阅读说明：本技术 大肥菇菌丝体胞内多糖在制备免疫调节药品、保健品或食品中的应用 (Application of intracellular polysaccharide of pleurotus ferulae mycelium in preparation of immunoregulation medicine, health-care product or food ) 是由 彭强 袁木荣 李文霞 袁芳廷 于 2021-01-22 设计创作，主要内容包括：本发明公开了大肥菇菌丝体胞内多糖在制备免疫调节药品、保健品或食品中的应用,经本发明研究表明,在细胞实验发现柴达木大肥菇菌丝体胞内多糖可显著提高小鼠脾细胞增殖率,促进小鼠巨噬细胞株RAW264.7分泌TNF-α和NO等免疫因子,证明大肥菇菌丝体胞内多糖具有很好的免疫活性调节功能,而非单纯地单向提高免疫活性,从而可防止过度免疫。在动物实验发现,大肥菇菌丝体胞内多糖可明显调节免疫抑制小鼠脾及胸腺指数、改善免疫抑制小鼠淋巴细胞水平,缓解免疫亢进小鼠腹腔巨噬细胞吞噬活性亢进和脾淋巴细胞体外增殖能力亢进降低,说明该多糖具有较好的双向免疫调节作用。从而可以将其作为安全可靠的免疫调节药物、保健品或食品。(The research of the invention shows that the intracellular polysaccharide of the pleurotus ferulae mycelium can obviously improve the proliferation rate of mouse splenocytes and promote mouse macrophage strain RAW264.7 to secrete immune factors such as TNF-alpha, NO and the like, and the intracellular polysaccharide of the pleurotus ferulae mycelium has a good immune activity regulating function, but not simply improves the immune activity in a single direction, thereby preventing over immunity. Animal experiments show that the intracellular polysaccharide of the pleurotus ferulae mycelium can obviously regulate the spleen and thymus indexes of immunosuppressive mice, improve the lymphocyte level of the immunosuppressive mice, relieve hyperfunction of macrophage phagocytosis activity of abdominal cavities of the mice with hyperfunction of the immunity and reduction of hyperfunction of lymphocyte proliferation capacity in vitro of the spleen, and the polysaccharide has better two-way immunoregulation effect. So that the compound can be used as safe and reliable immunoregulation medicine, health-care product or food.)

1. Application of intracellular polysaccharide of Agaricus blazei Murill mycelium in preparing immunoregulation medicine is provided.

2. The application of intracellular polysaccharide of Agaricus blazei Murill mycelium in preparing immunoregulation health products is provided.

3. Application of intracellular polysaccharide of Agaricus blazei Murill mycelium in preparing immunoregulation food is provided.

4. Use according to claim 1 or 2 or 3, characterized in that: the pleurotus ferulae mycelium intracellular polysaccharide is polysaccharide extracted from pleurotus ferulae mycelium cells.

5. Use according to claim 1 or 2 or 3, characterized in that: the immune regulation is bidirectional regulation comprising increasing immune activity and reducing immune activity.

Technical Field

The invention relates to the technical field of medicinal health-care products, in particular to application of boletus edulis mycelium polysaccharide in preparation of immune-related medicines, health-care products, foods and the like.

Technical Field

The chai-dalbergia macrorrhiza is a wild large edible fungus under the special ecological environment condition of the chai-dalbergia basin in the Qinghai-Tibet plateau, and has the characteristics of huge fruiting body, strong stress resistance, underground fruiting, low-temperature fruiting and the like due to the growth in a severe environment. The chai dalwood fat mushroom has rich nutrient components, the fruiting body and the mycelium of the chai dalwood fat mushroom not only contain a large amount of nutrient components such as protein and mineral elements, but also have higher polysaccharide content, have better activities such as anti-anoxia and the like, and have the effects of improving immunity, resisting anoxia, resisting oxidation, resisting fatigue and the like after being eaten for a long time. Therefore, many researches on fruiting bodies and mycelia of chai-da-mu-tou mushroom are currently conducted in the industry, including separation and purification methods of polysaccharides, eating, functional applications and the like. However, the research generally focuses on fruiting bodies of the stropharia rugoso-annulata and extracellular polysaccharides of the mycelia, and the research on the immune activity of the intracellular polysaccharides of the stropharia rugoso-annulata mycelia is not reported. Meanwhile, the application of the functions is aimed at improving immunity (namely increasing immune activity), which inevitably causes the problem that the user generates over immunity in some aspects, thereby causing new health problems.

Disclosure of Invention

The invention aims to provide a new application of intracellular polysaccharide of chaenomeles speciosa mycelium.

Specifically, the invention firstly provides the application of the intracellular polysaccharide of the pleurotus ferulae mycelium in the preparation of the immunoregulation medicine.

Secondly, the invention also provides the application of the intracellular polysaccharide of the pleurotus ferulae mycelium in the preparation of the immunoregulation health-care product.

Thirdly, the invention also provides the application of the intracellular polysaccharide of the pleurotus ferulae mycelium in the preparation of immunoregulation food.

Furthermore, the intracellular polysaccharide of the pleurotus ferulae mycelium is polysaccharide extracted from the pleurotus ferulae mycelium cells, is convenient to extract and can be directly realized by applying the prior art, and is a glycoprotein compound obtained by hot water extraction, ethanol precipitation, deproteinization and decoloration of the chaulmus ferulae mycelium.

Furthermore, the immunoregulation is bidirectional regulation comprising improving the immunocompetence and reducing the immunocompetence so as to enable the immunocompetence of the human body to be optimally matched with the functions of the human body, avoid over-immunity and change the application that the conventional fruiting body of the stropharia rugoso-annulata and mycelium extracellular polysaccharide only improve the immunocompetence in a single direction.

The research of the invention shows that the intracellular polysaccharide of the mycelium of the chaenomeles giganteus can obviously improve the proliferation rate of the splenocytes of mice and promote macrophage strains RAW264.7 of the mice to secrete immune factors such as TNF-alpha, NO and the like in cell experiments, and the intracellular polysaccharide of the mycelium of the chaenomeles giganteus has good immune activity regulating function, but not simply improves the immune activity in a single direction, thereby preventing over-immunity. Animal experiments show that the intracellular polysaccharide of the pleurotus ferulae mycelium can obviously regulate the spleen and thymus indexes of immunosuppressive mice, improve the lymphocyte level of the immunosuppressive mice, relieve hyperfunction of macrophage phagocytosis activity of abdominal cavities of the mice with hyperfunction of the immunity and reduction of hyperfunction of lymphocyte proliferation capacity in vitro of the spleen, and the polysaccharide has better two-way immunoregulation effect. So that the compound can be used as safe and reliable immunoregulation medicine, health-care product or food.

Drawings

FIG. 1 is a statistical graph of the effect of ABSP on the viability of RAW264.7 cells;

FIG. 2 is a micrograph (200) of RAW264.7 cells;

FIG. 3 is a statistical graph of the effect of ABSP on the phagocytic capacity of RAW264.7 cells.

In FIGS. 1 and 3, Conrol is a blank control (cell culture fluid), other groups are treated with different concentrations of Agrocybe aegerita polysaccharide (ABSP) (e.g., 25, 50, 100, 200, 400, 600, 800, 1000. mu.g/mL), and LPS is a positive control (lipopolysaccharide 0.1. mu.g/mL).

Detailed Description

The invention is further illustrated by the following specific embodiments in conjunction with the accompanying drawings:

example 1 Effect of Agaricus blazei mycelium intracellular polysaccharide (ABSP) on the Activity proliferation potency of macrophages

The CCK-8 method is adopted to detect the influence of the polysaccharide on the activity of RAW264.7 cells. RAW264.7 cells were cultured in RPMI1640 medium containing 1% diabody (penicillin and streptomycin) and 10% fetal bovine serum. The culture conditions were 37 ℃ and 5% CO2, and the subculture was performed every other day. When the cells were frozen, the cells were frozen in 10% DMSO/RPMI1640 medium and stored in a liquid nitrogen tank. Thawing at 37 deg.C for 1min, centrifuging at 1000r/min for 5min, discarding supernatant, replacing fresh RPMI1640 culture solution, and culturing in carbon dioxide incubator. Collecting cells in logarithmic growth phase, adjusting the concentration of cell suspension to 5 × 10⁴cells/mL were plated in 96-well plates at 100. mu.L/well. The 96-well plate with the cells was placed in a carbon dioxide incubator and cultured at 37 ℃ under 5% CO2 for 24 hours. Discarding the supernatant, washing with PBS for 2 times, adding 100 μ L of polysaccharide (25, 50, 100, 200, 400, 800, 1000 μ g/mL) diluted with RPMI1640 culture solution at different concentrations, and adding cell culture medium only to the negative control group; and adding LPS with the final mass concentration of 1 mu g/mL into the positive control group, culturing for 24h, adding 10 mu L of CCK-8 solution into each hole, and incubating for 1h in an incubator. The absorbance of each well was measured at 450nm using a microplate reader. Cell viability was calculated according to the following formula:

Cell viability％＝(ODsample－ODblank/ODcontrol－ODblank)×100％

as shown in figure 1, ABSP (> 400. mu.g/mL) can significantly promote the proliferation of RAW264.7 cells after the cells are treated with different concentrations of Agrocybe aegerita mycelium intracellular polysaccharide (ABSP) for 24 h. The proliferation condition of macrophages is an important index for reflecting the immune dynamic of the organism, and the improvement of the proliferation capacity of the macrophages has positive influence on the immune system of the organism. When the body is infected, the immune system is activated, and macrophages can be recruited from blood to an infected part and then rapidly proliferate, or can be activated in situ at the infected part and rapidly proliferate, so that the anti-infection effect is exerted. ABSP can promote cell proliferation in a dose-dependent manner, which indicates that ABSP has the activity of enhancing the immune function of the body.

Example 2 Effect of ABSP on macrophage morphology

The morphology of the cell can directly reflect the activity of the cell, and the toxic effect of the drug on the cell can be further judged. The RAW264.7 cells were treated with ABSP for 24h and then observed microscopically, and the micrograph is shown in FIG. 2. Most cells of the control group are circular and oval, and a small part of cells have tentacles; in the ABSP administration group, RAW264.7 cells were significantly increased in volume, and all cells were tentacle-differentiated to present an irregular morphology. The change of macrophage morphology can cause the change of corresponding functions, and the activated macrophages have the characteristics of enlarged volume, increased intracellular enzymes, increased inclusion and the like, and have obviously improved immunoregulation function. After the macrophages are treated by ABSP, the cells still adhere to the wall perfectly without the phenomena of shrinkage and floating, and the cell density shows an increasing trend. Thus, ABSP has no toxic effect on 264.7 cells at the concentration and can promote cell proliferation. In addition, the cells also have the phenomena of volume increase, shape change into long fusiform or prismatic, pseudopodia elongation and the like. It can be concluded that ABSP has potential immunomodulatory activity.

Example 3 Effect of ABSP on macrophage phagocytic function

The influence on the phagocytic capacity of the RAW264.7 cells is detected by adopting a phagocytic neutral red experiment. Collecting cells in logarithmic growth phase, adjusting cell concentration to 5 × 10⁴cells/mL were plated in 96-well plates at 100. mu.L/well. The 96-well plate loaded with the cells was placed in a carbon dioxide incubator at 37 ℃ with 5% CO₂Culturing for 24h under the condition. After discarding the supernatant and washing with PBS 2 times, 150. mu.L of polysaccharide (50, 100, 200. mu.g/mL) diluted with RPMI1640 medium was added, and 150. mu.L of LPS (0.1. mu.g/mL) and medium were used as positive and blank controls, respectively. After 24h of culture, discarding the cell culture medium, adding 100. mu.L of 0.1% neutral red physiological saline solution into each well, continuing to culture for 4h, decanting the supernatant, washing with PBS solution pre-warmed at 37 ℃ for 3 times, adding 150. mu.L of cell lysate (glacial acetic acid: ethanol 1:1) into each well, standing at 37 ℃ for 1h, shaking uniformly, and measuring 540nm with microplate readerThe absorbance was measured.

Macrophages exert immunoregulation functions through phagocytosis in vivo, and after resting macrophages are stimulated by immunoregulation drugs, the phagocytosis functions of the resting macrophages are enhanced, so that the phagocytosis functions of the macrophages can be measured to evaluate the immune activity of the drugs. Phagocytic activity of RAW264.7 cells was measured by the neutral red method, and the effect of ABSP on the phagocytic function of RAW264.7 cells was evaluated by measuring OD540nm after treating the cells with different concentrations of ABSP for 24 h. As shown in fig. 3, ABSP can increase phagocytic ability of RAW264.7 cells, and with increasing concentration of ABSP, the phagocytic ability of cells is further increased. After ABSP treatment, the neutral erythrophagocytic rate of macrophages was significantly increased and was dose-dependent. Therefore, ABSP can increase the phagocytic capacity of macrophages, thereby improving the immune function of the body.

Example 4 Effect of ABSP on immunosuppressed mice

Randomly dividing mice into 4 groups (blank control group, immunosuppression model group, polysaccharide low-dose group and polysaccharide high-dose group), each group contains 5 mice (n is 5), preparing immunologic hypofunction model by continuous 5d subcutaneous injection of cyclophosphamide 80mg/kg for other groups except control group, and preparing low-dose group (10 mg. kg.) of Agaricus blazei polysaccharide^-1·d^-1) And the high-dose group of the boletus edulis polysaccharide (30mg kg)^-1·d^-1) The drug is administrated by a gastric lavage way, and a blank control group and an immunosuppression model group are administrated by normal saline according to the weight of 0.1mL/10g and are continuously administrated for 10 days.

After administration for 10 days, the mice in each group are blood-collected and anticoagulated, and then the hemogram of the mice is detected by a hemocytometer. And (3) fasting the animals for 12 hours before the last administration without water prohibition, taking the spleen and the thymus after each group of animals die 24 hours after the administration is stopped, weighing the spleen and the thymus by dampness, and calculating the index of the spleen and the thymus according to a formula.

Spleen index (mg) spleen weight/body weight (g) thymus index (mg) thymus weight/body weight (g)

In each group of mice, at the 3 rd administration, 2% sheep red blood cells are injected intraperitoneally, 0.2mL of sensitization is injected into each mouse, the thickness of the plantar part of the right hind foot is measured after 5 days, then 20% sheep red blood cells are injected subcutaneously into a measurement part, the thickness of the plantar part of the right hind foot is measured after 24 hours, the average value is obtained after three times of measurement, the thickness (mm) of the plantar increase is calculated, and the DTH degree is expressed by the thickness of the plantar increase.

Mice in each group were sensitized at 3d by intraperitoneal injection of 0.2mL of 2% SRBC. 24 hours after the last administration, the animals were sacrificed, the mice were bled from the eyeballs, the serum was separated, the serum was diluted 200 times with physiological saline, lmL diluted serum was placed in the test tube, 0.5mL of 10% sheep red blood cells and lmL complement (diluted with physiological saline at l: 10) were added in sequence, and a control tube without serum was provided (replaced with physiological saline). Placing in a constant temperature water bath kettle at 37 deg.C, maintaining the temperature for 15min, terminating the reaction in ice bath, and centrifuging at 2000r/min for l0 min. And (3) putting lmL parts of supernatant and 3mL parts of physiological saline in a test tube, putting 0.25mL part of 10% SRBC in another test tube, fully mixing, standing for l0min, and measuring the optical density value of each tube at 540 nm.

Half hemolysis value HC₅₀(OD value of sample/OD value at half hemolysis of SRBC) × dilution factor

As seen from Table 1, compared with the blank control group, the spleen index and the thymus index of the mice in the immunosuppression model group are lower than those of the mice in the blank control group, and the difference is obvious. Compared with the model control group, the spleen index and the thymus index of the immunosuppressed mice are obviously improved in the low-dose group and the high-dose group.

Table 1 effect of ABSP on mouse organ index (X ± S, n ═ 5)

Group of	Dosage/mg.kg	Spleen index	Index of thymus
				Control group	—	4.39±0.75	3.29±0.74
Model set	—	2.29±0.47##	0.98±0.85##
				Low dose group	10	6.42±1.59**	2.86±0.47**
High dose group	30	8.68±1.53**	3.48±0.15**

Note:^##comparison with control group, P<0.01；^**Comparison with model groups, P<0.01

Effect of ABSP on humoral immune function in mice: as can be seen from Table 2, the serum hemolysin value of the model group mice is obviously reduced (P is less than 0.01) compared with that of the blank control group, the humoral immunity function of the immunosuppressed mice can be improved after the polysaccharide is administrated, and the antibody content in the peripheral blood of the immunosuppressed mice can be recovered to the normal level by the high and low dose of the polysaccharide.

Table 2 effects of ABSP on mouse serum hemolysin (X ± S, n ═ 5)

Group of	Dosage/mg.kg	Relative value of hemolysin
			Control group	—	378.36±41.43
Model set	—	225.17±56.38##
			Low dose group	10	309.15±39.49**
High dose group	30	339.25±41.29**

Note:^##comparison with control group, P<0.01；^**Comparison with model groups, P<0.01

Effect of ABSP on immune function of mouse cells: as shown in Table 3, after subcutaneous cyclophosphamide injection, the delayed hypersensitivity of mice in the immunosuppression model group is obviously lower than that of mice in the blank control group, which indicates that cyclophosphamide has obvious inhibitory effect on the cellular immune function of mice. After the immunosuppressive mice are administrated, the delayed hypersensitivity capacity of the immunosuppressive mice is obviously higher than that of an immunosuppressive group, and the delayed hypersensitivity capacity of the immunosuppressive mice can be restored to a normal level by high dosage.

Table 3 effects of ABSP on mouse serum hemolysin (X ± S, n ═ 5)

Group of	Dosage/mg.kg	Thickness difference/mm of foot pad
			Control group	—	2.93±0.18
Model set	—	2.19±0.19##
			Low dose group	10	3.19±0.24**
High dose group	30	3.29±0.16**

Note:^##comparison with control group, P<0.01；^**Comparison with model groups, P<0.01

Example 5 Effect of ABSP on immunosuppressed mice

Taking 50 healthy Kunming male mice, randomly dividing into 5 groups of 10 mice each, and respectively making into normal control group, immunity enhancing model group, immunity enhancing + Hericium giganteum polysaccharide [ low (10 mg. kg. polysaccharide)^-1·d^-1) High (30mg kg)^-1·d^-1)]Dose groups. Intramuscular injection of BCG vaccine to each mouse once a daySaline solution (containing 5mg of BCG) was injected continuously for 11 days to create a model of immune hyperactivity. The mice in the normal control group and the hyperfimmunity model group are perfused with deionized water (20 ml. kg) for 1 time every day^-1) And (3) intragastrically irrigating the other groups of mice with the pleurotus ferulae polysaccharide solution with the corresponding dose for 1 time every day for 11 days continuously.

Detecting the phagocytic activity of macrophages: on the 10 th day of the experiment, 1mL of 6% sterile soluble starch solution was injected into the abdominal cavity of each group of mice to cause the inflammatory reaction of the abdominal wall of the mice, and to promote the migration of macrophages. Mice body mass was measured on the morning of experiment 11 days, and then gavaged. Injecting 0.5mL of 3% chicken erythrocyte solution into abdominal cavity after 1h, killing the mouse by dislocation of cervical vertebra after 1h, soaking for 2min by 75% ethanol, cutting off abdominal skin, lifting peritoneum by forceps, injecting 2mL of pre-cooled physiological saline by an injector, gently rubbing abdominal part for 1min, sucking abdominal cavity flushing fluid to prepare a smear, randomly observing 100 macrophages by an oil lens after dyeing by a Reye dyeing method, counting the number of the macrophages engulfed with the chicken erythrocyte and the number of the phagocytized chicken erythrocyte, and respectively calculating phagocytosis percentage and phagocytosis index according to a formula of 'the number of the macrophages with phagocytosis/the total number of the counted macrophages multiplied by 100%' and 'the phagocytosis index is the total number of the chicken erythrocytes engulfed by the macrophages with the phagocytosis/the total number of the counted macrophages with the phagocytosis'.

In vitro proliferation capacity assay of mouse spleen lymphocytes: sucking abdominal cavity flushing fluid, cutting off peritoneum, taking out spleen under aseptic condition, placing in an aseptic culture dish, removing fat and fascia tissues, rinsing with Hank fluid, then crushing and uniformly mixing with a 1mL injector core, filtering the obtained cell suspension with a 200-mesh cell sieve, placing the filtrate in a 2mL centrifuge tube, centrifuging at 1000r/min for 3min, removing supernatant, adding 1mL hemolysin into each tube, uniformly mixing, standing for 3min, centrifuging at 1000r/min for 3min, removing supernatant, centrifuging and washing spleen lymphocyte sediment for 2 times by RPMI culture solution 1640; finally, the collected spleen lymphocytes are placed in a glass culture bottle, and a part of the spleen lymphocytes are taken out and subjected to 0.5 percent trypan blue staining counting to adjust the cell number to 5 multiplied by 10⁶mL, and the number of viable cells should be greater than 95%. Adding the spleen lymphocyte fluid of the mouse into a 96-hole cell culture plate according to 100 uL/hole, repeating the steps for 3 spleen lymphocytes of each mouse,blank group only contains culture solution, control group contains cells and culture solution, and experimental group is added with 100 μ L Con A (2.5 μ g/mL); culturing the cell culture plate in an incubator at 37 deg.C and 5% CO2 for 68 hr, taking out, adding 50 μ L MTT working solution into each well, culturing for 4 hr, slightly sucking out supernatant of culture solution from each well, and discarding

Then 150 mu L of dimethyl sulfoxide is added into each hole to dissolve the MTT formazan precipitate, a micro oscillator is used for mixing uniformly, and the optical density value (OD 570nm) of each hole is read at the wavelength of 570nm of an enzyme labeling instrument. Stimulation indices were calculated to reflect the proliferation of splenic lymphocytes in each group: stimulation index (experiment OD value-blank OD value)/(control OD value-blank OD value)

TABLE 4 Effect of ABSP on hyperimmune mice

Group of	Dosage/mg.kg	Macrophage phagocytosis index	Lymphocyte proliferation index
				Control group	—	0.41±0.06	1.37±0.16
Hyperfunction model group	—	0.68±0.08##	1.92±0.21##
				Low dose group	10	0.57±0.09**	1.63±0.15**
High dose group	30	0.47±0.09**	1.48±0.18**

As can be seen from Table 4, compared with the normal control group, the phagocytic activity of the abdominal macrophages of the mice in the hyperimmune model group is remarkably increased, and the proliferative capacity of the splenic lymphocytes is remarkably increased. Compared with an hyperfimmunity model group, the mouse abdominal cavity macrophage phagocytosis activity and the splenic lymphocyte in-vitro proliferation ability of each dosage group of ABSP are reduced, and the hyperfunction of the abdominal cavity macrophage phagocytosis activity and the splenic lymphocyte in-vitro proliferation ability of the hyperfimmunity mouse are relieved, so that the polysaccharide has a better immune down-regulation effect on the hyperimmunity.

The present invention has been described in detail, and it should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention.