Use of selenopolysaccharides for the preparation of immunostimulants

Abstract

The application belongs to the application field of selenium polysaccharide, and particularly relates to application of the selenium polysaccharide in preparation of an immune active agent, wherein the selenium polysaccharide is obtained by treating Cyclobalanopsis glauca with exogenous selenium, has good immune activity, can better promote proliferation and phagocytosis of RAW264.7 macrophages, and has a significant advantage in improving the release amount of NO, and the results show that the polysaccharide after exogenous selenium strengthening has better immune activity. Exogenous selenium strengthening obviously improves the characteristics of the selenium polysaccharide in Cyclobalanopsis glauca leaves, and has better potential in antioxidation and immune improvement, and provides data support for further research and utilization.

Description

TECHNICAL FIELD

[0001] The application belongs to the application field of selenium polysaccharides, and particularly relates to application of selenium polysaccharides in preparation of an immune active agent. BACKGROUND

[0002] Cyclocarya paliurus is a deciduous arbor plant of the Cyclocarya paliurus in the Juglandaceae family and is also a unique rare plant with medicinal and edible properties. Because Cyclocarya paliurus contains rich flavonoids, triterpenes, polysaccharides and other components, it has the effects of antioxidant, blood sugar reduction, anti-aging, beauty and weight loss, and is widely used in the production of functional foods and is widely recognized by consumers.

[0003] Selenium polysaccharides are an important organic selenium form of selenium in plants, and selenium polysaccharides have better biological activity than polysaccharides. At present, the research on selenium polysaccharides in selenium-rich Cyclocarya paliurus mainly focuses on their biological activity functions, such as antioxidant activity. In order to further expand the application range of selenium polysaccharides in selenium-rich Cyclocarya paliurus, further research is needed on other biological activities of selenium polysaccharides. SUMMARY

[0004] To solve the above technical problems, the application provides application of selenium polysaccharides in preparation of an immune active agent. The selenium polysaccharides in selenium-rich Cyclocarya paliurus can effectively improve the proliferation, phagocytic activity and NO release amount of macrophages, thereby improving the immune activity.

[0005] To achieve the above technical purposes, the technical solutions of the application are as follows.

[0006] The application provides, in a first aspect, application of selenium polysaccharides in preparation of an immune active agent. The selenium polysaccharides refer to polysaccharides in Cyclocarya paliurus that are selenized by exogenous selenium spraying on the leaves of Cyclocarya paliurus, and the selenium content of the selenium-rich Cyclocarya paliurus obtained is not less than 34 mg / kg, and the selenium polysaccharides are extracted from the leaves of the selenium-rich Cyclocarya paliurus.

[0007] The immune active agent refers to a preparation with immune regulation effect.

[0008] In the application, polysaccharides in Cyclocarya paliurus are selenized by exogenous selenium spraying, and the selenium content of the obtained selenium-rich Cyclocarya paliurus is not less than 34 mg / kg, and the selenium polysaccharides are extracted therefrom. It is found through in-vitro experiments on the extracted selenium polysaccharides that the selenium polysaccharides have immune regulation activity.

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

[0010] In another preferred embodiment, the selenium polysaccharides are 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 preparation with immunoregulatory effect refers to a preparation for improving the proliferation, phagocytosis activity and NO release amount of macrophages. It can better promote the proliferation and phagocytosis function of RAW264.7 macrophages, and has a significant advantage in improving the release amount of NO.

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

[0014] In another preferred embodiment, the immunoreactive agent is any one of a tablet, a powder, a granule, and an aqueous agent.

[0015] In another preferred embodiment, the immunoreactive agent further comprises a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient broadly refers to any component other than the active therapeutic ingredient. The excipient can be an inert substance, an inactive substance and / or a non-pharmaceutically active substance. The excipient can be used for various purposes, such as serving as a carrier, a vehicle, a diluent, a tablet aid and / or to improve the administration and / or absorption of the active substance.

[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 selenium polysaccharide is obtained by the following specific process:

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

[0019] The extract is passed through a macroporous resin, concentrated, mixed with a sevage reagent, centrifuged, 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 crude polysaccharide is activated with an anion exchanger, and then columned, eluted with water, concentrated, dialyzed and dried to obtain the selenium polysaccharide. The anion exchanger is DEAE-52 cellulose; and the elution is gradient elution with a NaCl solution with a concentration of 0 mol / L to 0.4 mol / L.

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

[0023] In the present application, the polysaccharide in Cyclocarya paliurus is selenized, so that the content of selenium polysaccharide is not less than 34 mg / kg, and the selenium polysaccharide obtained has good immunocompetence and can be used for preparing an immunocompetent agent.

[0024] The selenium polysaccharide in the present application has a molecular weight of 76.662 kDa and is composed of fucose, rhamnose, arabinose, galactose, glucose, xylose, galacturonic acid and glucuronic acid, so that it can better promote the proliferation and phagocytosis of RAW264.7 macrophages, and has a significant advantage in improving the release amount of NO. The results show that the exogenous selenium-strengthened Cyclocarya paliurus polysaccharide has better immunocompetence. Exogenous selenium-strengthening significantly improves the characteristics of selenium polysaccharide in Cyclocarya paliurus leaves, and has better potential in antioxidant and immune enhancement, which provides data support for further research and utilization. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The infrared characterization graph of the polysaccharide and the selenium polysaccharide in the selenium-enriched Cyclocarya paliurus is shown in the figure, wherein CPP-1 is the polysaccharide and Se-CPP-1 is the selenium polysaccharide.

[0026] Figure 2 The scanning electron microscope graph of different polysaccharides is shown in the figure, wherein A is the scanning electron microscope graph of the polysaccharide and B is the scanning electron microscope graph of the selenium polysaccharide.

[0027] Figure 3 The molecular weight distribution graph of Se-CPP-1 is shown in the figure.

[0028] Figure 4 The DPPH free radical scavenging capacity result graph of different polysaccharides is shown in the figure.

[0029] Figure 5 The ABTS free radical scavenging capacity result graph of different polysaccharides is shown in the figure.

[0030] Figure 6 The cell proliferation result graph of different polysaccharides is shown in the figure.

[0031] Figure 7 The cell phagocytosis result graph of different polysaccharides is shown in the figure.

[0032] Figure 8 The NO experiment result graph of different polysaccharides is shown in the figure. DETAILED DESCRIPTION

[0033] The present application will be described in detail below in conjunction with specific embodiments, but should not be understood as limiting the present application. If not specially stated, the technical means used in the following examples are conventional means familiar to those skilled in the art, and the materials, reagents, etc. used in the following examples, if not specially stated, can be obtained from commercial channels.

[0034] In recent years, polysaccharides isolated from Cyclocarya paliurus, their biological activities and structures have been reported a lot, but there are few related researches on selenium-rich Cyclocarya paliurus polysaccharides. The selenium polysaccharide in selenium-rich Cyclocarya paliurus is studied in the present application, and it is found that the selenium polysaccharide has better scavenging effect on DPPH and ABTS free radicals, and the in vitro cell experiment verifies its immunomodulatory activity, and the results show that the selenium polysaccharide can better promote the proliferation and phagocytosis of RAW264.7 macrophages, and has a significant advantage in improving the release amount of NO.

[0035] 1. Experimental material preparation

[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 is prepared, and selenium is sprayed on the surface of the leaves of Cyclocarya paliurus by foliar application of selenium for 15 days, and the selenium content in the leaves is determined to obtain the leaves of selenium-rich Cyclocarya paliurus 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 are placed in an electric heating air drying oven, dried at 65℃ to constant weight, and then crushed into powder by a pulverizer, and then passed through an 80-mesh sieve and placed in a self-sealing bag for standby.

[0040] 1) Cyclocarya paliurus polysaccharide extraction solution

[0041] The water extraction and alcohol precipitation method is used to extract selenium polysaccharide from the leaves of Cyclocarya paliurus, and the specific process is as follows:

[0042] 40 g of Cyclocarya paliurus powder is weighed and placed in a round-bottom flask, and deionized water is added according to the solid-liquid ratio of 1:25 g / mL, and then placed in a magnetic stirring water bath, and extracted at 90℃ for 2 h, and then slowly poured out the supernatant after standing, and then centrifuged at 10000 r / min for 15 min to discard the precipitate, and then poured out the supernatant to obtain a clear brown polysaccharide extraction solution.

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

[0044] Color removal: AB-8 macroporous resin was soaked in 80% ethanol for 3h, then the ethanol was poured out, and excess distilled water was added and repeatedly washed until the washing liquid was completely free of ethanol. A small amount of cotton was prepared in advance and placed at the bottom of a 40mm*40cm glass chromatography column. The soaked AB-8 macroporous resin was poured into the glass chromatography column at a uniform speed, the outlet valve of the chromatography column was opened, and the liquid in the column was discharged to allow the AB-8 macroporous resin to naturally settle to the upper end of the chromatography column 10cm. There were no macroporous resin particles floating in the chromatography column. At this time, the polysaccharide extract was slowly poured into the column, and a light yellow polysaccharide solution was seen flowing out, indicating that the polysaccharide was successfully removed from the color. The decolorized polysaccharide solution was concentrated using a rotary evaporator. The condenser water and vacuum pump were turned on, the device was checked for airtightness, the temperature was set to 55°C, and the rotation speed was 60r / min. The solution was concentrated to 200mL.

[0045] Protein removal: Prepare sevage reagent (chloroform:n-butanol = 4:1) 1 / 4 volume of polysaccharide extract to remove protein. Pour the polysaccharide extract and sevage reagent into a separatory funnel, shake vigorously for 10min, then stand for 2h. After the solution was clearly layered, centrifuge at 5000r / min for 10min, collect the supernatant, and repeat the sevage method 3 times to remove protein.

[0046] Alcohol precipitation and freeze-drying: After removing the protein from the polysaccharide solution, add 4 times the volume of 80% anhydrous ethanol to the polysaccharide extract in a beaker. A large amount of flocculent material can be seen in the solution. After overnight, centrifuge at 10000r / min for 15min, pour out the supernatant, and wash the precipitate with anhydrous ethanol 4 times. Then, take the precipitate for freeze-drying treatment to obtain the selenium-rich polysaccharide from Cyclocarpon sinense.

[0047] 3) Purification of Cyclocarpon sinense polysaccharide

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

[0049] Weigh 100g of DEAE-52 cellulose dry powder and dissolve it in ultrapure water. Stir with a glass rod for 3h. After removing the impurities on the surface of the solution, dry the water. Put it in a 0.5mol / L HCL solution and stir evenly for 2h. Then wash the solution to neutral with ultrapure water. Dry the water, then put it in a 0.5mol / L NaOH solution and stir evenly for 2h. Wash the solution to neutral with ultrapure water and dry the water. Put the activated cellulose in ultrapure water for standby. At this point, the DEAE-52 cellulose activation is complete.

[0050] Column packing: Fix a 40mm*40cm glass chromatography column on an iron stand, stuff a small amount of cotton into the bottom of the chromatography column, slowly pour in the activated cellulose, open the outlet valve, and repeatedly compact the column bed with ultrapure water, allowing it to settle naturally for 12 hours.

[0051] Sample loading and elution: Weigh 0.5 g of crude polysaccharide and dissolve it completely in 10 mL of ultrapure water. Centrifuge at 5000 r / min for 10 min. Slowly pour the supernatant along the inner wall of the chromatography column. Connect the inlet tube at the top of the chromatography column to a constant flow pump and adjust the flow rate to 10 mL / min. Start gradient elution with NaCl solutions of 0 mol / L, 0.05 mol / L, 0.1 mol / L, 0.2 mol / L, 0.3 mol / L, and 0.4 mol / L sequentially. Collect the eluent in 10 mL centrifuge tubes, collecting 40 tubes for each concentration. After elution, determine the polysaccharide content in the solution at 490 nm using the phenol-sulfuric acid method. Plot the elution curve. Combine the eluents under the main elution peaks and concentrate to 20 mL. Place the solution in a 3500 Da dialysis bag to remove small molecules and dialyze for 48 h, changing the ultrapure water 8 times during this period. Then freeze-dry to obtain purified selenium polysaccharide.

[0052] 3. Characterization of selenium polysaccharides

[0053] 1) Structural characterization

[0054] The polysaccharides and selenium polysaccharides obtained above were characterized by infrared spectroscopy, and the results were... Figure 1 As shown. From Figure 1 It can be seen that the -OH tensile vibration peak of Se-CPP-1 appears at 3350.1 cm⁻¹. -1 2929.34cm -1 The peaks for the CH stretching vibrations of the methyl and methylene groups are present at 1619.93 cm⁻¹, while the peak for the C=O stretching vibration of the carboxyl group occurs at 1619.93 cm⁻¹. -1 Additionally, 1415.74cm -1 The peak at 834.12 cm⁻¹ is a characteristic absorption peak for the angular vibration of CH. It can be seen that Se-CPP-1 after selenization modification possesses the infrared spectral characteristics of polysaccharides. -1 The new absorption peak at this location can be attributed to O-Se-O bonds. These results confirm that the selenium polysaccharide has been successfully selenized.

[0055] Scanning electron microscopy analysis was performed on CPP-1 and Se-CPP-1, and the results are as follows: Figure 2As shown, the results indicate that the introduction of exogenous selenium significantly affected the surface microstructure of CPP-1. At a magnification of 5.0 K, the surface of CPP-1 exhibited roughness and inhomogeneity, displaying an irregular honeycomb structure. In contrast, Se-CPP-1 exhibited an irregular sheet-like morphology with a smooth surface, forming a stark contrast to CPP-1. The particle size of Se-CPP-1 was significantly reduced, exhibiting a clustered, loose, and porous structure with an increased specific surface area. It is inferred that Se in Se-CPP-1 is likely the main site initiating hydrophobic binding. This binding caused the sugar chains to transition from an extended to a folded state, thereby leading to the reduction in particle size and significant morphological changes in Se-CPP-1.

[0056] 2) Molecular weight characterization

[0057] The molecular weight distribution of Se-CPP-1 was determined by high-performance gel permeation chromatography (HP-GPC) as follows: 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 exhibited relatively high homogeneity after separation and purification, and its molecular weight was determined to be 76.662 kDa.

[0058] 3) Monosaccharide composition

[0059] To further investigate the structural characteristics of CPP-1 and Se-CPP-1, the monosaccharide composition was analyzed in detail using high-permeability ion chromatography (HPIC). Given that sugar molecules may exist partially or entirely in anionic form under high-pH eluent conditions, the analysis was performed by detecting the current generated by the oxidation reaction of hydroxyl groups on the gold electrode surface. As shown in Table 1, a comparison with the retention times of standard monosaccharide derivatives revealed that both CPP-1 and Se-CPP-1 contain multiple 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). Glucose and arabinose are the main components of CPP-1 and Se-CPP-1, respectively. The relative contents of galacturonic acid in CPP-1 and Se-CPP-1 are 6.10% and 19.81%, respectively, demonstrating their characteristics as acidic polysaccharides.

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

[0061]

[0062] Note: Man represents mannose.

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

[0064] 1) DPPH radical scavenging capacity determination

[0065] Selenium polysaccharide and Vc were weighed at 10 mg each and dissolved in 10 mL of ultrapure water. After thorough mixing, the sample solution was diluted to 6 concentrations in proportion. The 10 mg / mL VC solution was prepared into 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 with the extraction solution for standby. According to the instructions, the above reagents were added and vortexed, and then placed in the dark at room temperature for 30 min. Each group was set up in triplicate, and the absorbance at 515 nm was recorded as Ablank, Atest, Acontrol, and Apositive control, respectively. Each test tube required a control tube. The positive control tube and the blank tube only needed to be measured 1-2 times. The DPPH radical scavenging rate calculation formula is as follows:

[0066] The positive control radical scavenging rate calculation formula is: DPPH radical scavenging rate DVC% = [(Ablank-Apositive control) ÷ Ablank] x 100%;

[0067] The sample radical scavenging rate calculation formula is: DPPH radical scavenging rate D% = [[Ablank-(Atest-Acontrol)] ÷ Ablank] x 100%.

[0068] The results are shown in Table 1. Figure 4 As can be seen from Table 1, the in vitro DPPH radical scavenging activity of CPP-1 and Se-CPP-1 is dose-dependent, and both show significant DPPH radical scavenging rates. When the concentration of CPP-1 and Se-CPP-1 is 6 mg / mL, the scavenging rates of CPP-1 and Se-CPP-1 are 56.02% and 72.06%, respectively. And in each concentration range, the DPPH radical scavenging rate of Se-CPP-1 is stronger than that of CPP-1.

[0069] 2) ABTS radical scavenging capacity determination

[0070] Take 10 mg of polysaccharide, selenium polysaccharide and Vc respectively, dissolve in 10 mL of ultrapure water, mix thoroughly, and dilute the sample solution into 6 concentrations, 1 mg / mL, 2 mg / mL, 3 mg / mL, 4 mg / mL, 5 mg / mL, 6 mg / mL. The 10 mmol / L VC solution is prepared into 0.4 mmol / L, 0.2 mmol / L, 0.1 mmol / L, 0.05 mmol / L, 0.025 mmol / L, 0.0125 mmol / L VC solution with the extraction solution. According to the instructions, add the above reagents, mix thoroughly, and stand at room temperature for 6 min. Measure the absorbance at 405 nm, and record as Ablank, Atest, Acontrol, Apositive control. The positive control tube and the blank tube only need to be measured 1-2 times.

[0071] The ABTS free radical scavenging rate calculation formula is as follows:

[0072] The free radical scavenging rate calculation formula of the positive control is: ABTS free radical scavenging rate DVC% = [(Ablank-Apositive control) ÷ Ablank] x 100%.

[0073] The free radical scavenging rate calculation formula of the sample is: ABTS free radical scavenging rate D% = [Ablank-(Atest-Acontrol)] ÷ Ablank x 100%.

[0074] The results are shown in Table 1. Figure 5 As shown in Table 1, in the concentration range of 1 mg / mL-2 mg / mL, the ABTS free radical scavenging rates of CPP-1 and Se-CPP-1 are not much different, which may be because the low concentration of CPP-1 and Se-CPP-1 solution cannot provide enough reducing groups to induce ABTS delocalization. But with the increase of the concentration of CPP-1 and Se-CPP-1, the ABTS free radical scavenging rates of the two gradually open up some gap, at 6 mg / mL, the ABTS free radical scavenging rates of CPP-1 and Se-CPP-1 are 15.78% and 38.96% respectively, which is basically consistent with the DPPH free radical scavenging trend of CPP-1 and Se-CPP-1, which can show that the ABTS free radical scavenging rate of Se-CPP-1 is better than that of polysaccharide.

[0075] 3) Cell proliferation experiment

[0076] When the cell growth density reaches about 80%, the concentration of RAW 264.7 macrophages is adjusted to 10 4ml. Access to 96-well plates with 100 uL per well for culture 12 h ~ 24 h, centrifugal to remove supernatant, except for the blank control group, and set CPP-1 and Se-CPP-1 concentration of 25 ug / mL, 50 ug / mL, 100 ug / mL, 200 ug / mL, 400 ug / mL, 800 ug / mL, each group set 6 holes, 100 uL per well, then mark and put into CO2 incubator. Continue to cultivate 24 h, discard the supernatant, add 100 uL CCK-8 per well, incubate for 2 h. Then use the microplate reader to measure the absorbance at 450 nm. The following public calculation of polysaccharide on cell survival rate:

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

[0078] Wherein, As represents the absorbance of the experimental hole; Ac represents the absorbance of the control hole; Ab represents the absorbance of the blank hole.

[0079] The results are shown in Figure 6 , after adding different concentrations of CPP-1 and Se-CPP-1 culture, RAW 264.7 cell activity is obviously enhanced. Compared with CPP-1, Se-CPP-1 can significantly enhance the proliferation of RAW264.7 cells, and the cell activity decreases at 800 ug / mL. Se-CPP-1 in the range of 25 ug / mL ~ 800 ug / mL, the cell survival rate is 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. Comprehensive consideration, the subsequent experiment is carried out at 25 ug / mL ~ 800 ug / mL.

[0080] 4) Cell phagocytosis experiment

[0081] The neutral red uptake method was used to determine the phagocytic ability of cells. The concentration of 25 ug / mL, 50 ug / mL, 100 ug / mL, 200 ug / mL, 400 ug / mL, 800 ug / mL of cyclocarya polysaccharide and selenium polysaccharide samples were inoculated in 96 well plates, and cultured for 24 h, then the supernatant was removed, and PBS was washed 1 ~ 2 times to remove non-adherent cells. Add 100 uL of neutral red solution to each well and incubate for 1 h, remove the supernatant, and wash with PBS 3 times to remove the neutral red solution. Then add 100 uL of pre-prepared cell lysate to each well, wherein the cell lysate is mixed by ethanol and acetic acid in a ratio of 1:1, and then the cells are incubated at 37℃ for 2 hours. After incubation, the absorbance at 490 nm was measured using a microplate reader, and the phagocytic ability was calculated according to the data obtained. The formula for calculating the phagocytic index is as follows:

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

[0083] Wherein, As represents the absorbance of the experimental hole; Ac represents the absorbance of the control hole; Ab represents the absorbance of the blank hole.

[0084] The results are shown in Table 1. Figure 7 As shown in Table 1, compared with the blank group and the CPP-1 group, the cells treated by the Se-CPP-1 group showed stronger phagocytic activity, and significantly stimulated the phagocytic activity of the macrophage RAW264.7 in the concentration range (P<0.05), which indicated that the Se-CPP-1 could enhance the phagocytosis of the macrophage RAW264.7. The phagocytic activity of the Se-CPP-1 was the largest at 25 μg / mL. The above experimental results showed that the selenium-modified polysaccharide could significantly enhance the activity of the macrophage RAW264.7.

[0085] 5) NO experiment

[0086] The content of NO secreted by the RAW264.7 macrophage was measured by using the Griess reagent, and the concentration of 25 ug / mL, 50 ug / mL, 100 ug / mL, 200 ug / mL, 400 ug / mL, 800 ug / mL of the CPP-1 and the Se-CPP-1 was inoculated in the 96-well plate, the RAW264.7 macrophage was stimulated for 48 h, wherein the density of the macrophage was 10 4 Individuals / ml; the blank control group and the negative control group were set. 50 uL of the supernatant of each well was transferred to a new 96-well plate, and then mixed with 50 uL of the Griess reagent, and incubated at room temperature for 10 min. The absorbance was measured at 540 nm by using the enzyme-labeled instrument, and the standard curve was drawn and the content of NO was calculated according to the kit instruction.

[0087] Results Figure 8 As shown in Table 1, under the stimulation of different concentrations of the two polysaccharides, the content of NO secreted by the mouse macrophage was significantly enhanced. It was found that the Se-CPP-1 greatly enhanced the amount of NO secreted by the macrophage RAW264.7. Compared with the blank group, under the stimulation of different concentrations of the Se-CPP-1 and the CPP-1, the maximum content of NO increased from 2.29±0.39 μm of the blank group to 40.75±0.96 μm (50 μg / mL) and 22.41±1.4 μm (400 μg / mL), respectively. This indicated that the Se-CPP-1 might stimulate the macrophage RAW264.7 to secrete more NO, and then played a role in immune regulation.

[0088] The above is only the preferred embodiment of the present application, and is not used to limit the present application, any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Use of a selenopolysaccharide for the preparation of an immunostimulant, characterized in that, The selenium polysaccharide refers to the polysaccharide in the greenbrier leaf being selenized by exogenous selenium spraying, obtaining selenium-rich greenbrier with selenium content not less than 34 mg / kg, and then extracting from the selenium-rich greenbrier leaf; The immunologically active agent refers to a preparation with immunoregulatory effect; 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%.

2. Use of selenopolysaccharide according to claim 1 for the preparation of an immunostimulant, characterized in that, The molecular weight of the selenium polysaccharide is 76.662 kDa.

3. An immunologically active agent, characterized in that, The immunologically active agent has the selenium polysaccharide as the only active ingredient.

4. The immunologically active agent of claim 3, wherein, The immunologically active agent is any one of a tablet, a powder, a granule, and an aqueous agent.

5. The immunotherapeutic agent of claim 3, wherein the agent is a vaccine. The immunologically active agent further comprises a pharmaceutically acceptable excipient.

6. The application of the selenium polysaccharide according to claim 1 in the preparation of immunomodulatory agents, characterized in that, The exogenous selenium is sodium selenite solution, and the concentration of the sodium selenite solution is 120 mg / L-160 mg / L.

7. The application of the selenium polysaccharide according to claim 1 in the preparation of immunomodulatory agents, characterized in that, The specific obtaining process of the selenium polysaccharide is as follows: The leaf blades of the selenium-rich greenbrier are crushed, and then water extraction and alcohol precipitation are performed to obtain an extraction liquid; the extraction liquid is passed through a macroporous resin, and then concentrated to obtain a concentrated liquid; the concentrated liquid is mixed with a sevage reagent, centrifuged, and the supernatant is taken; the supernatant is alcohol precipitated and freeze-dried to obtain polysaccharide; and the polysaccharide is purified to obtain the selenium polysaccharide. The immunologically active agent has the selenium polysaccharide as the only active ingredient. The immunologically active agent is any one of a tablet, a powder, a granule, and an aqueous agent. The immunologically active agent further comprises a pharmaceutically acceptable excipient. The exogenous selenium is sodium selenite solution, and the concentration of the sodium selenite solution is 120 mg / L-160 mg / L. The specific obtaining process of the selenium polysaccharide is as follows: The leaf blades of the selenium-rich greenbrier are crushed, and then water extraction and alcohol precipitation are performed to obtain an extraction liquid; the extraction liquid is passed through a macroporous resin, and then concentrated to obtain a concentrated liquid; the concentrated liquid is mixed with a sevage reagent, centrifuged, and the supernatant is taken; the supernatant is alcohol precipitated and freeze-dried to obtain polysaccharide; and the polysaccharide is purified to obtain the selenium polysaccharide.

Images