent white to dark red (guar gum, almond gum), amber (tragacanth gum, peach gum, galangal gum), light gray (karaya gum), dark brown, etc. In addition, gums obtained from plants are considered to be natural biopolymers, composed of polysaccharides as the main components, containing small amounts of proteins, phenolic compounds and inorganic elements (calcium, potassium, magnesium and sodium), etc. The functional properties and potential applications of natural gums depend largely on their structure and physicochemical properties, such as monosaccharide composition, molecular weight, glycosidic bond configuration, conformation and rheological properties. In this context, these macromolecular biopolymers have high industrial potential and are widely used in food, pharmaceuticals, cosmetics, water, energy and environmental fields. Therefore, the industrialization of natural gums is related to their performance as thickeners, gelling agents, emulsifiers, stabilizers, dietary fibers, and food coatings and films.

[0003] Elaeagnus angustifolia gum is the gum exuded from the stems and branches of the Elaeagnus angustifolia plant. Elaeagnus angustifolia is a desert tree species with tenacious vitality, distributed in North China, Northwest China and Liaoning. Elaeagnus angustifolia gum is rich in resources in Xinjiang, easy to collect, and widely used. It has multiple functions and innovative characteristics in the food industry. The main component of Elaeagnus angustifolia gum is polysaccharide, which is a macromolecule with extensive biological activity. In recent years, many researchers have conducted a large number of in-depth studies on polysaccharide components isolated from natural plants and found that polysaccharides not only have extensive biological and pharmacological activities, but also have the characteristics of bidirectional immunomodulation, pleiotropy and low toxicity and side effects. Natural plant polysaccharides often synergize with other immunomodulators in immunomodulation to jointly promote the secretion of cytokines. For example, synergizing with antioxidants such as vitamin C and vitamin E can reduce the damage of oxidative stress to immune cells and enhance the ability of immune cells to secrete cytokines. In addition, plant polysaccharides can also interact with certain Chinese medicine ingredients to exert better immunomodulatory effects, promote the rational secretion of cytokines, and maintain the body's immune balance. In the process of immune regulation, different cytokines have different effects, and natural plant polysaccharides can regulate the secretion balance of cytokines. On the one hand, it can promote the secretion of anti-inflammatory cytokines, such as interleukin-10 (IL-10), which helps to reduce excessive inflammatory responses and prevent immune damage. On the other hand, in the face of pathogen invasion, plant polysaccharides can induce the production of pro-inflammatory cytokines, such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), to enhance the body's anti-infection ability. In short, natural plant polysaccharides promote the secretion of cytokines through various pathways in the process of immune regulation, and play an important role in maintaining the body's immune balance and health. As a substance widely present in nature, polysaccharides have many biological activities, safety and low toxicity advantages, which will play a broad development prospect in clinical applications.

[0004] Elaeagnus angustifolia gum is a water-soluble high molecular polysaccharide, and its main components and structure are basically the same as those of gum arabic. Therefore, it can have a wide range of application value in the cosmetics, food, medicine and other industries like gum arabic. The relatively high price of gum arabic is determined by the fact that its production cannot be expanded, which limits its use. Therefore, it is of great significance to purify and develop Elaeagnus angustifolia gum, a special resource in Xinjiang, to meet market demand and enhance the competitiveness of gum arabic. At the same time, the industrialization of this project will improve the comprehensive utilization efficiency of Xinjiang's resources and play a positive role in promoting the development of the local economy. At present, the gum arabic used in my country's food industry is mainly imported. In other industries or fields, the application of gum also has great potential, so the development of domestic gum, especially its application research, is of great significance to my country's scientific progress and economic construction.

[0005] The 10 patent documents retrieved with the keyword of Elaeagnus elata polysaccharide and the 4 patent documents on preparation methods did not mention the research content related to the separation and purification of natural Elaeagnus elata polysaccharide, and no research on Elaeagnus elata polysaccharide has been reported. The Chinese patent document with application number 201610097695.9 discloses a method for preparing Elaeagnus elata polysaccharide, which to a certain extent analyzes the material basis of Elaeagnus elata polysaccharide, a traditional folk hair care cosmetics secret recipe in Xinjiang; the patent document with application number 200410078837.4 discloses a method for preparing Elaeagnus elata powder; the patent document with application number 02117954.9 discloses a method for refining and decolorizing Elaeagnus elata; the patent document with application number 94117408.5 involves the preparation of a natural gum hair care mousse; these four patents have the same raw materials as this patent and are the results of the previous work of this research group. The present invention is a further study based on the previous work of this subject. It adopts a wide range of applications, stable and effective water extraction and alcohol precipitation method to extract crude polysaccharides. On the one hand, it is different from and has advantages over the acetone extraction method involved in the application publication number 201610097695.9. On the other hand, the extraction method involved in this patent reduces the extraction cost and also reduces the pollution to the environment. It is suitable for large-scale, batch, and standardized production, and increases the industrialization of angustifolia gum polysaccharides, so as to better play its application in food and health products. In the present invention, the separation and purification of angustifolia gum polysaccharides uses DEAE agarose gel (DEAE Sepharose-FF) chromatographic column and cross-linked agarose gel (Superdex-200) chromatographic column filler that matches the characteristics and molecular weight of the polysaccharide. Its high adsorption capacity, good selectivity, high chemical stability, easy operation and reusability have become a commonly used separation tool in the fields of biochemistry, pharmacy and biotechnology. It can efficiently separate and purify angustifolia gum polysaccharides, and provides strong support for the separation and purification research and production of angustifolia gum homogeneous polysaccharides. However, the current research on Elaeagnus oleifera polysaccharides is very scarce, and there has been no research report on the separation and purification of uniform polysaccharides from Elaeagnus oleifera and its antioxidant / immune effects. The previous invention and research on Elaeagnus oleifera polysaccharides only stayed at the preliminary understanding and description of the crude extract of Elaeagnus oleifera, and the Elaeagnus oleifera polysaccharides had not been systematically separated and purified. Therefore, the present invention is to prepare a high-purity uniform polysaccharide from Elaeagnus oleifera, which contains an average distribution of similar chain lengths and is a uniform component with a certain weight range of high consistency. At the same time, its biological activity is preliminarily explored. On the one hand, it lays a solid foundation for the accurate structural analysis and subsequent research and application development of Elaeagnus oleifera uniform polysaccharides, and on the other hand, it provides a new way for the efficient utilization and development of Elaeagnus oleifera resources in Xinjiang, and provides a theoretical basis for the development and utilization of Xinjiang's characteristic medicinal plant resources. Summary of the invention

[0006] The purpose of the present invention is to provide a preparation method and use of jujube gum uniform polysaccharide, the method comprising: removing impurities from elata gum, crushing and screening, and then extracting with pure water; precipitating the extract with ethanol to obtain a precipitate as elata gum crude polysaccharide; loading the crude polysaccharide onto an ion exchange chromatography column for elution, collecting the polysaccharide in the water eluted portion; freeze-drying to obtain the elata gum neutral portion polysaccharide; loading the neutral portion polysaccharide onto a gel chromatography column for purification, eluting with water, collecting the peak top, and freeze-drying to obtain elata gum uniform polysaccharide. The obtained elata gum uniform polysaccharide is a white flaky flocculent, and the molecular weight is 158.8×10 4 Dalton, showing a smooth and sharp single peak, indicating high purity, its monosaccharide composition is rhamnose, arabinose, xylose, mannose and galactose, the molar ratio is: 3.240:48.842:2.633:5.729:32.734; the test shows that the uniform polysaccharide of Elaeagnus angustifolia gum has significant immunomodulatory and antioxidant activities. It can be used as an immunomodulatory component or / and antioxidant active component in the preparation of health products and foods, and has broad application prospects and market value.

[0007] The method for preparing a uniform polysaccharide of Elaeagnus angustifolia gum according to the present invention is carried out according to the following steps:

[0008] a. Wash the collected Elaeagnus angustifolia gum, dry it naturally, and then mechanically crush it for 60-80 seconds, sieve it through a 60-100 mesh sieve to separate impurities, and obtain Elaeagnus angustifolia gum powder;

[0009] b. Dissolve the elata gum powder obtained in step a in distilled water to prepare an elata gum aqueous solution with a concentration of 1.8-2%, stir magnetically for 8-12 hours at a temperature of 25-35° C., centrifuge for 10 minutes, separate and remove impurities, and obtain an elata gum solution;

[0010] c. Slowly add the Elaeagnus angustifolia gum solution obtained in step b to 95% ethanol at a volume ratio of 1:4 for alcohol precipitation, stirring at a uniform speed while adding, and standing in a refrigerator at 4°C for 8-12 hours after alcohol precipitation, then take out, centrifuge and filter, and wash the obtained precipitate with anhydrous ethanol at a volume ratio of 1:1 for 3-5 times, and collect the precipitate for later use;

[0011] d. After re-dissolving the precipitate collected in step c, add deionized water, dialyze with water and a dialysis bag with a pore size of 3500Da for 24 hours, and freeze-dry to obtain crude polysaccharide of Elaeagnus angustifolia.

[0012] e. Load the crude polysaccharide of Elaeagnus anhydrica onto a DEAE agarose anion exchange gel column, elute with water, collect the effective part eluted with water, follow up and detect the collected liquid with anthrone-sulfuric acid method, draw the elution curve, dialyze with water and a dialysis bag with a pore size of 3500Da for 24 hours, and freeze-dry to obtain the polysaccharide of the neutral part eluted with Elaeagnus anhydrica;

[0013] f. Dissolve the neutral part polysaccharide of Elaeagnus angustifolia in water, centrifuge, take the supernatant, load it on a cross-linked agarose gel chromatography column, and elute with water; use anthrone-sulfuric acid method to track and detect the collected solution, draw an elution curve, collect the peak top, dialyze and dry, and obtain an average molecular weight of 158.8×10 4 Dalton, high purity, sugar content 53.54%, protein content less than 1%; monosaccharide composition: rhamnose, arabinose, xylose, mannose and galactose, molar ratio of 3.240:48.842:2.633:5.729:32.734, an elata gum uniform polysaccharide.

[0014] The use of the uniform polysaccharide of Elaeagnus angustifolia obtained by the method in preparing foods containing antioxidant active ingredients.

[0015] The uniform polysaccharide of Elaeagnus angustifolia obtained by the method is used in preparing health products with antioxidant active ingredients.

[0016] The use of the uniform polysaccharide of Elaeagnus angustifolia obtained by the method in preparing foods containing immunomodulatory ingredients.

[0017] The uniform polysaccharide of Elaeagnus angustifolia obtained by the method is used in preparing health products with immune regulation components.

[0018] The preparation method and use of a jujube gum uniform polysaccharide described in the present invention, the method separates and purifies from the elata gum to obtain a uniform polysaccharide with antioxidant and immunomodulatory activities, the preparation method is simple, no acid, alkali or other toxic and harmful reagents are used in the entire preparation process, the stability of the active structure of the elata gum uniform polysaccharide can be fully guaranteed, and it is suitable for large-scale production. Experiments have shown that the obtained elata gum uniform polysaccharide can effectively increase the secretion level of cytokines such as interleukin-6, interleukin-1β, tumor necrosis factor-α and nitric oxide in mouse monocytic leukemia macrophages (RAW264.7), and can be used as an immunomodulatory component for the preparation of health products or foods. It provides a basis for the development of antioxidant and / or immunomodulatory foods and health products, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the elution curve of the separation of the Elaeagnus angustifolia glue part using a DEAE agarose gel anion exchange column in the present invention;

[0020] Figure 2 This is the elution curve of the cross-linked agar gel chromatography column used to purify the homogeneous polysaccharide of Elaeagnus angustifolia.

[0021] Figure 3 This is the standard curve for the determination of the content of the homogeneous polysaccharide of Elaeagnus angustifolia gum in the present invention;

[0022] Figure 4 This is an ultraviolet scanning analysis diagram of the uniform polysaccharide of Elaeagnus angustifolia gum in the present invention;

[0023] Figure 5 This is an infrared spectrum test diagram of the uniform polysaccharide of Elaeagnus angustifolia in the present invention;

[0024] Figure 6 The HPLC gel chromatogram of the homogeneous polysaccharide of Elaeagnus angustifolia gum in the present invention;

[0025] Figure 7 This is a graph showing the results of gas chromatography-mass spectrometry testing of the standard substance in the experiment of the uniform polysaccharide and monosaccharide composition of Elaeagnus angustifolia gum in the present invention;

[0026] Figure 8 This is a graph showing the results of gas chromatography-mass spectrometry testing of samples in the experiment of uniform polysaccharide and monosaccharide composition of Elaeagnus angustifolia gum in the present invention;

[0027] Fig. 9 This is a graph showing the results of an experiment on the antioxidant activity of the homogeneous polysaccharide of Elaeagnus angustifolia in the present invention;

[0028] Fig.10 This is a graph showing the experimental results of the effect of the homogeneous polysaccharide of Elaeagnus angustifolia on the survival rate of mouse monocytic leukemia macrophages (RAW 264.7) stimulated by the present invention;

[0029] Fig.11 This is a graph showing the experimental results of the effect of the homogeneous polysaccharide of Elaeagnus angustifolia in the present invention on inducing the release of cytokines TNF-α, IL-6, IL-1β and NO in mouse monocytic leukemia macrophages (RAW 264.7). DETAILED DESCRIPTION

[0030] The present invention is further described in detail below in conjunction with examples and drawings, but the uses and purposes of these exemplary embodiments are only used to list the present invention and do not constitute any form of limitation on the actual protection scope of the present invention, nor do they limit the protection scope of the present invention to them.

[0031] Example 1

[0032] a. Wash the collected Elaeagnus angustifolia gum, dry it naturally, and then mechanically crush it for 60 seconds, sieve it through a 60-mesh sieve to separate impurities, and obtain Elaeagnus angustifolia gum powder;

[0033] b. Dissolve the elata gum powder obtained in step a in distilled water to prepare an elata gum aqueous solution with a volume concentration of 1.8%, stir magnetically for 8 hours at 25° C., centrifuge for 10 minutes, separate and remove impurities, and obtain an elata gum solution;

[0034] c. Slowly add the Elaeagnus angustifolia gum solution obtained in step b to 95% ethanol at a volume ratio of 1:4 for alcohol precipitation, stirring at a uniform speed while adding, and standing in a refrigerator at 4°C for 12 hours after alcohol precipitation, take out, centrifuge and filter, and wash the obtained precipitate with anhydrous ethanol by centrifugation for 3-5 times at a volume ratio of 1:1, and collect the precipitate for later use;

[0035] d. After re-dissolving the precipitate collected in step c, add deionized water, dialyze with water and a dialysis bag with a pore size of 3500Da for 24 hours, and freeze-dry to obtain crude polysaccharide of Elaeagnus angustifolia.

[0036] e. Load the crude polysaccharide of Elaeagnus anhydrica onto an anionic DEAE agarose ion exchange gel column, elute with water, collect the effective part eluted with water, follow up and detect the collected liquid with anthrone-sulfuric acid method, draw the elution curve, dialyze with water and a dialysis bag with a pore size of 3500Da for 24 hours, and freeze-dry to obtain the polysaccharide of the neutral part eluted with Elaeagnus anhydrica;

[0037] f. Dissolve the neutral part polysaccharide of Elaeagnus angustifolia in water, centrifuge, take the supernatant, load it on a cross-linked agarose gel chromatography column, and elute with water; use anthrone-sulfuric acid method to track and detect the collected solution, draw an elution curve, collect the peak top, dialyze and dry, and obtain an average molecular weight of 158.8×10 4 Dalton, high purity, sugar content 53.54%, protein content less than 1%; monosaccharide composition: rhamnose, arabinose, xylose, mannose and galactose, molar ratio of 3.240:48.842:2.633:5.729:32.734, an elata gum uniform polysaccharide.

[0038] Example 2

[0039] a. Wash the collected Elaeagnus angustifolia gum, dry it naturally, and then mechanically crush it for 70 seconds, sieve it through an 80-mesh sieve to separate impurities, and obtain Elaeagnus angustifolia gum powder;

[0040] b. Dissolve the elata gum powder obtained in step a in distilled water to prepare an elata gum aqueous solution with a volume concentration of 2%, stir magnetically for 10 hours at a temperature of 30° C., centrifuge for 10 minutes, separate and remove impurities, and obtain an elata gum solution;

[0041] c. Slowly add the Elaeagnus angustifolia gum solution obtained in step b to 95% ethanol at a volume ratio of 1:4 for alcohol precipitation, stirring at a uniform speed while adding, and standing in a refrigerator at 4°C for 10 hours after alcohol precipitation, then take out, centrifuge and filter, and wash the obtained precipitate 4 times with anhydrous ethanol at a volume ratio of 1:1, and collect the precipitate for later use;

[0042] d. After re-dissolving the precipitate collected in step c, add deionized water, dialyze with water and a dialysis bag with a pore size of 3500Da for 24 hours, and freeze-dry to obtain crude polysaccharide of Elaeagnus angustifolia.

[0043] e. Load the crude polysaccharide of Elaeagnus anhydrica onto a DEAE agarose anion exchange gel column, elute with water, collect the effective part eluted with water, follow up and detect the collected liquid with anthrone-sulfuric acid method, draw the elution curve, dialyze with water and a dialysis bag with a pore size of 3500Da for 24 hours, and freeze-dry to obtain the polysaccharide of the neutral part eluted with Elaeagnus anhydrica;

[0044] f. Dissolve the neutral part polysaccharide of Elaeagnus angustifolia in water, centrifuge, take the supernatant, load it on a cross-linked agarose gel chromatography column, and elute with water; use anthrone-sulfuric acid method to track and detect the collected solution, draw an elution curve, collect the peak top, dialyze and dry, and obtain an average molecular weight of 158.8×10 4 Dalton, high purity, sugar content 53.54%, protein content less than 1%; monosaccharide composition: rhamnose, arabinose, xylose, mannose and galactose, molar ratio of 3.240:48.842:2.633:5.729:32.734, an elata gum uniform polysaccharide.

[0045] Example 3

[0046] a. Wash the collected Elaeagnus angustifolia gum, dry it naturally, and then mechanically crush it for 80 seconds, sieve it through a 100-mesh sieve to separate impurities, and obtain Elaeagnus angustifolia gum powder;

[0047] b. Dissolve the elata gum powder obtained in step a in distilled water to prepare an elata gum aqueous solution with a concentration of 2%, stir magnetically for 12 hours at 35° C., centrifuge for 10 minutes, separate and remove impurities, and obtain an elata gum solution;

[0048] c. Slowly add the Elaeagnus angustifolia gum solution obtained in step b to 95% ethanol at a volume ratio of 1:4 for alcohol precipitation, stirring at a uniform speed while adding, and standing in a refrigerator at 4°C for 12 hours after alcohol precipitation, then take out, centrifuge and filter, and wash the obtained precipitate with anhydrous ethanol by centrifugation 5 times at a volume ratio of 1:1, and collect the precipitate for later use;

[0049] d. After re-dissolving the precipitate collected in step c, add deionized water, dialyze with water and a dialysis bag with a pore size of 3500Da for 24 hours, and freeze-dry to obtain crude polysaccharide of Elaeagnus angustifolia.

[0050] e. Load the crude polysaccharide of Elaeagnus anhydrica onto a DEAE agarose anion exchange gel column, elute with water, collect the effective part eluted with water, follow up and detect the collected liquid with anthrone-sulfuric acid method, draw the elution curve, dialyze with water and a dialysis bag with a pore size of 3500Da for 24 hours, and freeze-dry to obtain the polysaccharide of the neutral part eluted with Elaeagnus anhydrica;

[0051] f. Dissolve the neutral part polysaccharide of Elaeagnus angustifolia in water, centrifuge, take the supernatant, load it on a cross-linked agarose gel chromatography column, and elute with water; use anthrone-sulfuric acid method to track and detect the collected solution, draw an elution curve, collect the peak top, dialyze and dry, and obtain an average molecular weight of 158.8×10 4 Dalton, high purity, sugar content 53.54%, protein content less than 1%; monosaccharide composition: rhamnose, arabinose, xylose, mannose and galactose, molar ratio of 3.240:48.842:2.633:5.729:32.734, an elata gum uniform polysaccharide.

[0052] Example 4

[0053] Determination of the physical and chemical properties of uniform polysaccharides from Elaeagnus angustifolia gum:

[0054] Determination of neutral polysaccharide content in homogeneous polysaccharide of Elaeagnus angustifolia

[0055] Preparation of 6% phenol solution: accurately weigh 30 g of phenol, dissolve it in distilled water and dilute to 500 mL to obtain 6% phenol solution;

[0056] Standard curve drawing: accurately weigh 20 mg of standard glucose in a 500 mL volumetric flask, add water to the mark, dissolve and mix, and then dilute to a 0.2 mg / ml glucose standard solution; take 0.4 mL, 0.36 mL, 0.3 mL, 0.2 mL, 0.16 mL and 0.1 mL respectively, make up to 0.4 mL with water, then add 0.2 mL 6% phenol and 1.0 mL concentrated sulfuric acid, repeat three groups, let stand for 10 minutes, shake well, and measure the absorbance at 490 nm after leaving at room temperature for 20 minutes (Table 1). Use 2.0 mL of water and follow the same steps as the blank group. The horizontal axis is the number of micrograms of polysaccharide, and the vertical axis is the absorbance value. The standard curve ( Figure 3 );

[0057] Sample content determination: The homogeneous polysaccharide of Elaeagnus angustifolia gum was prepared into 0.2 mg / ml, and 3 groups of samples were taken. The average value of the 3 groups of data was obtained according to the standard curve. The total sugar content of the homogeneous polysaccharide of Elaeagnus angustifolia gum was calculated according to the standard curve.

[0058] Table 1. Standard absorbance values ​​of the homogeneous polysaccharide content determination experiment of Elaeagnus angustifolia

[0059]

[0060] According to the calculation, the content of neutral polysaccharide in the homogeneous polysaccharide of Elaeagnus angustifolia gum is 53.54%;

[0061] The purity and molecular weight of the homogeneous polysaccharide of Elaeagnus angustifolia gum were determined by high performance liquid gel chromatography (HPGPC):

[0062] The chromatographic columns are KS-804 and KS-805 connected in series; the mobile phase is 0.2 mol / L sodium chloride aqueous solution; column temperature: 40°C; flow rate: 0.6 mL / min; injection volume: 50 μL;

[0063] Take 2 mg of each of Dextran P-series standard dextran P-5, P-10, P-20, P-50, P-100, P-200, P-400 and P-800 with known molecular weight, and prepare them into 2 mg / mL solutions with 0.2 mol / L sodium chloride aqueous solution as mobile phase; centrifuge at 10000 rpm for 10 min; take the supernatant and inject 20 μL each time; make a standard curve with the logarithm of molecular weight versus retention time;

[0064] Take 2 mg of the sample to be tested, and prepare a 2 mg / mL solution with a 0.2 mol / L sodium chloride aqueous solution as the mobile phase; centrifuge at 10000 rpm for 10 min; take the supernatant, inject 20 μL, and perform high performance liquid gel chromatography test; according to the measured sample retention time, the average molecular weight of the sample can be obtained from the standard curve; the drawing and processing of the standard curve and the calculation of the molecular weight are automatically completed by the GPC software;

[0065] The molecular weight of the homogeneous polysaccharide of Elaeagnus angustifolia gum obtained by high performance liquid gel chromatography (HPGPC) was 158.8×10 4 Dalton;

[0066] Figure 6 The high performance liquid gel chromatography (HPGPC) diagram of the homogeneous polysaccharide of Elaeagnus angustifolia gum is shown in FIG. Figure 6 It can also be explained that the homogeneous polysaccharide of Elaeagnus angustifolia gum presents a smooth and sharp single peak after high performance liquid gel chromatography, indicating that the purity is high:

[0067] The obtained polysaccharide is a uniform polysaccharide;

[0068] Analysis of monosaccharide composition of homogeneous polysaccharide of Elaeagnus angustifolia gum:

[0069] Weigh 2 mg of polysaccharide sample and place it in a 5 mL ampoule, add 1 mL of 2 mol / L trifluoroacetic acid solution to dissolve it, and then place it in an oven at 121°C for hydrolysis for 120 min. After taking it out and letting it cool to room temperature, draw 100 μL of the hydrolyzate into a 20 mL stoppered test tube, neutralize it with 100 μL of 2 mol / L sodium hydroxide solution, and add 10 μL 2mg / mL inositol (internal standard), then add 1mL of newly prepared 0.5mol / L sodium borohydride-dimethyl sulfoxide solution, vortex mix, and reduce in a water bath at 40°C for 90min; after the reaction is completed, add 100μL of acetic acid dropwise to neutralize until no bubbles are generated, then add 1mL of acetic anhydride and 200μL of 1-methylimidazole, react in a water bath at 40°C for 10min, add distilled water to mix to terminate the reaction, and then add 1mL of chloroform to extract the acetylated product; after stratification, absorb the upper aqueous layer, add 2mL of distilled water to continue washing the lower chloroform layer, and after washing 3 times, absorb the lower solution, dry it over anhydrous sodium sulfate, place it in a gas chromatography-mass spectrometry injection bottle, sort the samples, and perform GC-MS detection and analysis;

[0070] GC conditions: DM-5MS (60mx0.25 mmx0.25 um) column, He carrier, flow rate 1mL / min; injection port ion source temperature 250°C;

[0071] Program temperature rise: start at 140°C, quickly increase to 192°C at 4°C / min, then slowly increase to 204°C at 1°C / min, keep the temperature for 4 min; then slowly increase to 215°C at 1°C / min, keep the temperature for 4 min; then increase to 250°C at 5°C / min, keep the temperature for 9 min, a total of 60 min;

[0072] Mass spectrometry: EI source, 70 eV, source temperature 230 °C, quadrupole 150 °C, scanning range: m / z = 40-500, mass spectrometry on time 8-60 min;

[0073] According to the GC-MS results, compared with the standard Figure 7 , followed by rhamnose, fucose, arabinose, xylose, mannose, glucose and galactose, and the sugar composition of the homogeneous polysaccharide of Elaeagnus angustifolia gum was obtained. Figure 8 , followed by rhamnose, arabinose, xylose, mannose and galactose, according to the peak area Figure 8 The molar ratio of rhamnose:arabinose:xylose:mannose:galactose is 3.240:48.842:2.633:5.729:32.734.

[0074] Example 5

[0075] Antioxidant activity determination of homogeneous polysaccharides of Elaeagnus angustifolia gum: 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity:

[0076] Take 300 μL of 0.25-5.0 mg / mL uniform polysaccharide solution of Elaeagnus angustifolia in a 1.5 mL centrifuge tube, add the same volume of 0.2 mmol / L DPPH methanol solution, mix well, react at room temperature in the dark for 30 min, transfer to a 96-well plate after the reaction, and record the absorbance at 517 nm with an ELISA reader using water as a blank control. Repeat three times for each point. The DPPH free radical scavenging activity is calculated as follows:

[0077]

[0078] Among them, A i represents the absorbance of 100 μL DPPH free radical + 100 μL polysaccharide solution, A0 represents the absorbance of 100 μL DPPH free radical + 100 μL methanol, A j Represents the absorbance of 100 μL polysaccharide solution + 100 μL methanol, and three parallels were set for each experimental point;

[0079] 2,2'-Azino-diammonium salt (ABTS) free radical scavenging activity:

[0080] Take 50 μL of the homogeneous polysaccharide solution of Elaeagnus angustifolia and mix it with 150 μL of ABTS·free radical solution at an absorbance of 0.78 at 734 nm. After reacting at room temperature in the dark for 10 minutes, water is used as a blank control and the absorbance is tested at 730 nm. Each experimental point is repeated 3 times. The ABTS·free radical scavenging activity is calculated according to the formula;

[0081]

[0082] Among them A i : Absorbance of 50 μL water and 150 μL ABTS·radical solution, A j : absorbance of 50 μL sample and 150 μL ABTS·free radical solution; A0: absorbance of 50 μL sample solution and 150 μL water, 3 parallels are set for each experimental point;

[0083] Hydroxyl Radical Scavenging Activity:

[0084] Take 70 μL of sample solution with a concentration of 0.2-2.0 mg / mL, mix with 60 μL salicylic acid-ethanol 6 mmol / L solution and 0.1% H2O2 5 μL solution, react at room temperature for 30 min, use VC as positive control and water as blank control, measure absorbance at 510 nm, repeat each experiment 3 times, the calculation formula of OH radical scavenging activity is as follows, set up 3 parallels for each experimental point;

[0085]

[0086] It was measured that the obtained uniform polysaccharide of Elaeagnus angustifolia gum exhibited dose-dependent antioxidant activity and could be used as an antioxidant active ingredient in the preparation of health foods or pharmaceutical preparations.

[0087] Example 6

[0088] Effect of homogeneous polysaccharide of Elaeagnus angustifolia on the immune activity of mouse monocytic leukemia macrophages (RAW 264.7):

[0089] Effect of homogeneous polysaccharide of Elaeagnus angustifolia on the survival rate of mouse monocytic leukemia macrophages (RAW 264.7) (thiazolidine blue colorimetric method (MTT method)):

[0090] Cell culture: RAW264.7 cells were cultured in DMEM high-glucose medium containing 10% FBS and 1% double antibody at 37°C in a 5% CO2 cell culture incubator. The medium was changed every 2-3 days and the cells were subcultured at a ratio of 1:3. The cell survival rate was detected by MTT method: When RAW264.7 cells grew to 70%-80% confluence, they were digested with 0.25% trypsin and the cell density was adjusted to 5×10 3 cell / mL, inoculated in a 96-well plate, and cultured for 18 hours; set up 200, 400, 800, and 1600 μg / ml of Elaeagnus angustifolia polysaccharide administration groups, and set up a blank group (containing only culture fluid) and a normal group (without polysaccharide), with 3 replicates in each group. After the cells are fully attached to the wall, the liquid in the wells is discarded, only culture fluid is added to the blank group, and 200, 400, 800, and 1600 μg / ml of Elaeagnus angustifolia polysaccharide are added to the administration group, and culture is continued for 24 hours. After the culture is completed, MTT solution (generally one-tenth of the volume of the cell culture fluid) is added to each well, and the culture plate is returned to the incubator for continued incubation for about 4 hours, so that MTT is reduced to insoluble blue-purple crystalline formazan by mitochondrial dehydrogenase in living cells;

[0091] Dissolve formazan: Carefully aspirate the culture medium in the wells to avoid aspirating formazan crystals, add 150 μl of dimethyl sulfoxide to each well, and shake the culture plate to fully dissolve the formazan crystals;

[0092] Colorimetric assay: Use a microplate reader to measure the absorbance value (OD value) of each well at a specific wavelength of 490 nm;

[0093] Result calculation: Cell survival rate calculation formula:

[0094]

[0095] The level of cytokine secretion by mouse mononuclear leukemia macrophages (RAW 264.7) induced by Elaeagnus angustifolia gum (determined by enzyme-linked immunosorbent assay):

[0096] Macrophages in the logarithmic growth phase were taken at a rate of 5×10 4 The cells were inoculated at a density of 100 μL / mL in a 96-well plate, 200 μL per well, and incubated in an incubator at 37°C and 5% CO2. After the cells were completely attached to the wall, the supernatant was gently discarded and divided into a blank control group. Each group had 3 parallel wells. After 24 hours of intervention, the supernatant was collected and centrifuged at 1000 r / min and 4°C for 10 minutes to remove impurities and cell debris for detection;

[0097] Add the standard working solution to the first two columns of wells in turn, add each concentration of working solution to two wells in parallel, 100μL to each well, add the sample to be tested to the other wells, 100μL to each well, cover the ELISA plate, incubate in an incubator at 37°C for 90min, discard the liquid, spin dry, and do not wash; add 100μL of biotinylated antibody working solution to each well, mix the ELISA plate, cover it with a film, incubate in an incubator at 37°C for 1h, spin off the liquid in the well, add 350μL of washing solution to each well, soak for 1-2min, and remove the liquid in the ELISA plate. The plate was patted dry on thick absorbent paper, and the washing step was repeated 3 times. 100 μL of enzyme conjugate working solution was added to each well, and the plate was covered with a film. The plate was incubated in an incubator at 37°C for 30 min. The liquid in the well was discarded, the plate was dried, and the plate was washed 5 times. 90 μL of substrate solution was added to each well, and the plate was covered with a film. The plate was incubated in an incubator at 37°C in the dark for about 15 min. When a clear gradient appeared in the standard well, the reaction was terminated immediately. 50 μL of stop solution was added to each well to terminate the reaction, and the optical density OD value of each well was immediately measured at a wavelength of 450 nm using an ELISA reader.

[0098] Determination of results: Calculate the average OD value of each set of duplicate wells, and subtract the OD value of the blank well from the average OD value of each standard as the correction value; draw a standard curve of the four-parameter logistic function with concentration as the horizontal axis and OD value as the vertical axis, and remove the value of the blank group when drawing the graph.

[0099] Finally, it is necessary to explain here that the above embodiments are only used to further explain the technical solution of the present invention in detail, and cannot be understood as limiting the scope of protection of the present invention. Some non-essential improvements and adjustments made according to the contents of the above embodiments belong to the scope of protection of the present invention.

Claims

1. A method for preparing a uniform polysaccharide of Elaeagnus angustifolia gum, characterized in that: The method proceeds in the following steps: a. Wash the collected Elaeagnus angustifolia gum, dry it naturally, and then mechanically crush it for 60-80 seconds, sieve it through a 60-100 mesh sieve to separate impurities to obtain Elaeagnus angustifolia gum powder; b. Dissolve the elata gum powder obtained in step a in distilled water to prepare an elata gum aqueous solution with a concentration of 1.8-2%, stir magnetically for 8-12 hours at a temperature of 25-35°C, centrifuge for 10 minutes, separate and remove impurities, and obtain an elata gum solution; c. Slowly add the Elaeagnus angustifolia gum solution obtained in step b to 95% ethanol at a volume ratio of 1:4 for alcohol precipitation, stirring at a uniform speed while adding, and standing in a refrigerator at a temperature of 4°C for 8-12 hours, then taking out, centrifuging and filtering, and washing the obtained precipitate with anhydrous ethanol by centrifugation for 3-5 times at a volume ratio of 1:1, and collecting the precipitate for later use; d. After re-dissolving the precipitate collected in step c, add deionized water, dialyze with water and a dialysis bag with a pore size of 3500Da for 24 hours, and freeze-dry to obtain crude polysaccharide of Elaeagnus angustifolia. e. Load the crude polysaccharide of Elaeagnus anhydrica onto a DEAE agarose anion exchange gel column, elute with water, collect the water-eluted portion, track and detect the collected liquid using anthrone-sulfuric acid method, draw an elution curve, dialyze with water and a dialysis bag with a pore size of 3500Da for 24 hours, and freeze-dry to obtain the polysaccharide in the neutral portion eluted with Elaeagnus anhydrica; f. Dissolve the neutral part polysaccharide of Elaeagnus angustifolia in water, centrifuge, take the supernatant, load it on a cross-linked agarose gel chromatography column, elute with water, and follow up the collected solution with anthrone-sulfuric acid method to draw an elution curve, collect the peak top, dialyze and dry to obtain an average molecular weight of 158.8×10 4 Dalton, high purity, sugar content 53.54%, protein content less than 1%; monosaccharide composition: rhamnose, arabinose, xylose, mannose and galactose, molar ratio of 3.240:48.842:2.633:5.729:32.734, an elata gum uniform polysaccharide.

2. Use of the homogeneous polysaccharide of Elaeagnus angustifolia obtained by the method as claimed in claim 1 in the preparation of foods containing antioxidant active ingredients.

3. Use of the uniform polysaccharide of Elaeagnus angustifolia obtained by the method as claimed in claim 1 in the preparation of health products containing antioxidant active ingredients.

4. Use of the homogeneous polysaccharide of Elaeagnus angustifolia obtained by the method as claimed in claim 1 in the preparation of foods containing immunomodulatory ingredients.

5. Use of the uniform polysaccharide of Elaeagnus angustifolia obtained by the method as claimed in claim 1 in the preparation of health products containing immunomodulatory ingredients.

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