Application of russula griseocarnosa extract in preparation of medicine for inhibiting myeloid-derived suppressor cells

Abstract

The invention discloses an application of a russula griseocarnosa extract in preparation of a medicine for inhibiting myeloid-derived suppressor cells. The russula griseocarnosa extract is a russula griseocarnosa sporocarp polysaccharide extract, and the polysaccharide content of the russula griseocarnosa sporocarp polysaccharide extract is 30.41-60.12%. It is verified for the first time that the russula griseocarnosa extract can inhibit the spleen of a tumor-causing mouse from generating MDSCs, the proportion and number of MDSCs in blood and tumor tissue of the tumor-causing mouse are inhibited, and the content and activity of CD3 + T lymphocytes are improved, so that the immune response ability of an organism is improved. The compound is not only suitable for preparing anti-tumor drugs, but also suitable for preparing drugs for MDSCs related diseases such as inflammatory mediated tissue damage, infectious diseases and autoimmune diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of a gray-fleshed red mushroom extract in the preparation of myeloid-derived inhibitory cell inhibitory drugs. Background Technology

[0002] Myeloid-derived suppressor cells (MDSCs) are a highly heterogeneous group of immature myeloid cells produced by the myelopoietic pathway in the bone marrow and spleen, possessing strong immunosuppressive activity. Based on cell morphology and protein expression markers, MDSCs are divided into granulocytic or polymorphonuclear MDSCs (PMN-MDSCs) and monocytic MDSCs (M-MDSCs). In mice, MDSCs are defined as cells expressing CD11b and Gr-1, with Gr-1 containing two epitope antigens, Ly6G and Ly6C. M-MDSCs are defined as cells expressing CD11b. + Ly6ChiLy6G − PMN-MDSCs are defined as cells expressing CD11b. + Ly6CloLy6G + The cells. In humans, there is no expression of Ly6C or Ly6G; M-MDSC is defined as CD11b. + CD33 + CD14 + CD15 - HLA - DRlo / - PMN-MDSCs are defined as CD11b + CD33 + CD14 - CD15 + HLA - DR - Currently, a third type of MDSC has been discovered in the human body. Lacking the characteristics of monocytes or granulocytes, it may be a precursor to M-MDSCs and PMN-MDSCs, but further research is needed to fully understand or confirm this potential evolutionary relationship. This subgroup is defined as CD11b. + CD33 + CD14 - CD15 - HLA - DR - .

[0003] Studies have shown that macrophage-derived stem cells (MDSCs) proliferate extensively in the peripheral blood, spleen, and tumor tissue of patients with various types of cancer (such as breast cancer, lung cancer, colon cancer, liver cancer, prostate cancer, and head and neck cancer) or tumor-bearing mice, showing a positive correlation with tumor burden and a negative correlation with shorter survival and response to anti-tumor therapy. MDSCs have been proven to possess potent immunosuppressive activity. They suppress the innate and acquired immunity of the tumor-bearing organism through multiple pathways, including expressing immunosuppressive checkpoints such as PD-L1, CTLA-4, and CD155 to induce T cell dysfunction, consuming amino acids required by T cells, generating reactive nitrogen and reactive oxygen species to damage T cell function, inducing regulatory T cell (Treg) infiltration and proliferation, blocking NK cell cytotoxicity, and preventing T lymphocyte homing, thus creating an immunosuppressive environment and weakening immune function. In clinical animal model studies, various chemotherapeutic drugs such as 5-fluorouracil (5-FU) and gemcitabine significantly inhibit the number of MDSCs, and clinical research is currently underway to investigate their combination with PD-1 or PD-L1 inhibitors. Currently, the use of inhibitors targeting and inhibiting MDSCs, either alone or in combination with immune checkpoint inhibitors and chemotherapy drugs, is a very important approach and strategy in cancer clinical treatment. For example, gemcitabine, as a small molecule chemotherapy drug, clears CD3 along with MDSCs. + T cells. In patients or tumor-bearing mouse models, after MDSCs are eliminated, there are very few T cells in the body when immunotherapy is performed, and the treatment effect is generally poor. Therefore, it is of great significance and urgent to find drugs that eliminate MDSCs while preserving T lymphocytes.

[0004] Further research has revealed that MDSCs also proliferate and accumulate in large quantities at sites of inflammation and in lymphoid organs of various diseases, such as infections, autoimmune diseases, and inflammatory-mediated tissue damage, suppressing T cell responses and leading to immunosuppression. In psoriasis patients and psoriasis-like mouse models, MDSCs exhibit aggregated expansion, which is positively correlated with disease severity. However, tretinoin and polyethylene glycol ointment can improve symptoms by reducing the number of MDSCs and Th17 cells in mice. Studies have found that in hepatitis B virus infection, MDSCs inhibit T cell function in an arginase-dependent manner, which provides a new direction and target for viral therapy.

[0005] To date, there have been no reports of extracts from *Russula fulvidraco* acting as inhibitors of myeloid-derived suppressor cells. Summary of the Invention

[0006] To address the shortcomings and deficiencies of the existing technologies, the primary objective of this invention is to provide an application of *Russula fulvidraco* extract in the preparation of myeloid-derived inhibitory cell-suppressing drugs. This provides a new substance for biopharmaceutical manufacturing and promotes the development of the biopharmaceutical industry.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] The first objective of this invention is to provide the application of *Russula greifolia* extract in the preparation of myeloid-derived inhibitory cell inhibitory drugs. The *Russula greifolia* extract is a polysaccharide extract of the fruiting body of *Russula greifolia*, with a polysaccharide content of 30.41-60.12% by mass. The preparation method of the *Russula greifolia* extract includes the following steps: *Russula greifolia* fruiting bodies are pulverized and extracted with pure water at 90-110°C; the filtrate is collected by filtration; the residue is extracted again with pure water; the filtrates are combined and concentrated to obtain a concentrate; 0.5-4 times the volume of anhydrous ethanol is added to the concentrate, the mixture is allowed to stand, and centrifuged to obtain a precipitate; the precipitate is washed and dissolved in pure water at 65-70°C; after removing the ethanol, the precipitate is filtered to obtain a filtrate; the filtrate is dialyzed and then freeze-dried to obtain the *Russula greifolia* extract.

[0009] Preferably, the extract of *Russula fulvidraco* is used in the preparation of myeloid-derived inhibitory cell suppressants and CD3-promoting agents. + Application in drugs that increase the content and activity of T lymphocytes.

[0010] Polysaccharide extract from the fruiting body of *Russula fulvidracoides* inhibits myeloid-derived suppressor cells while increasing the body's CD3 levels. + The content and activity of T lymphocytes can enhance the body's immune response.

[0011] Preferably, the myeloid-derived suppressor cells are myeloid-derived suppressor cells from blood, spleen, and / or tumors, and the CD3+ cells are... + T lymphocytes are CD3+ cells found in the blood, spleen, and / or tumors. + T lymphocytes.

[0012] Preferably, the extract of *Russula fulvidraco* inhibits myeloid-derived suppressor cells by suppressing the production of myeloid-derived suppressor cells in the body, clearing peripheral blood myeloid-derived suppressor cells, or inhibiting the recruitment of myeloid-derived suppressor cells at tumor sites.

[0013] Preferably, the dosage of the *Russula fulvidraco* extract is 100-200 mg / kg / day.

[0014] Preferably, the pulverization refers to pulverizing to 20-60 mesh, and in the extraction and repeated extraction, the amount of pure water added is 1g:15-25mL based on the mass of the fruiting body or filter residue of the gray-fleshed red mushroom, the extraction or repeated extraction time is 1-3h, and the concentration is vacuum concentration to 10%-20% of the combined filtrate volume.

[0015] Preferably, the 0.5-4 times volume of anhydrous ethanol is 4 times volume of anhydrous ethanol, the standing refers to standing overnight at 4°C, the centrifugation refers to centrifugation at 8000 rpm for 15 min, the washing refers to washing with cold ethanol at 0~10°C, the filtration after ethanol removal uses a 0.45μm filter membrane, and the dialysis refers to dialysis for 48 h using a 3500Da dialysis bag.

[0016] Preferably, the specific steps of the preparation method of the *Russula purpurea* extract are as follows:

[0017] 1) Crush the fruiting bodies of *Russula fulvidraco* to 40 mesh, add 20 times the mass of the raw material to pure water and extract for 2 hours at a temperature of 90 to 110°C; filter the obtained extract, collect the filtrate, add 20 times the mass of pure water to the residue, heat to 90 to 110°C and extract for another 2 hours, filter the obtained extract, combine the filtrates obtained after each extraction, and concentrate under vacuum to obtain a concentrate with a volume of 15% of the combined filtrate.

[0018] 2) While stirring, add 0.67-4 times the volume of anhydrous ethanol to the concentrate, mix thoroughly, let stand overnight at 4℃, centrifuge at 8000rpm for 15min to obtain the precipitate, and wash thoroughly with cold ethanol at 0-10℃; redissolve the precipitate with pure water at 65-70℃, concentrate under vacuum at 48-52℃ to evaporate the ethanol, centrifuge, filter through a 0.45μm filter membrane to obtain the filtrate; dialyze the filtrate through a 3500Da dialysis bag for 48 hours, then concentrate under vacuum again and freeze dry to obtain the extract of *Russula fulvidraco*.

[0019] The second objective of this invention is to provide a myeloid-derived inhibitory cell inhibitory drug, comprising a *Russula fulvidracoides* extract, wherein the *Russula fulvidracoides* extract is a polysaccharide extract of the fruiting body of *Russula fulvidracoides*, and the polysaccharide content is 30.41~60.12% by mass.

[0020] The molecular weight range of the extract from *Russula purpurea* is 0.5 × 10⁻⁶. 4 ~200×10 4 Da.

[0021] Preferably, the myeloid-derived inhibitory cell suppressor is an antitumor drug, an inflammatory-mediated tissue damage treatment drug, an autoimmune disease treatment drug, or an infectious disease treatment drug.

[0022] Compared with the prior art, the beneficial effects of the present invention are:

[0023] The extract of *Russula fulvidraco* of this invention can inhibit the production of MDSCs in the spleen of tumor-inducing mice, suppress the proportion and number of MDSCs in the blood and tumor tissue of tumor-inducing mice, and increase CD3 levels. +This method increases the content and activity of T lymphocytes, thereby enhancing the body's immune response. It is applicable not only to the preparation of anti-tumor drugs but also to the preparation of drugs for MDSC-related diseases such as inflammatory-mediated tissue damage, infectious diseases, and autoimmune diseases. Attached Figure Description

[0024] Figure 1 This is a graph showing the effect of RGI80 extract from *Russula purpurea* in Example 2 on inhibiting tumor growth in a 4T1 xenograft mouse model.

[0025] Figure 2 The graph shows the flow cytometry results of the reduction in the proportion and number of MDSCs in the blood of mice in the treatment group by the extract of *Russula gracilis* in Example 2.

[0026] Figure 3 The graph shows the flow cytometry results of the reduction in the proportion and number of MDSCs in the spleen of mice in the treatment group by the extract of *Russula gracilis* in Example 2.

[0027] Figure 4 The graph shows the flow cytometry results of the reduction in the proportion and number of MDSCs in the tumor tissue of mice in the treatment group by the extract of *Russula fulvidraco* in Example 2.

[0028] Figure 5 The extract of *Russula purpurea* in Example 2 promotes CD3... + Flow cytometry results of the proportion and number of T cells in the blood circulation of mice in the treatment group.

[0029] Figure 6 The extract of *Russula purpurea* in Example 2 promotes CD3... + Flow cytometry results showing the proportion and number of T cells in the spleen, a peripheral lymphoid organ, of mice in the treatment group.

[0030] Figure 7 This is the standard curve for polysaccharides. Detailed Implementation

[0031] The present invention will now be described in further detail with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0032] Example 1: Preparation and polysaccharide content determination of *Russula purpurea* extract

[0033] 1. Preparation of extract from *Russula purpurea*

[0034] The fruiting bodies of *Russula fulvidraco* were pulverized to 40 mesh, and then extracted for 2 hours at 100°C with approximately 20 times the mass of the raw material using pure water. The resulting extract was filtered, and the filtrate was collected. Pure water solvent was added to the residue in approximately 20 times the mass of the residue, and the mixture was heated to 100°C for another 2 hours. The resulting extract was filtered, and the filtrates obtained from each extraction were combined and concentrated under vacuum to obtain a 15% concentrate.

[0035] Extract 1: While stirring, add 0.67 times the volume of anhydrous ethanol to the concentrate, mix thoroughly, and let stand overnight at 4°C. Centrifuge at 8000 rpm for 15 min to obtain the precipitate, and wash thoroughly with cold ethanol at 4°C. Redissolve the precipitate with pure water at 65-70°C, concentrate under vacuum at 50°C to evaporate the ethanol, centrifuge at 8000 rpm for 15 min, collect the supernatant, filter through a 0.45 μm filter membrane, and obtain the filtrate. Dialyze the filtrate using a dialysis bag (3500 Da) for 48 hours, then concentrate again under vacuum and freeze-dry to obtain the RGI-40 extract sample from *Russula fulvidraco*, for later use.

[0036] Extract 2: While stirring, add anhydrous ethanol (1 volume of concentrate) to the concentrate, mix thoroughly, and let stand overnight at 4°C. Centrifuge at 8000 rpm for 15 min to obtain the precipitate, and wash thoroughly with cold ethanol at 4°C. Redissolve the precipitate with pure water at 65-70°C, concentrate under vacuum at 50°C to evaporate the ethanol, centrifuge at 8000 rpm for 15 min, collect the supernatant, filter through a 0.45 μm filter membrane, and obtain the filtrate. Dialyze the filtrate using a dialysis bag (3500 Da) for 48 hours, then concentrate again under vacuum and freeze-dry to obtain RGI-50 of the *Russula ovata* extract, for later use.

[0037] Extract 3: While stirring, add 1.5 times the volume of anhydrous ethanol to the concentrate, mix thoroughly, and let stand overnight at 4°C. Centrifuge at 8000 rpm for 15 min to obtain the precipitate, and wash thoroughly with cold ethanol at 4°C. Redissolve the precipitate with pure water at 65-70°C, concentrate under vacuum at 50°C to evaporate the ethanol, centrifuge at 8000 rpm for 15 min, collect the supernatant, filter through a 0.45 μm filter membrane, and obtain the filtrate. Dialyze the filtrate using a dialysis bag (3500 Da) for 48 hours, then concentrate again under vacuum and freeze-dry to obtain the RGI-60 extract sample of *Russula fulvidraco*, for later use.

[0038] Extract 4: While stirring, add 2.33 times the volume of anhydrous ethanol to the concentrate, mix thoroughly, and let stand overnight at 4°C. Centrifuge at 8000 rpm for 15 min to obtain the precipitate, and wash thoroughly with cold ethanol at 4°C. Redissolve the precipitate with pure water at 65-70°C, concentrate under vacuum at 50°C to evaporate the ethanol, centrifuge at 8000 rpm for 15 min, collect the supernatant, filter through a 0.45 μm filter membrane, and obtain the filtrate. Dialyze the filtrate using a dialysis bag (3500 Da) for 48 hours, then concentrate again under vacuum and freeze-dry to obtain the RGI-70 extract sample of *Russula fulvidraco*, for later use.

[0039] Extract 5: While stirring, add 4 times the volume of anhydrous ethanol to the concentrate, mix thoroughly, and let stand overnight at 4°C. Centrifuge at 8000 rpm for 15 min to obtain the precipitate, and wash thoroughly with cold ethanol at 4°C. Redissolve the precipitate with pure water at 65-70°C, concentrate under vacuum at 50°C to evaporate the ethanol, centrifuge at 8000 rpm for 15 min, collect the supernatant, filter through a 0.45 μm filter membrane, and obtain the filtrate. Dialyze the filtrate using a dialysis bag (3500 Da) for 48 hours, then concentrate under vacuum again and freeze-dry to obtain the RGI-80 extract sample of *Russula fulvidraco*, for later use.

[0040] 2. Polysaccharide content determination

[0041] The content of crude polysaccharides in edible fungi was determined by the phenol-sulfuric acid method in accordance with the "Agricultural Industry Standard of the People's Republic of China NY / T 1676-2008".

[0042] 1) Dextran standard solution curve

[0043] Prepare concentrated sulfuric acid; 5% phenol solution (prepare fresh and handle in the dark); 0.1 mg / mL dextran standard solution.

[0044] Prepare standard glucose solutions of different concentrations. Add 1.0 mL of phenol solution, then quickly add 5.0 mL of sulfuric acid, let stand for 10 min, and then place the test tubes in a 30℃ water bath for 20 min. Measure the absorbance at 490 nm. Construct a standard curve with dextran mass concentration on the x-axis and absorbance values ​​on the y-axis. The polysaccharide standard curve is shown below. Figure 7 As shown.

[0045] 2) Sample determination

[0046] Prepare polysaccharide samples with concentrations of 0.05 mg / mL, 0.1 mg / mL, and 0.2 mg / mL, repeat the standard curve determination procedure, and measure the absorbance at 490 nm.

[0047] according to Figure 7The polysaccharide content was calculated using the formula. The results, shown in Table 1, indicate that the polysaccharide content of the RGI extract of *Russula grebea* ranged from 30.41% to 60.12% by mass.

[0048] Example 2:

[0049] This embodiment verifies that the RGI40 and RGI80 extracts of *Russula ovata* obtained in Example 1 can reduce the content of MDSCs in tumor-bearing mice and improve the tumor microenvironment. The *Russula ovata* extract was also measured to inhibit myeloid-derived suppressor cells and increase CD3+. + CD8 + Increase the number and activity of T cells to enhance the body's immune response.

[0050] 1 Experimental Methods

[0051] 1.1 Preparation of 4T1 mouse breast cancer cells

[0052] Four T1 cells were taken from the liquid nitrogen storage tank, revived, activated and cultured in DMEM medium containing 1% penicillin, 1% streptomycin and 10% fetal bovine serum (FBS) and cultured in a 37°C, 5% CO2 cell culture incubator until the logarithmic growth phase.

[0053] 1.2 Establishment of a 4T1 subcutaneous xenograft model

[0054] The mice were female BALB / c mice (5-7 weeks old, weighing 20±2g) obtained from the Guangdong Provincial Experimental Animal Center. They were acclimatized for one week before the experiment. The 4T1 cells in the logarithmic growth phase were digested, counted, and adjusted to a cell density of 5×10⁶. 5 Cells / mL, 100μL subcutaneously implanted into mice. One week after modeling, mice were randomly assigned to three groups (control model group, RGI40 group, and RGI80 group), with 10 mice in each group. A normal control group was also set up.

[0055] 1.3 Administration

[0056] The control group received only physiological saline (0.9% sodium chloride aqueous solution). RGI40 / RGI80 groups: RGI40 and RGI80 extracts of *Russula fulvidraco* were administered intraperitoneally every other day at a dose of 100 mg / kg / day. The treatment period was 4 weeks, and tumor length was recorded.

[0057] The mice were sacrificed the day after the last administration, and blood, spleen, and tumors were collected using heparin sodium tubules. The samples were preserved according to the experimental objectives described below.

[0058] 1.4 Flow cytometry detection of MDSCs and CD3 + CD8 + T cells

[0059] 1) Single-cell preparation of blood samples

[0060] After drug administration, peripheral blood was collected using a blood collection tube containing sodium heparin via ocular sampling. 60 μL of blood was collected, and 2 mL of erythrocyte lysis buffer (ACK) was added. The mixture was vortexed and incubated in the dark for 10 min. The cells were centrifuged at 350 × g, 4 °C for 5 min, the supernatant was discarded, and the cells were washed twice with 2 mL of PBS buffer, the supernatant was discarded again, and the cell pellet was obtained. The cells were resuspended in PBS buffer to obtain a single-cell suspension, which was then placed on ice for later use.

[0061] 2) Preparation of spleen cell single-cell suspension

[0062] After drug administration, the four groups of tumor-bearing mice were euthanized by cervical dislocation. The euthanized BalB / C mice were sterilized with alcohol in a laminar flow hood for 5 minutes. After removal, the spleen was cut along the midline of the abdomen, washed with pre-chilled PBS, and transferred to 1.5 mL centrifuge tubes containing RPMI 1640. The cells were then homogenized using a rubber homogenizer. The homogenate was filtered through a 70 μM cell filter, and the filter was washed with PBS. The cells were centrifuged at 350 g for 7 min, the supernatant was discarded, and the cells were collected as a pellet. The cells were resuspended in 2 mL of erythrocyte lysis buffer, homogenized, and lysed at room temperature for 10 min. The cells were then centrifuged at 350 g for 7 min, the supernatant was discarded, and the cells were resuspended in PBS and washed. The cells were then centrifuged at 350 g for 7 min, the supernatant was discarded, and the cells were resuspended in PBS buffer to obtain a single-cell suspension, which was then stored on ice for later use.

[0063] 3) Preparation of single-cell suspension of tumor tissue

[0064] After drug administration, the four groups of tumor-bearing mice were euthanized by cervical dislocation. The euthanized BalB / C mice were sterilized with alcohol in a laminar flow hood for 5 minutes. After removal, the tumor tissue was cut along the midline of the abdomen and cut into small pieces (approximately 2-4 mm) in ice-cold PBS. The tissue fragments were transferred to 5 mL of tissue digestion solution (1640 medium containing w / v 0.1% collagenase type I, 0.2% dispase type I, 1% DNase I stock) and incubated at 37°C for 30 min, vortexing every 5 min (continuous observation during this process until tissue dissociation). 10 mL of FACS buffer was added, and the mixture was filtered through a 70 μm cell filter, followed by rinsing the filter with 10-15 mL of FACS buffer. The mixture was centrifuged at 350 g for 5 min at 4°C, and the supernatant was discarded (gently poured out), retaining the pellet. 1 mL of erythrocyte lysis buffer was added, and the pellet was incubated at room temperature for 3 min. Immediately place the sample on ice and resuspend it in 10 mL of FACS buffer to neutralize the erythrocyte lysis buffer. Pipette the sample and then filter it through a 40 μm cell filter into a 50 mL tube. Centrifuge at 350 g for 5 min at 4 °C. Discard the supernatant (gently pour it out), resuspend the sample in PBS buffer to obtain a single-cell suspension, and place it on ice for later use.

[0065] 4) Flow cytometry

[0066] The spleen and tumor single-cell suspensions were centrifuged and resuspended in 100 μL PBS. Negative controls and single-positive staining controls were set up. The single-positive staining included cell surface markers of MDSCs and T cells: CD45, CD11b, Gr-1, CD3, and CD8. +The antibodies involved in the experiment included: Anti-mouse CD45-APC-eFluor 780 (30-F11) (Thermo Fisher Scientific, 47-0451-82), Anti-mouse CD11b-APC (M1 / 70) (Thermo Fisher Scientific, 17-0112-82), Anti-mouse CD3e-FITC (145-2C11) (Thermo Fisher Scientific, 11-0031-82), Anti-mouse GR-1-PE-Cyanine7 (RB6-8C5) (Thermo Fisher Scientific, 25-5931-82), Anti-mouse CD8-PE (53-6.7) (Thermo Fisher Scientific, 12-0081-82), and Anti-mouse CD16 / CD32 (BD, 553141).

[0067] Prepare a mixed antibody combination of 40 samples: Take 5 μL of Anti-mouse CD45-APC-eFluor 780, 20 μL of Anti-mouse CD3e-FITC, 10 μL of Anti-mouse CD8-PE, 5 μL of Anti-mouse CD11b-APC and 5 μL of Anti-mouse GR-1-PE-Cyanine7 and dilute them into 1000 μL of FACS buffer to prepare the antibody mixture. Place it on ice for later use.

[0068] The experimental steps are briefly described below:

[0069] (1) Resuspension 2-5×10 6 Cells were in 20 μL PBS.

[0070] (2) Add 25 μL of 7-ADD (BD, 559925) reagent, incubate on ice for 30 min, and protect from light.

[0071] (3) Add 100 μL of PBS to each tube, centrifuge at 350 g for 5 min at 4 °C, and gently pour out the supernatant.

[0072] (4) Add 25 μL of Anti-Mouse CD16 / CD32 dilution buffer (2 μL diluted to 23 μL FACS buffer), incubate on ice for 30 min, protected from light.

[0073] (5) Add 25 μL of antibody mixture and incubate on ice for 30 min in the dark.

[0074] (6) Add 100 μL of PBS to each tube, centrifuge at 350 g for 5 min at 4 °C, and gently pour out the supernatant.

[0075] (7) Add 100 μL of FACS buffer, transfer to a flow cytometry tube, add 300 μL of FACS buffer, and then perform the detection.

[0076] 4) Calculation and Analysis

[0077] The above data were calculated and analyzed using Prism 6.07 and FlowJo.

[0078] 2 Experimental Results

[0079] 2.1 RGI40 and RGI80 extracts from *Russula fulvidraco* inhibited tumor growth in 4T1 xenograft mouse models.

[0080] like Figure 1 As shown in Table 2, the experimental data indicate that, compared with the control group, *Russula ovata* extract effectively inhibited tumor growth in mice, demonstrating significant efficacy at a dose of 100 mg / kg / day administered every other day. Furthermore, this dose range is acceptable for humans.

[0081] 2.2 Extracts from *Russula fulvidraco* reduced the proportion of MDSCs cells in the blood, spleen, and tumors of tumor-bearing mice in the treatment group.

[0082] like Figure 2 As shown, flow cytometry analysis demonstrated that, compared with the control model group, the extract of *Russula greifolia* reduced the proportion and number of MDSCs in the blood of mice in the treatment group.

[0083] like Figure 3 As shown, flow cytometry analysis demonstrated that, compared with the control model group, the extract of *Russula gracilis* reduced the proportion and number of MDSCs in the spleen of mice in the treatment group.

[0084] like Figure 4 As shown, flow cytometry analysis demonstrated that, compared with the control model group, the extract of *Russula fulvidraco* reduced the proportion and number of MDSCs in the tumor tissues of the treated mice.

[0085] 2.3 Extracts from *Russula purpurea* increased CD3 levels in the blood and spleen of tumor-bearing mice in the treatment group. + T cell ratio

[0086] like Figure 5 As shown, flow cytometry analysis demonstrated that, compared to the control group, *Russula purpurea* extract promoted CD3 activation. + The proportion and number of T cells in the blood circulation of mice in the treatment group increased.

[0087] like Figure 6 As shown, flow cytometry analysis demonstrated that, compared to the control group, *Russula purpurea* extract promoted CD3 activation. + The proportion and number of T cells in the spleen, a peripheral lymphoid organ, increased in the treatment group mice.

[0088] The above results demonstrate that the extract of *Russula fulvidraco* of this invention can inhibit the production of MDSCs in the spleen of tumor-inducing mice, suppress the proportion and number of MDSCs in the blood and tumor tissue of tumor-inducing mice, and increase CD3 levels. + This method increases the content and activity of T lymphocytes, thereby enhancing the body's immune response. It is applicable not only to the preparation of anti-tumor drugs but also to the preparation of drugs for MDSC-related diseases such as inflammatory-mediated tissue damage, infectious diseases, and autoimmune diseases.

[0089] Table 1 Polysaccharide content sample Extraction rate / % Total sugar in extract / % RGI40 1.4 60.12 RGI50 1.9 50.87 RGI60 2.1 43.12 RGI70 2.9 33.68 RGI80 3.2 30.41

[0090] Polysaccharide extraction rate = polysaccharide mass / fruiting body dry weight × 100%.

[0091] Table 2. Inhibition rate of *Russula ovata* extract on tumor growth Group Tumor inhibition rate (%) Model group -- RGI40 37.2 RGI80 55.12

[0092] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Use of an extract of Russula griseocarnata in the preparation of a drug for inhibiting myeloid-derived suppressor cells, characterized in that, The grey-pulverized Russula extract is a Russula crassa fruiting body polysaccharide extract, and the polysaccharide content is 30.41-60.12% by mass. The preparation method of the Russula crassa extract comprises the following steps: crushing the Russula crassa fruiting body, adding pure water at a temperature of 90-110 ℃ to extract, filtering to collect the filtrate, adding pure water to the residue to repeat the extraction, combining the filtrates, concentrating to obtain a concentrated solution, adding 0.5-4 times the volume of anhydrous ethanol to the concentrated solution, standing, centrifuging to obtain a precipitate, washing the precipitate, dissolving the precipitate in 65-70 ℃ pure water, filtering after removing the ethanol, dialyzing the filtrate, and freeze-drying to obtain the Russula crassa extract.

2. Use according to claim 1, characterized in that, For the preparation of myeloid-derived suppressor cell inhibition and promotion of CD3 + The use of the drug with increased T lymphocyte content and activity.

3. Use according to claim 2, characterized in that, The myeloid-derived suppressor cells are myeloid-derived suppressor cells in the blood, spleen and / or tumor, the CD3 + T lymphocytes are CD3 + T lymphocytes in the blood, spleen and / or tumor.

4. Use according to claim 1 or 2, characterized in that, The Russula crassa extract can inhibit the production of myeloid-derived suppressor cells in the body, remove peripheral blood myeloid-derived suppressor cells, or inhibit the recruitment of myeloid-derived suppressor cells at the tumor site.

5. Use according to claim 1 or 2, characterized in that, The dosage of the Russula crassa extract is 100-200 mg / kg / day.

6. Use according to claim 1, characterized in that, The crushing refers to crushing to 20-60 mesh. In the extraction and the repeated extraction, the amount of pure water added is 1 g:15-25 mL of the material liquid ratio based on the mass of the Russula crassa fruiting body or the residue, and the extraction or repeated extraction time is 1-3 h. The concentration is vacuum concentration to 10%-20% of the volume of the combined filtrate.

7. The use according to claim 1, characterized in that, The 0.5-4 times the volume of anhydrous ethanol is 4 times the volume of anhydrous ethanol. The standing refers to standing overnight at 4 ℃. The centrifuging refers to centrifuging at 8000 rpm for 15 min. The washing refers to washing with 0-10 ℃ cold ethanol. The filtering after removing the ethanol refers to filtering with a 0.45 μm filter membrane. The dialysis refers to dialysis for 48 h with a 3500 Da dialysis bag.

8. The use according to claim 1, characterized in that, The specific steps of the preparation method of the Russula crassa extract are as follows: 1) crushing the Russula crassa fruiting body to 40 mesh, adding 20 times the mass of the raw material to extract for 2 hours at a temperature of 90-110 ℃, filtering the obtained extract to collect the filtrate, adding 20 times the mass of the raw material to the residue to extract for 2 hours at a temperature of 90-110 ℃, filtering the obtained extract, combining the filtrates obtained after each extraction, and vacuum concentrating to obtain a concentrated solution with a volume of 15% of the combined filtrate; 2) adding 0.67-4 times the volume of anhydrous ethanol to the concentrated solution while stirring, fully mixing, standing overnight at 4 ℃, centrifuging at 8000 rpm for 15 min to obtain a precipitate, washing the precipitate with 0-10 ℃ cold ethanol, redissolving the precipitate in 65-70 ℃ pure water, vacuum concentrating to evaporate the ethanol at a temperature of 48-52 ℃, centrifuging, filtering with a 0.45 μm filter membrane to obtain a filtrate, dialyzing the filtrate with a 3500 Da dialysis bag for 48 h, vacuum concentrating and freeze-drying to obtain the Russula crassa extract.

9. A myeloid-derived suppressor cell inhibiting drug, characterized by, The Russula crassa extract is a Russula crassa fruiting body polysaccharide extract, and the polysaccharide content is 30.41-60.12% by mass.

10. The medicament according to claim 9, characterized in that, The myeloid-derived suppressor cell inhibiting drug is an anti-tumor drug, an inflammatory-mediated tissue damage treatment drug, an autoimmune disease treatment drug, or an infectious disease treatment drug.

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