Application of red date polysaccharide in enhancing immunoregulation ability

Abstract

The invention belongs to the technical field of red date polysaccharide application, and discloses application of red date polysaccharide in preparation of a product for enhancing immune regulation ability, and the product is chewable tablets or capsules. The chewable tablet or capsule takes red date polysaccharide as a main active ingredient, and active ingredients and / or preparation auxiliary materials which do not influence the red date polysaccharide are added; the preparation process conditions of the red date polysaccharide are determined through single factor and response surface experiments, and in-vitro experiments verify that the red date polysaccharide can enhance the proliferation ability and phagocytic ability of macrophages, so that the immunoregulation ability can be enhanced, and the components are safe.

Description

Technical Field

[0001] The present invention relates to the technical field of application of red date polysaccharides, and in particular to the application of red date polysaccharides in enhancing immune regulation ability. Background Art

[0002] In the human health maintenance system, the immune system acts as a loyal guardian, shouldering the important task of identifying and eliminating pathogens and maintaining a stable internal environment. Market demand for immunomodulatory health products continues to grow. While chemically synthesized drugs such as coenzyme Q10 can boost immune indicators in the short term, they can also cause gastrointestinal dysfunction, heart palpitations, and allergic reactions. Hormonal preparations (such as those containing estrogen) disrupt endocrine balance, leading to risks such as menstrual disorders and breast hyperplasia. Some natural ingredient health supplements (such as excessive vitamins A and D) can easily cause nutritional imbalances and even liver and kidney toxicity. These issues highlight the urgency of developing safe and effective immune-enhancing products.

[0003] Jujubes, a traditional Chinese food and medicine, are known as "natural vitamin pills." They are rich in carbohydrates, vitamins, minerals, and bioactive ingredients. Jujube polysaccharides, as the primary active ingredient, have garnered significant attention in recent years. Jujube polysaccharides have a complex structure, primarily composed of monosaccharides such as arabinose, galactose, and glucose linked by glycosidic bonds. Differences in molecular weight, monosaccharide composition, and glycosidic bond type determine their bioactivity. Studies have confirmed that jujube polysaccharides can regulate intestinal flora, protect the liver, lower lipids, and replenish blood and qi. However, studies examining their ability to enhance immune regulation have been limited. Summary of the Invention

[0004] The present invention aims to provide an application of red date polysaccharide in enhancing immune regulation ability. Through single factor and response surface experiments, the preparation process conditions of red date polysaccharide are determined. Through in vitro experiments, it is verified that red date polysaccharide can enhance the proliferation and phagocytic ability of macrophages, enhance immune regulation ability, and the ingredients are safe.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] Application of red date polysaccharides in the preparation of products that enhance immune regulation ability.

[0007] Furthermore, the product uses red date polysaccharide as the main active ingredient, and adds active ingredients and / or preparation excipients that do not affect each other.

[0008] Furthermore, the red date polysaccharide is prepared by the following method:

[0009] S1. Extraction of red date polysaccharides

[0010] Weigh red date powder, add distilled water according to the solid-liquid ratio, ultrasonicate, extract in a water bath, filter the extract, concentrate in vacuo, add 95% ethanol, precipitate with alcohol, and centrifuge to obtain red date polysaccharide precipitate, which is freeze-dried to obtain crude red date polysaccharide;

[0011] S2. Isolation and purification of crude polysaccharides from red dates

[0012] The freeze-dried crude red date polysaccharide was dispersed in distilled water and centrifuged at high speed. The supernatant was loaded onto a cellulose anion exchange column pre-equilibrated with distilled water, and then gradually gradient eluted with NaCl solutions of different concentrations to collect the purified polysaccharide components, which were freeze-dried for later use. The freeze-dried powder was then redissolved in distilled water and loaded onto a gel column. The column was eluted with distilled water to collect the purified polysaccharide components and freeze-dried.

[0013] Furthermore, in S1, the ratio of red date powder to distilled water was 1:30, the ultrasonic treatment time was 20 min, the water bath temperature was 70°C, the extraction time was 3 h, the alcohol precipitation time was 12 h, the centrifugal speed was 5000 rpm, and the centrifugal time was 10 min.

[0014] Furthermore, the ratio of red date powder to distilled water is 50:1 (weight: volume); the cellulose anion exchange column is a DEAE-52 cellulose anion exchange column, the exchange column has a size of 2.6×50 cm, and the flow rate is 1 ml / min; the concentrations of the NaCl solution are 0, 0.1 M, and 0.3 M, respectively.

[0015] Furthermore, the ratio of freeze-dried powder to distilled water was 5:1 (weight:volume); the gel column had a size of 1.6×60 cm, and the flow rate was 0.5 ml / min.

[0016] The beneficial effects of the technical solution are:

[0017] The present invention uses polysaccharides extracted from red dates to regulate the body's immune function, has no side effects, and is easily absorbed. The preparation process conditions for the polysaccharides were determined through single-factor and response surface experiments. In vitro experiments revealed that polysaccharides can enhance the proliferation and phagocytic capacity of macrophages. In vivo experiments also revealed that polysaccharides can improve abnormal immune organ indices induced by cyclophosphamide and promote the secretion of IgA and IgM in serum, thereby exerting immunomodulatory activity. Specifically, the experiment showed that jujube polysaccharide can significantly increase the cell viability of macrophages RAW264.7 at a concentration of 6.25-200 μg / mL (p<0.05), indicating that jujube polysaccharide has a value-added effect on macrophages RAW264.7 and can enhance cellular immune activity. In addition, the cell viability of macrophages RAW264.7 is greater at concentrations of 12.5, 25, and 50 μg / mL. Therefore, the optimal concentrations of jujube polysaccharide for RAW264.7 cells are determined to be 12.5, 25, and 50 μg / mL. When different concentrations (12.5, 25, and 50 μg / mL) of red date polysaccharide (HP2-1) were applied to macrophages RAW264.7, red date polysaccharide could significantly improve the phagocytic activity of RAW264.7 cells in a dose-dependent manner (p<0.05), indicating that red date polysaccharide can improve the immune capacity of RAW264.7 cells by enhancing the phagocytic ability of RAW264.7 cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is the DEAE-52 elution curve of red date polysaccharide in Example 1 of the present invention;

[0019] Figure 2 This is a bar graph showing the effect of different concentrations of HP2-1 on RAW264.7 cell viability in Example 2 of the present invention;

[0020] Figure 3 This is a bar graph showing the effects of different concentrations of HP2-1 on the phagocytic activity of RAW2664.7 cells in Example 2 of the present invention. DETAILED DESCRIPTION

[0021] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments:

[0022] Example 1

[0023] 1. Preparation of Red Date Polysaccharides

[0024] (1) Extraction of red date polysaccharides

[0025] Accurately weigh an appropriate amount of red date powder, add distilled water at a solid-liquid ratio of 1:30, ultrasonically treat for 20 minutes, extract in a 70°C water bath for 3 hours, filter the extract, vacuum concentrate, add four times the volume of 95% ethanol, precipitate for 12 hours, centrifuge at 5000 rpm for 10 minutes to obtain red date polysaccharide precipitate, and freeze-dry the precipitate to obtain crude red date polysaccharide.

[0026] (2) Isolation and purification of crude polysaccharides from red dates

[0027] The freeze-dried crude red jujube polysaccharide extract (300 mg) was dispersed in 6 ml of distilled water and centrifuged using a TDL-40B high-speed centrifuge; the supernatant was loaded onto a DEAE-52 cellulose anion exchange column (2.6×50 cm) pre-equilibrated with distilled water; then, a stepwise gradient elution was performed using NaCl solution from low concentrations (0, 0.1 M, 0.3 M) at a flow rate of 1 ml / min; the main purified polysaccharide components were collected based on the absorbance at 490 nm and freeze-dried for later use.

[0028] The lyophilized powder (25 mg) was redissolved in 5 ml of distilled water and loaded onto a Sephadex G-100 gel column (1.6 × 60 cm) and eluted with distilled water at a flow rate of 0.5 ml / min. The main purified polysaccharide fractions were collected based on the absorbance at 490 nm and lyophilized for further analysis.

[0029] 2. Experimental Results

[0030] (1) Experimental results on the optimization of the preparation process of red jujube polysaccharides

[0031] Design expert 11.0 predicted the optimal extraction conditions for red date polysaccharide yield to be: solid-liquid ratio of 1:28.5 g / mL, ultrasonic time of 18.8 min, water bath temperature of 69.0°C, and water bath time of 2.83 h. Considering the actual experimental conditions, the extraction conditions were adjusted to a solid-liquid ratio of 1:30 g / mL, ultrasonic time of 20 min, water bath temperature of 70°C, and water bath time of 3 h.

[0032] (2) Experimental results of separation and purification of crude jujube polysaccharides

[0033] Polysaccharides are rich in alcoholic hydroxyl groups and are usually weakly acidic. Therefore, a weak anion exchanger, cellulose DEAE-52, was selected for purification. The crude polysaccharides of red dates were eluted in sequence with 0, 0.1, and 0.3 mol / L NaCl solutions through a DEAE-52 anion exchange column. The elution curve is shown in Figure 2. Figure 1As shown in the figure, when the concentration of the elution solution NaCl solution was 0 and 0.1 mol / L, the elution effect was better. These two red date polysaccharide fractions were collected and named red date polysaccharide fraction 1 (HP1) and red date polysaccharide fraction 2 (HP2) according to the elution order. The red date polysaccharide fraction (HP2) was further purified using dextran G-100 to obtain red date polysaccharide fraction HP2-1.

[0034] Example 2

[0035] Study on the immune activity of red date polysaccharides

[0036] 1. Experimental steps

[0037] (1) Effect of red date polysaccharides on the proliferation of macrophage RAW 264.7 cells

[0038] The concentration of the cell suspension in the logarithmic growth phase was adjusted to 1×105cell / mL using complete medium, inoculated into a sterile 96-well plate (100μL), and cultured at 37℃, 5% CO2 for 24h. The supernatant was discarded, and 100μL of different concentrations of HP2 (12.5μg / mL, 25μg / mL, 50μg / mL, prepared with DEME medium) were added as experimental groups. DEME medium and 1μg / mL lipopolysaccharide (LPS) were used as blank control and positive control, respectively. The cells were incubated for 24h, the supernatant was discarded, 100μL MTT solution (0.5mg / mL) was added to each well, and the cells were cultured for another 4h at 37℃, 5% CO2. The supernatant was discarded, 150μL DMSO was added to each well, and the cells were shaken in the room temperature for 10min. The absorbance of each well was measured at 570nm using a microplate reader.

[0039] (2) Experiment on the phagocytic activity of red date polysaccharides on macrophage RAW 264.7 cells

[0040] The concentration of the logarithmic growth phase cell suspension was adjusted to 1×105 cells / mL using complete medium and inoculated into a sterile 96-well plate (100 μL). The cells were cultured at 37°C and 5% CO2 for 24 hours. The supernatant was discarded, and 100 μL of different concentrations of red jujube polysaccharides (prepared with DEME medium) were added to each well as experimental groups. DEME medium and 1 μg / mL lipopolysaccharide (LPS) were used as blank and positive controls, respectively. The cells were incubated for 24 hours, the supernatant was discarded, and 100 μL of 0.1% neutral red saline solution was added to each well. The cells were cultured for another 4 hours at 37°C and 5% CO2. The supernatant was discarded, and the cells were washed several times with PBS until colorless. 100 μL of cell lysis buffer was added to each well and the cells were cultured for another 1 hour to completely lyse the cells. The absorbance of each well was measured at 540 nm using a microplate reader.

[0041] 2. Experimental results

[0042] (1) Effect of red date polysaccharides on the viability of macrophage RAW264.7 cells

[0043] Different concentrations (6.25, 12.5, 25, 50, 100, 200 μg / mL) of jujube polysaccharide were used to act on macrophage RAW264.7 cells for 24 hours. The cell viability was detected by MTT method to investigate the effect of jujube polysaccharide on the viability of macrophage RAW264.7 cells. The results are as follows Figure 2 As shown in the figure, compared with the blank control group (con) RAW264.7 cells, red date polysaccharides at a concentration of 6.25-200 μg / mL can significantly increase the cell viability of macrophage RAW264.7 (p<0.05), indicating that red date polysaccharides have a value-added effect on macrophage RAW264.7 and can enhance cellular immune activity. In addition, red date polysaccharides have a greater cell viability of macrophage RAW264.7 at concentrations of 12.5, 25, and 50 μg / mL. Therefore, the concentrations of red date polysaccharides acting on RAW264.7 cells were determined to be 12.5, 25, and 50 μg / mL.

[0044] (2) Effect of red date polysaccharide (HP2-1) on the phagocytic activity of macrophages RAW264.7

[0045] Phagocytic activity can measure the ability of macrophages to activate immune function. Different concentrations (12.5, 25, 50 μg / mL) of red jujube polysaccharide (HP2-1) were applied to macrophages RAW264.7 for 24 hours. The phagocytic activity of RAW264.7 cells was determined by neutral red method. The effect of red jujube polysaccharide on the phagocytic activity of RAW264.7 cells was investigated. The results are as follows: Figure 3 As shown in the figure, compared with the normal control group (NC) RAW264.7 cells, red date polysaccharides can significantly improve the phagocytic activity of RAW264.7 cells in a dose-dependent manner (p<0.05). This shows that red date polysaccharides can improve the immune capacity of RAW264.7 cells by enhancing their phagocytic ability.

[0046] According to the above experimental results, the red date polysaccharide prepared in Example 1 can be used as the main active ingredient to prepare health products, such as capsules or chewable tablets, to enhance human immunity.

[0047] For example, when formulated in capsule form, the active ingredient of red jujube polysaccharide is added as needed, including fillers (such as starch, microcrystalline cellulose, etc.), disintegrants (such as sodium carboxymethyl starch), and lubricants (such as magnesium stearate). Red jujube polysaccharide generally accounts for 30%-80% of the capsule content; fillers, such as starch, generally account for 10%-50% of the capsule content; disintegrants, such as sodium carboxymethyl starch, generally account for 2%-10% of the capsule content; and lubricants, such as magnesium stearate, generally account for 0.5%-3% of the capsule content.

[0048] When prepared in the form of chewable tablets, the active ingredients of red date polysaccharide include fillers (such as sorbitol, mannitol, etc.), binders (such as hydroxypropyl methylcellulose), sweeteners (such as aspartame), lubricants (calcium stearate), etc. as needed. Red date polysaccharide accounts for 25%-35% of the total weight of the chewable tablet, fillers account for 30%-40% of the total weight, binders account for 2%-5% of the total weight, sweeteners generally account for 0.05%-0.2% of the total weight, and lubricants are generally used in an amount of 0.5%-1% of the total weight.

[0049] In summary, the present invention uses polysaccharides extracted from red dates to regulate the body's immune function, has no side effects, and is easily absorbed. The preparation process conditions for red date polysaccharides were determined through single-factor and response surface experiments. In vitro experiments revealed that red date polysaccharides can enhance the proliferation and phagocytic capacity of macrophages. In vivo experiments revealed that red date polysaccharides can improve abnormal immune organ indices caused by cyclophosphamide and promote the secretion of IgA and IgM in serum, thereby exerting immunomodulatory activity. Specifically, the experiment showed that jujube polysaccharide can significantly increase the cell viability of macrophages RAW264.7 at a concentration of 6.25-200 μg / mL (p<0.05), indicating that jujube polysaccharide has a value-added effect on macrophages RAW264.7 and can enhance cellular immune activity. In addition, the cell viability of macrophages RAW264.7 is greater at concentrations of 12.5, 25, and 50 μg / mL. Therefore, the optimal concentrations of jujube polysaccharide for RAW264.7 cells are determined to be 12.5, 25, and 50 μg / mL. When different concentrations (12.5, 25, and 50 μg / mL) of red date polysaccharide (HP2-1) were applied to macrophages RAW264.7, red date polysaccharide could significantly improve the phagocytic activity of RAW264.7 cells in a dose-dependent manner (p<0.05), indicating that red date polysaccharide can improve the immune capacity of RAW264.7 cells by enhancing the phagocytic ability of RAW264.7 cells.

[0050] The above is only an embodiment of the present invention, and common knowledge such as the specific technical solutions or characteristics in the solution is not described in detail here. It should be pointed out that for those skilled in the art, without departing from the technical solution of the present invention, several variations and improvements can be made, which should also be regarded as the scope of protection of the present invention, and these will not affect the effect of the implementation of the present invention and the practicality of the patent. The scope of protection required by this application shall be based on the content of its claims, and the specific implementation methods and other records in the description can be used to interpret the content of the claims.

Claims

1. Application of red date polysaccharides in the preparation of products that enhance immune regulation ability.

2. The use according to claim 1, characterized in that: The product uses red date polysaccharide as the main active ingredient, and is added with active ingredients and / or preparation excipients that do not affect each other.

3. The use according to claim 1, characterized in that: The red date polysaccharide is prepared by the following method: S1. Extraction of red date polysaccharides Weigh red date powder, add distilled water according to the solid-liquid ratio, ultrasonicate, extract in a water bath, filter the extract, concentrate in vacuo, add 95% ethanol, precipitate with alcohol, and centrifuge to obtain red date polysaccharide precipitate, which is freeze-dried to obtain crude red date polysaccharide; S2. Isolation and purification of crude polysaccharides from red dates The freeze-dried crude red date polysaccharide was dispersed in distilled water and centrifuged at high speed. The supernatant was loaded onto a cellulose anion exchange column pre-equilibrated with distilled water, and then gradually gradient eluted with NaCl solutions of different concentrations to collect the purified polysaccharide components, which were freeze-dried for later use. The freeze-dried powder was then redissolved in distilled water and loaded onto a gel column. The column was eluted with distilled water to collect the purified polysaccharide components and freeze-dried.

4. The use according to claim 3, characterized in that: In S1, the ratio of red date powder to distilled water was 1:30, the ultrasonic treatment time was 20 min, the water bath temperature was 70 °C, the extraction time was 3 h, the alcohol precipitation time was 12 h, the centrifugal speed was 5000 rpm, and the centrifugal time was 10 min.

5. The use according to claim 3, characterized in that: The ratio of red date powder to distilled water was 50:1 (weight: volume); the cellulose anion exchange column was a DEAE-52 cellulose anion exchange column with a size of 2.6×50 cm and a flow rate of 1 ml / min; the concentrations of the NaCl solutions were 0, 0.1 M, and 0.3 M, respectively.

6. The use according to claim 3, characterized in that: The ratio of lyophilized powder to distilled water was 5:1 (weight:volume); the gel column had a size of 1.6×60 cm, and the flow rate was 0.5 ml / min.

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