New application of moss phenol glucoside

Abstract

The invention belongs to the technical field of medicines, and particularly discloses application of moss phenol glucoside in preparation of medicines for preventing and / or treating tumors. According to the method, activated CD8 + T cells, MC38-OVA cells and traditional Chinese medicine monomeric compounds are co-cultured, the traditional Chinese medicine monomeric compounds capable of effectively promoting the anti-tumor activity of the CD8 + T cells are selected by utilizing a high-content screening technology, and finally, the experiment is carried out by taking the moss phenol glucoside as a research object. In-vitro experiment results show that the moss phenol glucoside can significantly promote T cell activation, promote release of cell factors such as IFN-gamma and TNF-alpha, and enhance the killing effect of T cells on tumors. A mouse in-vivo tumor-bearing experiment result shows that the moss phenol glucoside can promote the anti-tumor activity of T cells so as to inhibit tumor growth, has no obvious toxic or side effect, can be used for preparing the medicine for preventing and / or treating tumors, and has a potential clinical application prospect and a relatively high economic value.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to the use of orcinol glucoside in preparing medicines for preventing and / or treating tumors. Background Art

[0002] Malignant tumors are a major global health problem, and their incidence and mortality rates continue to rise. In my country, the incidence and mortality rates of colorectal cancer are both on the rise, and have become the focus of medical research and clinical practice. In recent years, immunotherapy has gradually become a research hotspot due to its forward-looking and specific nature. Immunotherapy is divided into two types: specific and non-specific. The former enhances the immune system's attack on tumors by activating or inhibiting clear targets, while the latter improves the body's immune capacity as a whole. However, tumor cells have mechanisms to evade immune killing, such as regulating antigen expression, reducing the expression level of HLA-I molecules, and changing antigen processing and presentation mechanisms. In order to induce an effective anti-tumor response, it is necessary to ensure that tumor antigens are absorbed by dendritic cells and cross-presented to CD8 + T cells, and antigens must also be directly presented by the tumor in order to be recognized and killed by specific T cells. However, tumors often use a variety of escape mechanisms to circumvent these two key steps. Therefore, enhancing the antigen presentation function of tumor cells is the focus of current immunotherapy.

[0003] T cell activation is crucial in cancer treatment. It is not only key to initiating anti-tumor immune responses but also possesses the ability to kill and eliminate tumor cells. Activated T cells can release cytotoxins or induce programmed cell death to eliminate tumor cells, a crucial mechanism in cancer treatment. Furthermore, activated T cells can form memory T cells, which persist in the body for a long time and are reactivated when cancer recurs. This immune memory effect also helps prevent cancer recurrence. Furthermore, activated T cells can overcome tumor escape mechanisms and effectively respond to tumor cells that evade the immune system.

[0004] Currently, colorectal cancer treatment primarily relies on surgery, supplemented by chemotherapy and immunotherapy, with the goal of improving surgical resection rates, reducing recurrence rates, and increasing survival rates. However, clinically, the five-year survival rate for colorectal cancer has not significantly improved. Monomer compounds in traditional Chinese medicine (TCM) are single chemical components meticulously extracted and purified from complex TCM systems and represent a significant achievement in the modernization of TCM. These compounds not only retain the active ingredients of TCM but also possess well-defined chemical structures and significant biological activity, paving the way for the internationalization and standardization of TCM. In the field of anti-tumor drugs, TCM monomer compounds have demonstrated unique advantages. For example, artemisinin, extracted from Artemisia annua, has become a globally recognized antimalarial drug and has also attracted considerable attention for its anti-tumor activity. Monomers in TCM, such as paclitaxel and camptothecin, have also demonstrated excellent results in the treatment of various cancers and have become clinically important anti-cancer drugs. Looking ahead, the development prospects of TCM monomer compounds in the anti-tumor field are promising. With the continuous advancement of modern science and technology, the in-depth exploration of TCM resources, and the accelerated integration of multiple disciplines, further breakthroughs in the research of TCM monomer compounds in the anti-tumor field are expected. At the same time, the combined application of Chinese medicine monomer compounds and Western medicine will also become a new research hotspot, providing cancer patients with more and more effective treatment options.

[0005] To date, researchers' focus on Traditional Chinese Medicine (TCM) treatments has primarily focused on the anti-tumor effects of TCM monomers themselves, often as an adjunct to Western medicine, to alleviate symptoms, enhance immune function, and reduce side effects. However, relatively little research has been conducted on the role of TCM monomers in immunotherapy. We hope to identify one or more TCM monomers that could play a role in immunotherapy, not only promoting surface antigen presentation on tumor cells, enabling better recognition by T cells and their immune response, but also effectively activating and enhancing T cell immune activity. This would provide new insights and directions for the treatment of malignant tumors, combining the wisdom of traditional Chinese medicine with advances in modern immunology to bring greater hope and possibilities to cancer patients. Summary of the Invention

[0006] The main technical problem solved by the present invention is to provide a drug for inhibiting tumor growth by activating and promoting the anti-tumor activity of T cells, which contains a pharmaceutically effective amount of orcinol glucoside.

[0007] At the same time, the present invention provides a new application of orcinol glucoside.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0009] The new use of orcinol glucoside is specifically: the use of orcinol glucoside in the preparation of drugs for preventing and / or treating tumors.

[0010] Orcinol glucoside is also known as orcinol glucoside, molecular formula: C 13 H 18 O7, molecular weight: 286.28, CAS registration number: 21082-33-7, chemical structure is as follows:

[0011]

[0012] As a preferred embodiment of the present invention, orcinol glucoside is used in the preparation of a drug for inhibiting tumor growth by activating and promoting the anti-tumor activity of T cells.

[0013] As a preferred embodiment of the present invention, the application includes but is not limited to one or more of the following aspects:

[0014] (1) Application of orcinol glucoside in the preparation of drugs for promoting T cell proliferation and activation;

[0015] (2) Application of orcinol glucoside in the preparation of drugs for promoting T cell cytokine release;

[0016] (3) Application of orcinol glucoside in the preparation of drugs that promote T cells to kill tumor cells.

[0017] Specifically, the cytokines are mainly factors that can directly kill tumor cells, including but not limited to IFN-γ, TNF-α, etc.

[0018] As a preferred embodiment of the present invention, the application includes but is not limited to one or more of the following aspects:

[0019] (1) Application of orcinol glucoside in the preparation of drugs that promote immune cell infiltration into tumor tissues;

[0020] (2) Application of orcinol glucoside in the preparation of drugs for activating the body's anti-tumor immune response;

[0021] (3) Application of orcinol glucoside in the preparation of drugs for inhibiting tumor growth.

[0022] Specifically, the immune cells include but are not limited to CD8 + T cells.

[0023] Specifically, the promotion of immune cell infiltration into tumor tissue includes but is not limited to increasing the number of immune cells (such as CD8 + T cells) ratio.

[0024] Specifically, the inhibition of tumor growth includes but is not limited to reducing tumor volume, reducing tumor weight, etc.

[0025] Specifically, the tumors include but are not limited to colorectal cancer, endometrial cancer, hepatocellular carcinoma, pancreatic cancer, lung adenocarcinoma, esophageal cancer, breast cancer, low-grade glioma, renal papillary cell carcinoma, nodal Parisa myofibroblastoma, neuromuscular aberrant tumor, desmoid tumor, juvenile angiofibroma, enamel-type craniopharyngiocarcinoma, hepatoblastoma, pancreatic ductal carcinoma, biliary tract cancer, cerebral hemangioma, Merkel cell carcinoma of the skin, squamous cell carcinoma of the skin, melanoma in situ, basal cell carcinoma of the skin, etc.

[0026] A medicine for preventing and / or treating tumors, wherein the effective component of the medicine comprises orcinol glucoside.

[0027] As a preferred embodiment of the present invention, the content of orcinol glucoside in the drug is a pharmaceutically effective amount, and can be any value within the range of 0.01 wt% to 99.99 wt%.

[0028] As a preferred embodiment of the present invention, the drug further comprises other pharmacological ingredients, including but not limited to other anti-tumor or auxiliary anti-tumor pharmacological ingredients, for combined therapy or auxiliary therapy.

[0029] As a preferred embodiment of the present invention, the drug further comprises a pharmaceutically acceptable carrier, including but not limited to solvents, excipients, preservatives, stabilizers, emulsifiers, salts for regulating osmotic pressure, buffers, and the like.

[0030] As a preferred embodiment of the present invention, the dosage form of the drug is a pharmaceutically acceptable dosage form, including but not limited to powder injection, injection, tablet, pill, capsule, spray, dispersion, etc.

[0031] As a preferred embodiment of the present invention, the administration route of the drug is a pharmaceutically acceptable route, including but not limited to oral, intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, and transdermal, intranasal or oral inhalation.

[0032] As a preferred embodiment of the present invention, the dosage of the drug is a pharmaceutically acceptable dosage.

[0033] Beneficial effects of the present invention:

[0034] The present invention selects mouse colorectal cancer MC38 cell line and expresses OVA 257-264 The peptide sequence was transfected into MC38 cells to form an MC38 cell line that can stably express the surface pattern antigen OVA. Then, 1280 Chinese medicine monomer compounds were screened at 2μM using this cell line to exclude monomer compounds that had obvious inhibitory or lethal effects on the growth of the cell line. Then, spleen cells were taken from the spleen of OT-1 mice and CD8+ T cells, activated with IL-2, to obtain cells that can recognize OVA 257-264 Peptide fragments and activated CD8 + T cells, which then activate CD8 + T cells were co-cultured with MC38-OVA cells and the remaining Chinese medicine monomer compounds, and high-content screening technology was used to select the compounds that could effectively promote CD8 + A team of researchers investigated the effects of traditional Chinese medicine monomer compounds on T cell anti-tumor activity. Six compounds with significant T cell anti-tumor activity were selected from a library of 1,280 TCM monomer compounds. Orcinol glucoside was chosen as the research subject for in vitro and in vivo experiments. In vitro results demonstrated that orcinol glucoside significantly promoted T cell activation and the release of cytokines such as IFN-γ and TNF-α, thereby enhancing T cell anti-tumor activity. In vivo tumor-bearing mouse studies demonstrated that orcinol glucoside promoted T cell anti-tumor activity and subsequently inhibited tumor growth, including reductions in tumor volume and weight, without significant side effects, demonstrating a high safety profile. Furthermore, previous studies have revealed the pharmacokinetics and pharmacological activities of orcinol glucoside, which is absorbed unchanged from the intestine into the bloodstream, with specific distribution in renal tissue. Absorption is significantly increased during states of deficiency and coldness. Orcinol glucoside also exhibits antioxidant, anti-osteoporotic, antidepressant, and antianxiety properties. In summary, orcinol glucoside can be used to prepare drugs that inhibit tumor growth by activating and promoting the anti-tumor activity of T cells, and has potential clinical application prospects and high economic value.

[0035] The drug for preventing and / or treating tumors provided by the present invention comprises a pharmaceutically effective amount of orcinol glucoside and a pharmaceutically acceptable carrier or excipient, and can be prepared into a pharmaceutical dosage form that is convenient for clinical use and can exert the best therapeutic effect, and has certain pharmaceutical prospects and commercial value. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 These are the in vitro experimental results in the experimental example.

[0037] Figure 2 These are the in vivo experimental results in the experimental examples.

[0038] In the accompanying drawings, the significance analysis symbols include * indicating p < 0.05, ** indicating p < 0.01, and *** indicating p < 0.001.

[0039] To more clearly illustrate the technical solutions to be protected by the present invention, the drawings in the embodiments or experimental examples are briefly introduced above. It should be understood that the above drawings should not be construed as limiting the scope of protection of the present invention. Those skilled in the art can also derive other relevant drawings based on these drawings without inventive effort. DETAILED DESCRIPTION

[0040] The technical solution of the present invention will be clearly and completely explained below with reference to specific embodiments and experimental examples.

[0041] Those skilled in the art should understand that the following embodiments and experimental examples are intended only to illustrate the technical solutions and effects of the present invention and should not be construed as limiting the scope of protection of the present invention. Based on the following embodiments, other technical solutions obtained by those skilled in the art without inventive effort, such as those obtained through modification, deformation, or simple substitution, are also within the scope of protection of the present invention.

[0042] Unless otherwise specified, the raw materials, reagents, and equipment used in the examples or experimental examples are all commercially available products.

[0043] Example 1

[0044] This embodiment provides a new use of orcinol glucoside, specifically: use of orcinol glucoside in the preparation of a drug for preventing and / or treating tumors. Specifically, orcinol glucoside inhibits tumor growth by activating and promoting the anti-tumor activity of T cells.

[0045] Orcinol glucoside is also known as orcinol glucoside, molecular formula: C 13 H 18 O7, molecular weight: 286.28, CAS registration number: 21082-33-7, chemical structure is as follows:

[0046]

[0047] Specifically, the tumors include but are not limited to colorectal cancer, endometrial cancer, hepatocellular carcinoma, pancreatic cancer, lung adenocarcinoma, esophageal cancer, breast cancer, low-grade glioma, renal papillary cell carcinoma, nodal Parisa myofibroblastoma, neuromuscular aberrant tumor, desmoid tumor, juvenile angiofibroma, enamel-type craniopharyngiocarcinoma, hepatoblastoma, pancreatic ductal carcinoma, biliary tract cancer, cerebral hemangioma, Merkel cell carcinoma of the skin, squamous cell carcinoma of the skin, melanoma in situ, basal cell carcinoma of the skin, etc.

[0048] Example 2

[0049] This embodiment provides a new use of orcinol glucoside, including but not limited to one or more of the following aspects:

[0050] (1) Application of orcinol glucoside in the preparation of drugs for promoting T cell proliferation and activation;

[0051] (2) Application of orcinol glucoside in the preparation of drugs for promoting T cell cytokine release;

[0052] (3) Application of orcinol glucoside in the preparation of drugs that promote T cells to kill tumor cells.

[0053] Specifically, the cytokines are mainly factors that can directly kill tumor cells, including but not limited to IFN-γ, TNF-α, etc.

[0054] Example 3

[0055] This embodiment provides a new use of orcinol glucoside, including but not limited to one or more of the following aspects:

[0056] (1) Application of orcinol glucoside in the preparation of drugs that promote immune cell infiltration into tumor tissues;

[0057] (2) Application of orcinol glucoside in the preparation of drugs for activating the body's anti-tumor immune response;

[0058] (3) Application of orcinol glucoside in the preparation of drugs for inhibiting tumor growth.

[0059] Specifically, the immune cells include but are not limited to CD8 + T cells.

[0060] Specifically, the promotion of immune cell infiltration into tumor tissue includes but is not limited to increasing the number of immune cells (such as CD8 + T cells) ratio.

[0061] Specifically, the inhibition of tumor growth includes but is not limited to reducing tumor volume, reducing tumor weight, etc.

[0062] Example 4

[0063] This embodiment provides a drug for preventing and / or treating tumors, comprising a pharmaceutically effective amount of orcinol glucoside, and a pharmaceutically acceptable oil-soluble solvent, a stabilizer, and a preservative, for preparing the drug into an injection dosage form.

[0064] In other embodiments of the present invention, the drug further comprises other medicinal ingredients for combined treatment or adjuvant treatment of colorectal cancer.

[0065] In other embodiments of the present invention, the dosage form of the drug is other pharmaceutically acceptable dosage forms, such as capsules, tablets, etc., which contain other pharmaceutically acceptable carriers.

[0066] In other embodiments of the present invention, the dosage of the drug is a pharmaceutically acceptable dosage, and the usage and dosage of the drug should be determined according to the condition of the disease and can be followed according to the doctor's advice.

[0067] Experimental example

[0068] 1. Compound Screening

[0069] First, the mouse colorectal cancer MC38 cell line was selected and the CD8 + OVA peptides that can be specifically recognized by T cells (i.e., OVA 257-264 The sequence of peptide fragment was transfected into MC38 cells to form an MC38 cell line that can stably express the surface pattern antigen OVA, namely the MC38-OVA cell line. 1280 Chinese herbal medicine monomer compounds were screened at 2μM using this cell line, and 42 Chinese herbal medicine monomer compounds were screened out to have a significant inhibitory or lethal effect on the growth of the MC38-OVA cell line. These 42 compounds were subsequently excluded. Splenocytes were taken from the spleen of OT-1 mice, and CD8 + T cells, using IL (interleukin)-2 to activate T lymphocytes, thereby obtaining activated CD8 + T cells. + T cells were co-cultured with MC38-OVA cells and different Chinese medicine monomer compounds, and high-content screening was used to select the compounds that could effectively promote CD8 + A Chinese herbal monomer compound with T cell anti-tumor activity was selected. Six Chinese herbal monomer compounds that significantly promoted T cell anti-tumor activity were ultimately screened from a library of 1,280 Chinese herbal monomer compounds. After literature screening and repeated experimental verification, the Chinese herbal monomer compound orcinol glucoside was selected as the research subject for subsequent experiments. Screening experiments demonstrated that orcinol glucoside could promote T cell anti-tumor activity at a concentration of 2 μM and had no effect on tumor cell growth at this concentration. Figure 1 A shows the status of cells under co-culture conditions.

[0070] 2. In vitro experiments

[0071] 1. CFSE detection of T cell proliferation

[0072] (1) Isolation and activation of T cells: The isolated CD8 +T cells were divided into three groups for activation. Two groups received 0.5 μg / mL of anti-mouse CD3 antibody, 0.5 μg / mL of anti-mouse CD28 antibody, and 100 ng / mL of mouse IL-2 to fully activate the T cells. The remaining group served as a control group and received only 100 ng / mL of mouse IL-2 to evaluate the T cell status under the influence of IL-2 alone.

[0073] (2) Collection and pretreatment of T cells: After 48 hours of activation culture, the three groups of T cells were collected into 15 mL centrifuge tubes and centrifuged at 2000 rpm for 5 minutes to precipitate the cells. Under light-proof conditions, CFSE (carboxyfluorescein diacetate succinimidyl ester) labeling solution was added to the cell pellet, and the cells were then placed in a 37°C environment and incubated in the dark for 10 minutes to label the activated T cells. Afterwards, about 10 mL of complete cell culture medium (containing serum) was immediately added to the centrifuge tube, and the CFSE labeling reaction was terminated by gently inverting and mixing, and the tube was centrifuged again at 2000 rpm for 5 minutes to remove excess CFSE.

[0074] (3) Treatment and culture after CFSE labeling: Add 8 mL (5-10 mL) of complete cell culture medium to the cell pellet after centrifugation and incubate at 37°C for 5 min to promote the stable retention of CFSE in the cells and allow unreacted CFSE to enter the culture medium.

[0075] (4) The cells were washed twice with complete culture medium to remove residual CFSE and other impurities. Afterwards, orcinol glucoside was added to one of the two groups activated with antibodies and interleukins at a final concentration of 2 μM, while the remaining two groups remained as is or added with corresponding solvent controls. Finally, all three groups of cells were placed in an incubator and cultured for 5 days in the dark. Flow cytometry was used to detect the proliferation of T cells after the addition of orcinol glucoside, and the results were as follows: Figure 1 shown.

[0076] from Figure 1 B shows that after adding 2 μM of orcinol glucoside to the T cell culture system, the proliferation of T cells was significantly accelerated, which indicates that orcinol glucoside has a direct proliferation-promoting effect on T cells.

[0077] 2. Clone formation experiment

[0078] (1) Cell preparation and inoculation: MC38-OVA cells in the logarithmic proliferation phase were selected for digestion to ensure that the cells were evenly dispersed and viable. Subsequently, the cells were accurately counted and inoculated into 6-well cell culture plates at a density of 1000 cells per well. Experimental and control groups were set up. Orcinol glucoside was added to each well of the experimental group and its working concentration was adjusted to 2 μM, while an equal volume of DMSO solution was added to each well of the control group to simulate the effect of the solvent in the experimental group.

[0079] (2) Cell culture and treatment: Place the inoculated 6-well plate under appropriate cell culture conditions for 10 days. After the cells have fully grown and may form clonal colonies, carefully remove the culture medium and supernatant from each well to remove non-adherent cells and metabolic waste. Then, gently wash the cells three times with pre-chilled PBS buffer.

[0080] (3) Cell fixation: Add 1 mL of 4% paraformaldehyde solution to each well and let it stand at room temperature for 20 min.

[0081] (4) Cell staining: After fixation, thoroughly wash away the paraformaldehyde solution again with PBS buffer. Subsequently, add an appropriate amount of crystal violet staining solution to each well to clearly visualize the morphology of the cell clones. After staining at room temperature for 5 minutes, rinse the cells several times with PBS buffer until the excess staining solution is completely removed.

[0082] (5) Photography, statistics and graphing: Use a high-resolution scanner to take pictures of the cell clones in each well, use ImageJ to count and calculate the number of clones formed in the pictures, and the obtained data is used for subsequent data analysis. GraphPad is used to draw a graph to intuitively show the difference in clone formation ability between the experimental group and the control group. The results are shown in Figure 2. Figure 1 shown.

[0083] from Figure 1 As shown in Figure C, after the MC38-OVA cell line was exposed to 2 μM orcinol glucoside, the number of colonies formed did not show a significant difference compared to the control group (DMSO alone was added as a solvent control). This finding directly indicates that at the tested concentration, orcinol glucoside did not inhibit or promote the proliferation of MC38-OVA tumor cells, that is, orcinol glucoside itself did not have a significant effect on the proliferation of this cell line.

[0084] 3. Flow cytometry detection of CD8 + T cell intracellular factors

[0085] (1) CD8 + The density of T cells was precisely adjusted to 1×10 6cell / mL, add the activator combination: 0.5μg / mL anti-mouse CD3, 0.5μg / mL anti-mouse CD28 and 100ng / mL mouse IL-2 to the cell culture system to stimulate and activate CD8 + T cells. Orcinol glucoside was added to a final concentration of 2 μM, and the activated T cells were treated with the drug for 48 hours.

[0086] (2) Add protein transport inhibitors to each well to a final concentration of 1 μg / mL, and then return the cells to the incubator for a further 4 h.

[0087] (3) Anti-mouse CD8 BV605 antibody was added to the cell suspension, incubated on ice in the dark for 30 min, then 1 mL of PBS was added and centrifuged at 2000 rpm for 5 min.

[0088] (4) The cells obtained by centrifugation were treated with 200 μL of fixative for 30 min, and then 800 μL of membrane permeabilization agent was added for membrane permeabilization. After membrane permeabilization, the cells were centrifuged again at 2000 rpm for 5 min.

[0089] (5) Intracellular factor staining: Use a membrane permeabilization agent to dilute intracellular factor antibodies such as IFN-γ and TNF-α, and add the diluted antibodies to the fixed and permeabilized cells, and incubate on ice in the dark for 30 minutes.

[0090] (6) Detect the stained cells using flow cytometry and analyze CD8 + The expression of T cell surface markers and the content of IFN-γ and TNF-α factors in cells were analyzed. Figure 1 shown.

[0091] from Figure 1 D shows that orcinol glucoside treatment can significantly promote CD8 + T cells release IFN-γ and TNF-α factors, which enhance the ability of T cells to release key intracellular effector molecules.

[0092] 4. Co-culture system to explore the immune killing effect of compounds with similar structures to traditional Chinese medicine monomers on tumor cells

[0093] (1) Compound screening strategy: By searching the compound library and reviewing relevant literature, we identified Chinese herbal monomer compounds with similar structural features (i.e., sharing a common parent ring) to orcinol glucoside. After rigorous comparison and screening, we finally selected 6 Chinese herbal monomer compounds, including pyroside, homoarbutin, salirepin, citrusin C, tachioside, and α-arbutin, for the experiment. All of them showed significant structural similarity to orcinol glucoside.

[0094] (2) Cell culture and inoculation: MC38-OVA cells in the logarithmic proliferation phase were selected as experimental subjects and digested to ensure uniform cell dispersion and high cell viability. Subsequently, using precise cell counting methods, cells were evenly inoculated into 6-well cell culture plates at a density of 300,000 cells per well for cell culture.

[0095] (3)CD8 + Isolation and in vitro activation of T cells: OT-1 mice were sacrificed by spinal dislocation, spleen cells were collected and ground, and red blood cell lysis buffer was added to obtain a pure cell suspension. + The T cell isolation kit successfully isolates T cells from a cell suspension, and then activates them by adding appropriate amounts of interleukin and OVA peptide to achieve a high level of activation. The activated T cells are then cultured under appropriate conditions.

[0096] (4) Construction of the co-culture system: When the density of MC38-OVA cells reached about 70% of the well plate area, the cells in one well were digested and counted to determine the number of cells in each well. Subsequently, activated T cells and the screened TCM monomer compounds were added to the wells at a ratio of 0.3:1 to form an experimental group. To ensure the reliability of the experimental results, the experiment was repeated three times for each TCM monomer compound. The concentration of the TCM monomer compound was strictly controlled so that its final working concentration reached 2 μg / mL.

[0097] (5) Using high-content screening technology, we took photos and counted cells, and analyzed the anti-tumor immune activity of compounds with similar structures to orcinol glucoside. The results are as follows: Figure 2 shown.

[0098] from Figure 2As can be seen in Figure 4, the survival rate of tumor cells treated with different TCM monomer compounds did not show significant differences compared to the blank control group, suggesting that these compounds may have no or minimal effect on promoting anti-tumor immune activity. This experimental result further confirms that the promoting effect of orcinol glucoside on anti-tumor immune activity is primarily due to its unique structural features, rather than the role played by its shared parent ring structure.

[0099] 3. In vivo experiments

[0100] 1. Mouse tumor-bearing experiment

[0101] (1) MC38 cells in the logarithmic growth phase were selected and subjected to standard digestion treatment. They were then washed three times with pre-cooled PBS. After each wash, the cells were centrifuged at 800 rpm for 5 min at 4°C to remove residual culture medium and impurities. Subsequently, the cells were accurately counted and the cell density was adjusted to 2×10 6 The cells were plated at 4% paraformaldehyde (PNA) and 4% paraformaldehyde (PNA) at 4% paraformaldehyde (PNA) for 200 μL and placed on ice to maintain cell viability for subsequent subcutaneous inoculation.

[0102] (2) Ten 8-week-old C57BL / 6 male mice were selected and inoculated subcutaneously with 200 μL of 2×10 6 A cell suspension of MC38 cells was prepared to construct a subcutaneous tumor model.

[0103] (3) When the tumor volume reaches about 40-50 mm 3 At the time of the experiment, mice were randomly divided into two groups, with 5 mice in each group. The mice in the experimental group received daily intraperitoneal injections of 10 mg / kg body weight of orcinol glucoside, and the injection solution was a mixture of orcinol glucoside and corn oil; the control group received a mixture of DMSO and corn oil in equal volumes and proportions as a placebo. The first day of injection was set as the 7th day of the experiment (D7), and the weight and tumor size of the mice were recorded every 2 days thereafter, and the drug was administered continuously for 13 days. On the 13th day (D19), the mice were killed, the tumors were dissected out, and their size and weight were measured to obtain dynamic statistical graphs of tumor volume, and the tumor inhibition rate was calculated (the formula is as follows). The results are as follows: Figure 2 shown.

[0104] Tumor inhibition rate = (1-average tumor weight of experimental group / average tumor weight of control group) × 100%.

[0105] (4) After killing the mice, the heart, liver, spleen, lung, kidney, and tumor tissues were quickly removed and fixed in 4% paraformaldehyde to preserve tissue morphology and structure. Subsequently, the tumor tissue was embedded and 7 μm thick tissue sections were prepared using the frozen section technique. The sections were adhered to glass slides and labeled.

[0106] from Figure 2 B shows that the tumor volume and weight of the mice in the experimental group were significantly lower than those in the control group, revealing the significant inhibitory effect of orcinol glucoside on tumor growth, with an average inhibition rate of 61.70%.

[0107] from Figure 2 As shown in Figure C, the tumor growth rate of the experimental group mice was significantly slower than that of the control group after administration, and subsequent observations showed effective control of tumor volume. This suggests that orcinol glucoside has a significant inhibitory effect on tumor progression.

[0108] 2. Fluorescent staining of frozen sections

[0109] (1) Wash the slices of the experimental group and the control group in water.

[0110] (2) Antigen retrieval: Use EDTA antigen retrieval solution to treat the slices in a boiling water bath for 20 minutes, then let them cool naturally, wash them with PBS three times, each time for 5 minutes, and then use an immunohistochemistry pen to draw a circle around the tissue to form a heat-stable hydrophobic isolation zone surrounding the tissue sample.

[0111] (3) Antibody incubation: Add diluted anti-mouse CD8 antibody to the tissue at a ratio of 1:300, incubate at 4°C overnight, and then wash with PBS three times, each time for 5 minutes.

[0112] (4) Sealing: Wipe off excess water, add DAPI to the slide under light-proof conditions, and perform a full scan using the intelligent pathology imaging analysis system. The results are as follows: Figure 2 shown.

[0113] from Figure 2 D shows that CD8 + The proportion of T lymphocytes increased significantly, indicating that orcinol glucoside mainly enhances the anti-tumor response of the immune system, especially promotes the activation of cytotoxic CD8 + T cell infiltration and activation to inhibit tumor growth.

[0114] 3. HE staining experiment

[0115] (1) Water washing: Place the frozen sections of the heart, liver, spleen, lung, and kidney of the experimental and control mice in a pure water environment and gently shake them using a water washing device placed on a water platform to effectively remove excess embedding agent on the slide until the surface of the section is clean and free of residue.

[0116] (2) Gently remove excess water from the slice, then use an immunohistochemistry pen to accurately draw a circle around the tissue sample to form a heat-stable and hydrophobic isolation zone.

[0117] (3) Hematoxylin staining: Accurately add an appropriate amount of hematoxylin staining solution into the formed heat-stable hydrophobic isolation area, and control the staining time to 4 minutes. After staining, gently rinse the section under running water for 3 minutes to remove excess staining solution. Use absorbent paper to gently absorb the residual water on the edge of the section.

[0118] (4) Eosin staining: Add an appropriate amount of eosin stain to the sections that have been stained with hematoxylin and washed with water, and control the staining time to 1 minute. After staining, place the sections under running water and wash them three times, each washing time for 3 minutes to completely remove unbound dye. After washing, use absorbent paper to gently absorb excess water from the edges of the sections.

[0119] (5) The slides were sealed with cedar oil and then scanned with an intelligent pathological imaging analysis system to obtain high-resolution and high-definition image data to examine the histomorphological changes of the mouse heart, liver, spleen, lung, and kidney and to evaluate the biosafety of orcinol glucoside. The results are as follows: Figure 2 shown.

[0120] from Figure 2 E It can be seen that no significant difference was found between the experimental group and the control group through frozen section and HE staining, which confirmed that orcinol glucoside had no obvious toxic side effects on the main organs of mice at the above dosage, showing good biocompatibility and safety.

[0121] 4. Experimental Conclusion

[0122] To study the immunomodulatory effects of orcinol glucoside on colorectal cancer, the present invention used an in vitro co-culture system to co-culture OT-1 mouse T cells, orcinol glucoside, and the MC38-OVA cell line. It was found that orcinol glucoside significantly enhanced the T cell-specific killing ability against MC38 tumor cells. This finding suggests that orcinol glucoside may fight tumors by activating T cell-mediated immune responses.

[0123] Furthermore, the present invention directly treated MC38-OVA tumor cells with orcinol glucoside, but the results showed that the drug did not directly produce cytotoxic effects on tumor cells. Subsequently, it was observed through CFSE proliferation experiments that orcinol glucoside could significantly promote the proliferation ability of T cells. Intracellular factor staining analysis revealed that orcinol glucoside could stimulate T cells to release IFN-γ and TNF-α key immune effector molecules, which play a vital role in anti-tumor immunity. At the same time, through co-culture of traditional Chinese medicine monomer compounds with similar structures to orcinol glucoside, it was confirmed that the promoting effect of orcinol glucoside on anti-tumor immune activity was due to its unique structural characteristics, rather than the role played by its shared parent ring structure.

[0124] To verify the in vivo effects found in vitro, the present invention conducted a subcutaneous tumor-bearing experiment in mice and administered intraperitoneal injection of orcinol glucoside. By regularly monitoring the changes in tumor volume in mice and performing statistical analysis, it was confirmed that orcinol glucoside can also effectively inhibit the growth of MC38 tumors in vivo. + T cell immunofluorescence staining analysis showed that CD8 + The infiltration of T cells increased significantly, which directly proves that orcinol glucoside can promote the infiltration of immune cells and the activation of anti-tumor immune response in vivo.

[0125] Finally, by evaluating the histomorphological changes of the main organs of mice, the present invention confirmed that orcinol glucoside effectively inhibited tumors without causing obvious adverse effects on the physiological health of mice, demonstrating its good biosafety and application potential.

[0126] Although the technical solution of the present invention has been described in detail above using general descriptions, specific implementation methods, and experimental examples, it should be noted that the embodiments and experimental examples are only intended to illustrate the technical solution and technical effects of the present invention and should not be construed as limiting the scope of protection of the present invention. Simple variations, modifications, or improvements based on the technical concept of the present invention are all within the scope of protection claimed by the present invention.

Claims

1. A new use of orcinol glucoside, characterized in that: Application of orcinol glucoside in preparing medicine for preventing and / or treating tumors.

2. The novel use according to claim 1, characterized in that: Application of orcinol glucoside in the preparation of drugs for inhibiting tumor growth by activating and promoting the anti-tumor activity of T cells.

3. The new use according to claim 1, characterized in that: The application includes but is not limited to one or more of the following aspects: (1) Application of orcinol glucoside in the preparation of drugs for promoting T cell proliferation and activation; (2) Application of orcinol glucoside in the preparation of drugs for promoting T cells to release cytokines; (3) Application of orcinol glucoside in the preparation of drugs that promote T cells to kill tumor cells.

4. The new use according to claim 3, characterized in that: The cytokines include but are not limited to IFN-γ and TNF-α.

5. The new use according to claim 1, characterized in that: The application includes but is not limited to one or more of the following aspects: (1) Application of orcinol glucoside in the preparation of drugs that promote immune cell infiltration into tumor tissues; (2) Application of orcinol glucoside in the preparation of drugs for activating the body's anti-tumor immune response; (3) Application of orcinol glucoside in the preparation of drugs for inhibiting tumor growth.

6. The new use according to claim 5, characterized in that: The said promoting immune cell infiltration into tumor tissue includes but is not limited to increasing the proportion of immune cells in tumor tissue; And / or, the immune cells include but are not limited to CD8 + T cells; And / or, the inhibition of tumor growth includes but is not limited to reducing tumor volume and reducing tumor weight.

7. The new use according to claim 1, characterized in that: The tumors include, but are not limited to, colorectal cancer, endometrial cancer, hepatocellular carcinoma, pancreatic cancer, lung adenocarcinoma, esophageal cancer, breast cancer, low-grade glioma, renal papillary cell carcinoma, nodal parisha myofibroblastoma, neuromuscular aberrant tumor, desmoid tumor, juvenile angiofibroma, enamel-type craniopharyngiocarcinoma, hepatoblastoma, pancreatic ductal carcinoma, biliary tract cancer, cerebral hemangioma, skin Merkel cell carcinoma, skin squamous cell carcinoma, melanoma in situ, and skin basal cell carcinoma.

8. A drug for preventing and / or treating tumors, characterized in that: The active ingredients of the medicine include orcinol glucoside.

9. The drug according to claim 8, characterized in that: The content of orcinol glucoside in the drug is the effective amount, and can be arbitrarily taken in the range of 0.01wt%-99.99wt%.

10. The drug according to claim 8, characterized in that: The drug may also include other pharmacological ingredients, including but not limited to other anti-tumor or auxiliary anti-tumor pharmacological ingredients, for combined therapy or auxiliary therapy; And / or, the drug further comprises a pharmaceutically acceptable carrier, including but not limited to a solvent, an excipient, a preservative, a stabilizer, an emulsifier, a salt for adjusting osmotic pressure, and a buffer; and / or, the dosage form of the drug is a pharmaceutically acceptable dosage form, including but not limited to powder injection, injection, tablet, pill, capsule, spray, dispersion; And / or, the administration route of the drug is a pharmaceutically acceptable route, including but not limited to oral, intradermal, subcutaneous, intramuscular, intraperitoneal, intravenous, and transdermal, intranasal or oral inhalation.

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