Application of selenium polysaccharide in preparation of immunoactivator

Abstract

The invention belongs to the field of selenium polysaccharide application, and particularly relates to application of selenium polysaccharide in preparation of an immunocompetence agent.Cyclocarya paliurus is treated through exogenous selenium, and the obtained selenium polysaccharide has good immunocompetence, can better promote proliferation and phagocytic functions of RAW264.7 macrophages, has remarkable advantages in the aspect of increasing the NO release amount, and has good application prospects. The result shows that the polysaccharide strengthened by the exogenous selenium has better immunocompetence. Exogenous selenium strengthening obviously improves the characteristics of selenium polysaccharide in the cyclocarya paliurus leaves, has better potential in the aspects of oxidation resistance and immunity improvement, and provides data support for further research and utilization.

Description

Technical Field

[0001] The invention belongs to the field of application of selenium polysaccharides, and specifically relates to the application of selenium polysaccharides in the preparation of immunologically active agents. Background Art

[0002] Cyclocaryapaliurus is a deciduous tree plant of the genus Cyclocaryapaliurus in the Juglandaceae family. It is also a unique rare plant with both medicinal and edible properties. Because it is rich in flavonoids, triterpenes, polysaccharides and other ingredients, it has antioxidant, blood sugar-lowering, anti-aging, beauty and weight loss effects. It is widely used in the production of functional foods and is widely recognized by consumers.

[0003] Selenium polysaccharides are important organic forms of selenium in plants, and selenium polysaccharides have better biological activity than polysaccharides. At present, the research on selenium polysaccharides in selenium-enriched Cyclocarya paliurus mainly focuses on its biological activity functions, such as antioxidant activity. In order to further expand the application scope of selenium polysaccharides in selenium-enriched Cyclocarya paliurus, further research is needed on other biological activities of selenium polysaccharides. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides the use of selenium polysaccharides in the preparation of immunoactive agents. The selenium polysaccharides in selenium-enriched Cyclocarya paliurus can effectively increase the proliferation, phagocytic activity and NO release of macrophages, thereby improving immune activity.

[0005] To achieve the above technical objectives, the technical solution of the present invention is as follows.

[0006] The first aspect of the present invention provides the use of selenium polysaccharide in the preparation of an immunologically active agent, wherein the selenium polysaccharide is obtained by spraying exogenous selenium on the leaves of Cyclocarya paliurus to selenize the polysaccharide in the Cyclocarya paliurus to obtain a selenium-enriched Cyclocarya paliurus with a selenium content of not less than 34 mg / kg, and then extracting the selenium polysaccharide from the leaves of the selenium-enriched Cyclocarya paliurus;

[0007] The immunologically active agent refers to a preparation having an immunomodulatory effect.

[0008] The invention adopts exogenous selenium spraying to selenize polysaccharides in Cyclocarya paliurus, obtains selenium-enriched Cyclocarya paliurus with a selenium content of not less than 34 mg / kg, extracts selenium polysaccharides, and conducts in vitro experiments on the extracted selenium polysaccharides, finding that the selenium polysaccharides have immunomodulatory activity.

[0009] In another preferred embodiment, the molecular weight of the selenopolysaccharide is 76.662 kDa.

[0010] In another preferred embodiment, the selenium polysaccharide is composed of the following monosaccharides in terms of mass percentage:

[0011] Fucose 0.75%, rhamnose 11.61%, arabinose 25.06%, galactose 32.15%, glucose 6.37%, xylose 2.48%, galacturonic acid 19.81%, glucuronic acid 1.77%, total 100%.

[0012] In another preferred embodiment, the immunomodulatory agent refers to an agent that improves the proliferation, phagocytic activity and NO release of macrophages. It can better promote the proliferation and phagocytic function of RAW264.7 macrophages, and also has significant advantages in increasing the release of NO.

[0013] The second aspect of the present invention provides an immunologically active agent, wherein the selenium polysaccharide is the only active ingredient in the immunologically active agent.

[0014] In another preferred embodiment, the immunologically active agent is in the form of any one of tablets, powders, granules, and aqueous solutions.

[0015] In another preferred embodiment, the immunologically active agent further comprises a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients broadly refer to any component other than the active therapeutic ingredient. Excipients can be inert substances, inactive substances and / or non-pharmaceutically active substances. Excipients can be used for various purposes, such as as carriers, vehicles, diluents, tablet adjuvants and / or to improve the administration and / or absorption of active substances.

[0016] In another preferred embodiment, the exogenous selenium is a sodium selenite solution, and the concentration of the sodium selenite solution is 120 mg / L to 160 mg / L.

[0017] In another preferred embodiment, the specific process of obtaining the selenium polysaccharide is as follows:

[0018] The leaves of the selenium-rich Cyclocarya paliurus are crushed, extracted with water and precipitated with alcohol to obtain an extract;

[0019] The extract is passed through a macroporous resin and concentrated to obtain a concentrated solution; the concentrated solution is mixed with a sevage reagent, centrifuged, and the supernatant is taken, and the supernatant is precipitated with alcohol and freeze-dried to obtain a crude polysaccharide; and the crude polysaccharide is purified to obtain the selenium polysaccharide.

[0020] The specific process of the purification is as follows:

[0021] The anion exchanger and the crude polysaccharide are activated, the activated polysaccharide is loaded into a column, eluted with water, the eluate is collected, concentrated, dialyzed, and dried to obtain the selenium polysaccharide. The anion exchanger is DEAE-52 cellulose; the elution is carried out by gradient elution with a NaCl solution of 0 mol / L to 0.4 mol / L.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] In the present invention, the polysaccharide in Cyclocarya paliurus is selenized to make the content not less than 34 mg / kg, so that the obtained selenium polysaccharide has good immune activity and can be used to prepare an immune active agent.

[0024] The molecular weight of the selenium polysaccharide in the present invention is 76.662 kDa, and it is composed of fucose, rhamnose, arabinose, galactose, glucose, xylose, galacturonic acid and glucuronic acid, so that it can better promote the proliferation and phagocytic function of RAW264.7 macrophages, and also has significant advantages in increasing the release of NO. The results show that the Cyclocarya paliurus polysaccharide strengthened with exogenous selenium has better immune activity. Exogenous selenium strengthening significantly improves the characteristics of selenium polysaccharides in Cyclocarya paliurus leaves, and has better potential in anti-oxidation and improving immunity, providing data support for further research and utilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the infrared characterization image of polysaccharides and selenium polysaccharides in selenium-rich Cyclocarya paliurus. In the image, CPP-1 is a polysaccharide and Se-CPP-1 is a selenium polysaccharide.

[0026] Figure 2 are scanning electron microscope images of different polysaccharides; among them, A is the scanning electron microscope image of polysaccharide, and B is the scanning electron microscope image of selenium polysaccharide.

[0027] Figure 3 This is the molecular weight distribution diagram of Se-CPP-1.

[0028] Figure 4 This is the result graph of the DPPH free radical scavenging ability of different polysaccharides.

[0029] Figure 5 This is the result graph of ABTS free radical scavenging ability of different polysaccharides.

[0030] Figure 6 The graph shows the cell proliferation results of different polysaccharides.

[0031] Figure 7 The diagram shows the results of cell phagocytosis of different polysaccharides.

[0032] Figure 8 The graph shows the NO experimental results of different polysaccharides. DETAILED DESCRIPTION

[0033] The present invention is described in detail below in conjunction with specific examples, but it should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0034] In recent years, there have been a lot of reports on the polysaccharides isolated from Cyclocarya paliurus and their biological activities and structures, but there are few studies on selenium-rich Cyclocarya paliurus polysaccharides. The present invention starts with selenium polysaccharides in selenium-rich Cyclocarya paliurus and finds that selenium polysaccharides have better scavenging effects on DPPH and ABTS free radicals. At the same time, its immunomodulatory activity is verified by cell in vitro experiments. The results show that selenium polysaccharides can better promote the proliferation and phagocytic function of RAW264.7 macrophages, and also have significant advantages in increasing the release of NO.

[0035] 1. Preparation of experimental materials

[0036] The Cyclocarya paliurus seedlings are two-year-old Cyclocarya paliurus seedlings grown in Wufeng Mountain, Yichang City, Hubei Province.

[0037] A sodium selenite solution with a concentration of 160 mg / L was prepared, and selenium was applied to the surface of Cyclocarya paliurus leaves by foliar application. The spraying was continued for 15 days, and the selenium content in the leaves was measured to obtain selenium-enriched Cyclocarya paliurus leaves with a selenium content of 36.55 mg / kg.

[0038] 2. Polysaccharide extraction

[0039] The leaves with a selenium content of 34.55 mg / kg were placed in an electric air drying oven and dried at 65°C to constant weight. The leaves were crushed into powder using a grinder, passed through an 80-mesh sieve, and placed in a ziplock bag for later use.

[0040] 1) Cyclocarya paliurus polysaccharide extract

[0041] Selenium polysaccharides were extracted from the leaves of Cyclocarya paliurus by water extraction and alcohol precipitation. The specific process is as follows:

[0042] Weigh 40 g of Cyclocarya paliurus powder and place it in a round-bottom flask, add distilled deionized water at a solid-liquid ratio of 1:25 g / mL, put it in a magnetic stirring water bath, extract at 90°C for 2 h, let it stand and slowly pour out the supernatant, centrifuge the supernatant at 10,000 r / min for 15 min, discard the precipitate, pour out the supernatant, and obtain a clear brown polysaccharide extract.

[0043] 2) Removal of impurities from Cyclocarya paliurus polysaccharide

[0044] Pigment removal: Soak the AB-8 macroporous resin in 80% ethanol for 3 hours, pour out the ethanol, add excess distilled water to rinse repeatedly until the washing liquid has no ethanol taste at all. Prepare a small amount of cotton in advance and place it at the bottom of a 40mm*40cm glass chromatography column. Pour the soaked AB-8 macroporous resin into the glass chromatography column at a uniform speed, open the valve at the outlet of the chromatography column, release the liquid in the column to allow the AB-8 macroporous resin to naturally settle to 10cm from the upper port of the chromatography column, and no macroporous resin particles float in the chromatography column. At this time, slowly pour the polysaccharide extract into the column and pass it through the column. Seeing the light yellow polysaccharide solution flowing out, it means that the polysaccharide has been successfully depigmented. Collect the decolorized polysaccharide solution, use a rotary evaporator to concentrate the solution, turn on the condensation water and vacuum pump, check the air tightness of the device, set the temperature to 55°C, the speed to 60r / min, and concentrate the solution to 200mL.

[0045] Protein removal: prepare sevage reagent (chloroform: n-butanol is 4:1) with 1 / 4 volume of polysaccharide extract to remove protein. Pour the polysaccharide extract and sevage reagent into a separatory funnel, shake vigorously for 10 minutes and let it stand for 2 hours. After the solution shows obvious stratification, centrifuge at 5000r / min for 10 minutes, collect the supernatant, and remove protein by sevage method 3 times.

[0046] Alcohol precipitation and freeze-drying: After removing the protein from the polysaccharide solution, add 80% anhydrous ethanol in a volume 4 times that of the polysaccharide extract into a beaker. A large amount of flocs can be seen in the solution. After overnight, centrifuge at 10,000 r / min for 15 minutes, pour out the supernatant, wash the precipitate with anhydrous ethanol 4 times, and then take the precipitate for freeze-drying to obtain selenium-rich polysaccharides from Cyclocarya paliurus.

[0047] 3) Purification of Cyclocarya paliurus polysaccharides

[0048] Activation of DEAE-52 cellulose: The anion exchanger DEAE-52 cellulose was used in the polysaccharide purification experiment.

[0049] Weigh 100g of DEAE-52 cellulose powder and dissolve it in ultrapure water. Stir it evenly with a glass rod and soak it for 3 hours. Remove impurities on the surface of the solution and drain the water. Put it in 0.5mol / L HCL solution, stir it evenly and soak it for 2 hours. Wash the solution with ultrapure water until it is neutral and drain the water. Put it in 0.5mol / LNaOH solution, stir it evenly with a glass rod and soak it for 2 hours. Wash the solution with ultrapure water until it is neutral and drain the water. Put the activated cellulose in ultrapure water for use. At this point, the activation of DEAE-52 cellulose is completed.

[0050] Column loading: Fix a 40mm*40cm glass chromatography column on an iron stand, prepare a little cotton and plug it into the bottom of the column, slowly pour in the activated cellulose, open the liquid outlet valve, repeatedly compact the column bed with ultrapure water, and let it settle naturally for 12 hours.

[0051] Sampling and elution: Weigh 0.5g of crude polysaccharide and fully dissolve it in 10mL ultrapure water, centrifuge at 5000r / min for 10min, and take the supernatant and slowly pour it along the inner wall of the chromatography column. Connect the water inlet pipe above the chromatography column to a constant flow pump, adjust the flow rate to 10mL / min, and start gradient elution with 0mol / L, 0.05mol / L, 0.1mol / L, 0.2mol / L, 0.3mol / L, and 0.4mol / L NaCl solutions in sequence. Collect the eluate in a 10mL centrifuge tube, and collect 40 tubes of eluate for each concentration. After the elution is completed, the polysaccharide content in the solution is determined at an absorbance of 490nm using the phenol-sulfuric acid method, draw an elution curve, combine and concentrate the eluate under the main elution peak to 20mL, put it into a 3500Da dialysis bag to remove small salt molecules, dialyze for 48h, replace ultrapure water 8 times during the period, and then freeze-dry to obtain purified selenium polysaccharides.

[0052] 3. Characterization of Selenium Polysaccharides

[0053] 1) Structural characterization

[0054] The polysaccharides and selenium polysaccharides obtained above were characterized by infrared spectroscopy. Figure 1 As shown. Figure 1 It can be seen that the -OH stretching vibration peak of Se-CPP-1 appears at 3350.1 cm -1 , 2929.34cm -1 There are CH stretching vibration peaks of methyl and methylene at 1619.93 cm -1 In addition, 1415.74cm -1 The angular vibration characteristic absorption peak of CH is at 834.12cm. It can be seen that Se-CPP-1 after selenization modification has the infrared spectrum characteristics of polysaccharides. -1 The new absorption peak at 1400 nm can be attributed to the O-Se-O bond. The above results confirm that the selenium polysaccharide has been successfully selenized.

[0055] Scanning electron microscopy analysis of CPP-1 and Se-CPP-1 showed Figure 2As shown, the results indicate that the introduction of exogenous selenium significantly affects the surface micromorphology of CPP-1. At a magnification of 5.0K, the surface of CPP-1 exhibits roughness and unevenness, presenting an irregular honeycomb structure. In contrast, Se-CPP-1 exhibits an irregular lamellar morphology with a smooth surface, which is in sharp contrast to CPP-1. The particle size of Se-CPP-1 is significantly reduced, and it aggregates in clusters, is loose and porous, and has an increased specific surface area. It is inferred that Se in Se-CPP-1 may be the main site for inducing hydrophobic binding. This binding causes the sugar chain to transition from an extended state to a folded state, which in turn causes the particle size of Se-CPP-1 to decrease and a significant change in morphology.

[0056] 2) Molecular weight characterization

[0057] The molecular weight distribution of Se-CPP-1 was measured by high performance gel chromatography (HP-GPC). Figure 3 As shown, from Figure 3 It can be seen that a single absorption peak appeared with a retention time of 26.082 min, which indicates that the Se-CPP-1 component showed a relatively high degree of homogeneity after separation and purification, and its molecular weight was determined to be 76.662 kDa.

[0058] 3) Monosaccharide composition

[0059] In order to further explore the structural characteristics of CPP-1 and Se-CPP-1, the monosaccharide composition was analyzed in detail by ion chromatography (HPIC) technology. Given that sugar molecules may exist partially or completely in anionic form under high pH eluent conditions, the analysis of sugars was achieved by detecting the current generated by the oxidation reaction of hydroxyl groups in the sugar molecule structure on the gold electrode surface. As shown in Table 1, after comparing the retention time with that of standard monosaccharide derivatives, it was found that both CPP-1 and Se-CPP-1 contained a variety of monosaccharide components, such as galactose (Gal), arabinose (Ara), rhamnose (Rha), glucose (Glc), xylose (Xyl), fucose (Fuc), galacturonic acid (Gal-UA), and glucuronic acid (Glc-UA). Among them, glucose and arabinose are the main components of CPP-1 and Se-CPP-1. The relative content of galacturonic acid in CPP-1 and Se-CPP-1 is 6.10% and 19.81%, respectively, showing its characteristics as an acidic polysaccharide.

[0060] Table 1 Monosaccharide composition and relative content of CPP-1 and Se-CPP-1

[0061]

[0062] Note: Man means mannose.

[0063] 4. In vitro activity and immune activity of selenium polysaccharides

[0064] 1) DPPH free radical scavenging ability determination

[0065] Weigh 10 mg of selenium polysaccharide and 10 mg of Vc respectively, dissolve in 10 mL of ultrapure water, and dilute the sample solution into 6 concentrations in proportion after thorough mixing. Use the extract of 10 mg / mL VC solution to prepare 0.3 mg / mL, 0.25 mg / mL, 0.125 mg / mL, 0.0625 mg / mL, 0.03125 mg / mL, and 0.015625 mg / mL VC solutions for standby use. Add the above reagents according to the instructions, vortex mix, and stand at room temperature and away from light for 30 minutes. Set up three replicates for each group, and record the absorbance at 515 nm as A blank, A determination, A control, and A positive control, respectively. A control tube is required for each determination tube. The positive control tube and the blank tube only need to be measured 1 to 2 times. The formula for calculating the DPPH free radical scavenging rate is as follows:

[0066] The free radical scavenging rate of the positive control was calculated as follows: DPPH free radical scavenging rate DVC% = [(A blank - A positive control) ÷ A blank] × 100%;

[0067] The free radical scavenging rate of the sample was calculated by the formula: DPPH free radical scavenging rate D% = [[A blank - (A determination - A control)] ÷ A blank] × 100%.

[0068] The results are as follows Figure 4 As shown, it can be seen that the in vitro DPPH free radical scavenging activities of CPP-1 and Se-CPP-1 are dose-dependent and both show significant DPPH free radical scavenging rates. When the concentrations of CPP-1 and Se-CPP-1 are 6 mg / mL, the scavenging rates of CPP-1 and Se-CPP-1 are 56.02% and 72.06%, respectively. In addition, within each concentration range, the scavenging rate of Se-CPP-1 on DPPH free radicals is stronger than that of CPP-1.

[0069] 2) Determination of ABTS free radical scavenging ability

[0070] Weigh 10 mg of polysaccharide, selenium polysaccharide and Vc respectively, dissolve in 10 mL of ultrapure water, and dilute the sample solution into 6 concentrations after thorough mixing, namely 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, and 6 mg / mL. Use the extract of 10 mmol / L VC solution to prepare 0.4 mmol / L, 0.2 mmol / L, 0.1 mmol / L, 0.05 mmol / L, 0.025 mmol / L, and 0.0125 mmol / L VC solutions for standby use. Add the above reagents according to the instructions, mix thoroughly, and stand at room temperature and away from light for 6 minutes. Measure the absorbance at 405 nm, and record them as A blank, A determination, A control, and A positive control, respectively. The positive control tube and blank tube only need to be measured 1 to 2 times.

[0071] The formula for calculating the free radical scavenging rate of ABTS is as follows:

[0072] The free radical scavenging rate of the positive control was calculated by the formula: ABTS free radical scavenging rate DVC% = [(A blank - A positive control) ÷ A blank] × 100%.

[0073] The free radical scavenging rate of the sample was calculated by the formula: ABTS free radical scavenging rate D% = [A blank - (A determination - A control)] ÷ A blank × 100%.

[0074] The results are as follows Figure 5 As shown in the figure, in the concentration range of 1 mg / mL to 2 mg / mL, the scavenging rates of CPP-1 and Se-CPP-1 for ABTS free radicals are not much different, which may be because low concentrations of CPP-1 and Se-CPP-1 solutions cannot provide enough reducing groups to induce ABTS delocalization. However, with the increase of the concentrations of CPP-1 and Se-CPP-1, the scavenging rates of the two for ABTS free radicals gradually widened. At 6 mg / mL, the scavenging rates of CPP-1 and Se-CPP-1 for ABTS free radicals were 15.78% and 38.96%, respectively, which is basically consistent with the scavenging trend of CPP-1 and Se-CPP-1 and DPPH free radicals, indicating that the scavenging rate of Se-CPP-1 for ABTS free radicals is better than that of polysaccharides.

[0075] 3) Cell proliferation assay

[0076] When the cell growth density reaches about 80%, adjust the RAW 264.7 macrophage concentration to 10 4 / ml. 100uL was inoculated into each well of a 96-well plate for 12h to 24h, and the supernatant was removed by centrifugation. In addition to setting up a blank control group, the concentrations of CPP-1 and Se-CPP-1 were set to 25ug / mL, 50ug / mL, 100ug / mL, 200ug / mL, 400ug / mL, and 800ug / mL. Six replicate wells were set up in each group, with 100uL in each well. Then, they were marked and placed in a CO2 incubator. After continuing to culture for 24h, the supernatant was discarded, 100uL CCK-8 was added to each well, and incubated for 2h. The absorbance was then measured at 450nm using an enzyme reader. The following formula was used to calculate the cell survival rate of polysaccharides:

[0077] Cell survival rate (%) = [(As-Ab) / (Ac-Ab) × 100%];

[0078] Where As represents the absorbance of the experimental well; Ac represents the absorbance of the control well; Ab represents the absorbance of the blank well.

[0079] The results are as follows Figure 6 As shown in the figure, after adding different concentrations of CPP-1 and Se-CPP-1, the viability of RAW 264.7 cells was significantly enhanced. Compared with CPP-1, Se-CPP-1 can significantly enhance the proliferation of RAW264.7 cells, and the cell viability decreases at 800μg / mL. In the range of 25μg / mL to 800μg / mL, the cell viability of Se-CPP-1 was 223.1%, 248.8%, 257.9%, 262.2%, 290.9% and 210.1%, respectively. The experimental results show that Se-CPP-1 can significantly promote cell proliferation and has a certain dose dependence. Considering comprehensively, 25μg / mL to 800μg / mL was used for subsequent experiments.

[0080] 4) Cell phagocytosis experiment

[0081] The neutral red uptake method was used to determine the phagocytic ability of cells. The polysaccharides and selenium polysaccharide samples of 25ug / mL, 50ug / mL, 100ug / mL, 200ug / mL, 400ug / mL, and 800ug / mL of Cyclocarya paliurus were inoculated in a 96-well plate. After culturing for 24 hours, the supernatant was removed and washed with PBS 1 to 2 times to remove non-adherent cells. 100uL of neutral red solution was added to each well for 1 hour of incubation, the supernatant was removed, and PBS was added to wash 3 times to remove the neutral red solution. Subsequently, 100uL of pre-prepared cell lysate was added to each well, in which the cell lysate was mixed with ethanol and acetic acid in a ratio of 1:1, and then the cells were placed in a 37°C environment and cultured for 2 hours. After the culture was completed, the absorbance at a wavelength of 490nm was measured using an enzyme reader, and the phagocytic ability was calculated based on the obtained data. The calculation formula of the phagocytic index is as follows:

[0082] Phagocytic index = (As-Ab) / (Ac-Ab);

[0083] Where As represents the absorbance of the experimental well; Ac represents the absorbance of the control well; Ab represents the absorbance of the blank well.

[0084] The results are as follows Figure 7 As shown in the figure, compared with the blank group and CPP-1 group, the cells treated with Se-CPP-1 group showed stronger phagocytic activity, and significantly stimulated the phagocytic activity of macrophages RAW264.7 in the concentration range (P < 0.05), indicating that Se-CPP-1 can enhance the phagocytic effect of macrophages RAW264.7. The phagocytic activity of Se-CPP-1 was the highest at 25 μg / mL. The above experimental results show that polysaccharides modified by selenium can significantly enhance the activity of macrophages RAW 264.7.

[0085] 5) NO experiment

[0086] The NO secretion of RAW 264.7 macrophages was measured by Griess reagent. CPP-1 and Se-CPP-1 at concentrations of 25ug / mL, 50ug / mL, 100ug / mL, 200ug / mL, 400ug / mL, and 800ug / mL were inoculated in 96-well plates to stimulate RAW 264.7 macrophages for 48h. The density of macrophages was 10 4 pcs / ml; set up blank control group and negative control group. Pipette 50uL of supernatant from each well and transfer to a new 96-well plate, then mix with 50uL Griess reagent, incubate at room temperature for 10min, measure the absorbance at 540nm with an enzyme reader, draw a standard curve according to the kit instructions and calculate the NO content.

[0087] result Figure 8 As shown in the results, under the stimulation of different concentrations of the two polysaccharides, the secretion of NO by mouse macrophages was significantly enhanced. The experiment found that Se-CPP-1 greatly enhanced the amount of NO secreted by macrophages RAW264.7. Compared with the blank group, under the stimulation of different concentrations of Se-CPP-1 and CPP-1, the maximum content of NO increased from 2.29±0.39μm in the blank group to 40.75±0.96μm (50μg / mL) and 22.41±1.4μm (400μg / mL), respectively. This indicates that Se-CPP-1 may play an immunomodulatory role by stimulating macrophages RAW264.7 to secrete more NO.

[0088] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. The use of selenium polysaccharide in the preparation of immunologically active agents, characterized in that: The selenium polysaccharide is obtained by spraying exogenous selenium on the leaves of Cyclocarya paliurus to selenize the polysaccharide in the Cyclocarya paliurus, thereby obtaining a selenium-enriched Cyclocarya paliurus with a selenium content of not less than 34 mg / kg, and then extracting it from the leaves of the selenium-enriched Cyclocarya paliurus; The immunologically active agent refers to a preparation having an immunomodulatory effect.

2. The use of selenium polysaccharide according to claim 1 in the preparation of an immunologically active agent, characterized in that: The molecular weight of the selenium polysaccharide is 76.662 kDa.

3. The use of selenium polysaccharide according to claim 1 in the preparation of an immunologically active agent, characterized in that: In terms of mass percentage, the selenium polysaccharide is composed of the following monosaccharides: Fucose 0.75%, rhamnose 11.61%, arabinose 25.06%, galactose 32.15%, glucose 6.37%, xylose 2.48%, galacturonic acid 19.81%, glucuronic acid 1.77%, total 100%.

4. The use of selenium polysaccharide in the preparation of an immunologically active agent according to claim 1, characterized in that: The preparation having immunomodulatory effect refers to a preparation that improves the proliferation, phagocytic activity and NO release of macrophages.

5. An immunologically active agent, characterized in that The immunologically active agent contains the selenium polysaccharide described in claim 1 as the only active ingredient.

6. The immunologically active agent according to claim 5, characterized in that: The immunologically active agent is any one of tablets, powders, granules and aqueous solutions.

7. The immunologically active agent according to claim 5, characterized in that The immunologically active agent also includes pharmaceutically acceptable excipients.

8. The use of selenium polysaccharide in the preparation of an immunologically active agent according to claim 1, characterized in that: The exogenous selenium is a sodium selenite solution, and the concentration of the sodium selenite solution is 120 mg / L to 160 mg / L.

9. The use of selenium polysaccharide in the preparation of an immunologically active agent according to claim 1, characterized in that: The specific process of obtaining the selenium polysaccharide is as follows: The leaves of the selenium-rich Cyclocarya paliurus are crushed, extracted with water and precipitated with alcohol to obtain an extract; The extract is passed through a macroporous resin and concentrated to obtain a concentrated solution; the concentrated solution is mixed with a sevage reagent, centrifuged, and the supernatant is taken, and the supernatant is precipitated with alcohol and freeze-dried to obtain polysaccharides; and the polysaccharides are purified to obtain the selenium polysaccharide.

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