Application of a strain of Tolycopersicon genus in anti-tumor drugs

Abstract

The present invention discloses an application of a Toxoplasma fungus in an anti-tumor drug. The Toxoplasma fungus has been deposited in the "China Center for Type Culture Collection" on April 20, 2023, with a deposit number of CCTCCM2023568. The present invention extracts the mycelium of the Toxoplasma fungus, and uses a mouse transplant tumor model experiment to find that the mycelium of the Toxoplasma fungus can effectively inhibit the growth of tumor cells through oral administration. Long-term use has no obvious toxicity to various organs, and can be used to prepare anti-tumor drugs, etc., with good anti-tumor effects.

Description

Technical Field

[0001] The invention relates to the technical field of medicines, and in particular to application of a Tolypocladium fungus in anti-tumor drugs. Background Art

[0002] Malignant tumors are a major disease that threatens human health. Currently, the first-line treatments for malignant tumors mainly rely on surgery, radiation, chemotherapy, and immunotherapy. Although surgery can quickly reduce the patient's tumor burden, it cannot effectively remove residual micro-tumors, which can easily lead to postoperative recurrence. Although radiation and chemotherapy are effective, they also have a destructive effect on normal human cells, causing adverse reactions such as decreased white blood cell counts, anemia, and digestive tract damage, making it difficult for patients to tolerate treatment. At the same time, due to the presence of mutagenic effects, it is also easy to induce new tumors. Immunotherapy mainly regulates the body's anti-tumor immune function through specific targeted drugs (such as immune checkpoint blockers, etc.), thereby achieving the purpose of clearing tumors. However, most of these drugs are monoclonal antibodies, which have complex preparation processes and are expensive. Before application, patients need to verify tumor targets in order to improve the targetedness of treatment and achieve satisfactory results. Therefore, immunotherapy has high specificity requirements, and there are currently no broad-spectrum tumor immunotherapy drugs.

[0003] Toxoplasma is a common fungus widely distributed in the natural environment, including soil, water, plants and animals. The diversity of Toxoplasma fungi is very rich, and more than 200 different Toxoplasma fungi have been identified. These strains have different morphological, physiological and biochemical characteristics, some of which have important biologically active components and enzyme production capabilities. CN200410041167.9 discloses a polysaccharide complex isolated from a culture solution of Toxoplasma sinensis and a preparation method thereof. The method uses a liquid culture medium to culture Toxoplasma sinensis, and then collects and concentrates the culture solution to obtain an extracellular polysaccharide complex in the culture solution. The polysaccharide complex has the effects of inhibiting tumor growth and metastasis, improving immune function, etc., and has a stable therapeutic effect, and is safe and reliable. So far, except for extracellular secretions, there has been no report that Toxoplasma fungi themselves have an inhibitory effect on tumors. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention aims to provide a strain of Tolypocladium for use in anti-tumor drugs to solve the problems mentioned in the background technology.

[0005] The present invention provides a strain of Toxicodendron fungus ( Tolypocladium sp. F56), the fungus was deposited in the China Center for Type Culture Collection on April 20, 2023, with the deposit number CCTCCM2023568.

[0006] The Tolypocladium fungus described in the present invention is separated and screened from wild Cordyceps sinensis.

[0007] The invention discloses an application of the Tolypocladium fungus in anti-tumor drugs.

[0008] Preferably, the mycelium extract of Toxoplasma fungus is used to prepare anti-tumor drugs.

[0009] Preferably, the method for preparing the mycelium extract of Toxoplasma fungus comprises the following steps:

[0010] S1. Drying and crushing mycelium of Toxoplasma fungus to obtain mycelium powder;

[0011] S2, adding deionized water to the mycelium powder and centrifuging after refluxing, collecting the first supernatant and precipitate for later use;

[0012] S3, adding deionized water to the precipitate again, refluxing and centrifuging, collecting the second supernatant and combining it with the first supernatant to obtain a mycelium extract of the genus Tolylcophyte.

[0013] Preferably, the mycelium drying temperature in step S1 is 60-70°C.

[0014] Preferably, in step S2, the mass volume ratio of mycelium powder to deionized water is 15-40 g:500 mL.

[0015] Preferably, the mass volume ratio of the precipitate to water in step S3 is 9-24 g:500 mL.

[0016] Preferably, the temperature of the reflux extraction in steps S2 and S3 is 90-110° C., and the extraction time is 0.5-1.5 h.

[0017] Preferably, the centrifugation time in steps S2 and S3 is 12 to 17 min, and the rotation speed is 7000 to 9000 r / min.

[0018] Preferably, the crude drug concentration of the mycelium extract of the genus Toxoplasma in the drug is 15 to 40 mg / mL.

[0019] In order to explore the anti-tumor effect of Toxoplasma fungi isolated in our laboratory, the experiment used a mouse transplant tumor model to explore the oral administration of different doses of mycelial extracts of Toxoplasma fungi ( Tolypocladium.spExtract, TSE) on the volume of transplanted tumors in tumor-bearing mice, the survival rate after medication, and the quality of transplanted tumors, which effectively confirmed that the fungal mycelium extract had a tumor inhibitory effect on tumor-bearing mice. In addition, this experiment also used normal mice to explore the effects of high-dose TSE on normal mouse body weight, organ index (thymus, spleen, liver, testis), blood routine [white blood cell count (WBC), lymphocyte count (Lymph), monocyte count (Mon), neutrophil count (Gran), red blood cell count (RBC), hemoglobin concentration (HGB), platelet count (PLT)], liver function [serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), serum albumin (ALB)], and renal function [serum urea nitrogen (BUN), serum creatinine (CR)] through oral administration, which effectively confirmed that TSE had no obvious adverse reactions on normal mice and its dose for tumor treatment was safe.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The mycelium extract of the Toxoplasma fungus of the present invention has a significant inhibitory effect on the growth of transplanted tumors in tumor-bearing mice, and at the same time, the therapeutic dose has no obvious adverse reactions on the general condition, blood system, major organs, liver and kidney functions of normal mice;

[0022] 2. The mycelium extract of the Toxoplasma fungus of the present invention is simple to prepare and has low cost. It can be used to prepare drugs for preventing and treating tumor growth and reducing the expression of tumor-related genes, and can be used to prepare immunomodulatory drugs for patients with immunosuppression caused by diseases such as tumors. It is inexpensive, easy to produce, safe and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The effect of oral administration of TSE on the survival rate of tumor-bearing mice;

[0024] Figure 2 The effect of oral TSE on the volume of transplanted tumors;

[0025] Figure 3 The effect of oral TSE on the weight of transplanted tumors;

[0026] Figure 4 The effect of oral high-dose TSE on the body weight of mice;

[0027] Figure 5 The effects of oral high-dose TSE on organ indexes of mice;

[0028] Figure 6 The effects of oral administration of high doses of TSE on peripheral blood counts of mice;

[0029] Figure 7 This study shows the effects of oral high-dose TSE on liver and kidney function in mice. DETAILED DESCRIPTION

[0030] The technical solution of this patent is further described in detail below in conjunction with the specific implementation methods. It should be pointed out that the following specific implementation methods are detailed descriptions of the preferred implementation methods of this patent, but this patent is not limited to the above implementation methods. Various changes can be made within the scope of knowledge possessed by ordinary technicians in this field without departing from the technical scope of this patent.

[0031] It should be noted that the experimental materials of this experiment are as follows:

[0032] 1. Tumor cell lines

[0033] Mouse colon cancer cell line CT26.WT, cultured in RPMI 1640 + 10% FBS medium, was purchased from the Cell Bank of the Chinese Academy of Sciences.

[0034] 2. Experimental Animals

[0035] Thirty-one SPF male BALB / c mice, with an average body weight of 21.1 ± 0.8 g, were purchased from Shanghai Xipulbikai Laboratory Animal Co., Ltd., with the laboratory animal production license number: SCXK (Shanghai) 2018-0006. Good ventilation was ensured during animal breeding, the room temperature was maintained at 24 ± 2 °C, the relative humidity was maintained at 60 ± 10%, and the lighting time was 12 h / d (6:00 AM to 18:00 P.M.).

[0036] 3. Main Reagents

[0037] 0.25% Trypsin-EDTA (1×) was purchased from Gibco; RPMI Medium 1640 (1×) culture medium was purchased from Gibco; fetal bovine serum (FBS) was purchased from Moregate, Australia; penicillin-streptomycin solution was purchased from Hyclone, USA, and diluted at 1:100 (v / v); PBS pH=7.4 (1×) was purchased from Gibco; universal tissue fixative (neutral) was purchased from Wuhan Saiweier Biotechnology Co., Ltd.; TSE was prepared by the Navy Special Medical Center.

[0038] 4. Main instruments

[0039] Low-speed desktop centrifuge (Shanghai Anting Scientific Instrument Factory); cell culture incubator (Thermo Fisher Scientific, USA); clean bench (Suzhou Antai Air Technology Co., Ltd.); inverted microscope (Chongqing Chongguang Industrial Co., Ltd.); electronic balance (Shanghai Yueping Scientific Instrument Co., Ltd.); analytical balance (Denver Instruments, USA); electronic vernier caliper (Qifeng).

[0040] Preparation of Tolycopersicon mycelial extract (TSE)

[0041] 1. Dry the fresh mycelium at 65°C to constant weight, crush it and pass it through a 100-mesh sieve to obtain dry mycelium powder.

[0042] 2. Accurately weigh 36 g of mycelium powder, add it to 500 mL of ddH2O, reflux extract at 100℃ for 1 h, cool to room temperature, centrifuge at 8000 r / min for 15 min, collect the first supernatant and precipitate for later use.

[0043] 3. Add 500 mL ddH2O to the precipitate obtained in step 3 and reflux extract again for 1 h. Cool the secondary extract to room temperature, centrifuge at 8000 r / min for 15 min, collect the second supernatant, and finally combine the first and second supernatants, add deionized water to 1 L, and the final TSE stock solution has a crude drug concentration of 36 mg / mL, which is set as the high-dose administration concentration. After adding purified water to dilute it in half, the crude drug concentration of the dilution is 18 mg / mL, which is set as the low-dose administration concentration. Example

[0044] Determination of the inhibitory effect of extracts from the mycelium of the genus Toxoplasma on tumors

[0045] 1. Experimental Methods

[0046] 1. Tumor cell expansion culture

[0047] When the cells grew to 90% confluence, they were digested with 0.25% Trypsin-EDTA, and 10 mL of fresh culture medium was pipetted into a cell suspension. The cells in each bottle were combined, and the total cell count was 5 × 10 7 .

[0048] 2. Preparation of Cell Suspension

[0049] Centrifuge at 1000 r / min for 5 min at room temperature to precipitate the cells, remove the supernatant completely, and resuspend the cells in 4 ml PBS to adjust the cell concentration to 7.5 × 10 6 / mL. Dispense into 31 1 mL centrifuge tubes, each containing 1.5 × 10 6 / 200 μL cell suspension.

[0050] 3. Tumor Cell Inoculation

[0051] The skin on the right shoulder and back of the mouse was prepared, the hair was shaved off, the skin was exposed, and the cell suspension was drawn up with a 1 mL syringe. Each mouse was subcutaneously inoculated with 1.5 × 10 6 / 200 μL cell suspension, 31 cells in total.

[0052] 4. Grouped medication

[0053] About 10 days after tumor cell implantation, when the tumor volume reaches 150-300 mm 3 At , it indicated that the tumor-bearing mouse model was successfully prepared and they were randomly divided into a model group (10 mice), a low-dose group (11 mice) and a high-dose group (10 mice) to start the drug treatment experiment.

[0054] (1) Model group: deionized water was administered orally at a volume of 10 mL / kg, once a day for 14 consecutive days;

[0055] (2) High-dose group (TSE 360 mg / kg): oral administration dose was 360 mg / kg body weight, volume was 10 mL / kg body weight, once a day, for 14 consecutive days;

[0056] (3) Low-dose group (TSE 180 mg / kg): The oral administration dose was 180 mg / kg body weight in a volume of 10 mL / kg body weight, once a day for 14 consecutive days.

[0057] 5. Monitoring of Transplanted Tumor Growth and Mouse Survival Status

[0058] After inoculation, the tumor growth was observed. After the treatment started, the long diameter (L) and the transverse diameter (W) of the transplanted tumor were measured with a vernier caliper every 3 days according to the formula: [V = (L × W 2 ) / 2] Calculate the tumor volume. Observe and record the survival status of tumor-bearing mice every day and draw a survival curve.

[0059] 6. Processing of materials

[0060] On the 14th day after inoculation, the mice were anesthetized and killed by intraperitoneal injection of 0.2 mL of 10% chloral hydrate. The back skin was opened to examine the tumor formation, and the tumor was isolated and weighed and recorded.

[0061] 7. Data Processing

[0062] SPSS 22.0 software was used for statistical analysis. The Log Rank method was used for animal survival analysis after irradiation. One-way analysis of variance was used to test the significance of the data in each group.

[0063] 2. Test indicators and results

[0064] (1) Survival rate of tumor-bearing mice after administration

[0065] The survival of animals in each group after administration was observed and the survival rate was calculated ( Figure 1 ).

[0066] Results: 14 days after administration, the survival rate of the model group was 20%, the survival rate of the low-dose group was 72.7%, and the survival rate of the high-dose group was 60%. The survival rates of the low-dose group and the high-dose group were significantly higher than those of the model group (* P < 0.05), indicating that oral administration of TSE can significantly improve the survival rate of tumor-bearing mice.

[0067] (2) Transplanted tumor volume after drug administration

[0068] The transplanted tumor volume of each group of animals was monitored after administration ( Figure 2 Results: 3 to 12 days after administration, the volume of transplanted tumors in the high-dose group was significantly lower than that in the model group (* P < 0.05 or ** P < 0.01); 6 to 12 days after administration, the volume of transplanted tumors in the low-dose group was significantly lower than that in the model group (** P < 0.01), indicating that oral administration of TSE could significantly reduce the volume of transplanted tumors.

[0069] (3) Transplanted tumor weight after drug administration

[0070] The weight of transplanted tumors in each group of animals was measured 14 days after administration ( Figure 3 ).

[0071] Results: The weight of transplanted tumors in the high-dose group and the low-dose group were lower than that in the model group, indicating that oral administration of TSE can reduce the weight of transplanted tumors. Example

[0072] Long-term toxicological evaluation of oral extracts from the mycelium of the fungus Toxoplasma

[0073] 1. Experimental Methods

[0074] 1. Grouped medication

[0075] Twenty mice were randomly divided into two groups, namely the control group (10 mice) and the high-dose group (10 mice). They were given normal diet and drinking water before the experiment, and the oral toxicology experiment of the drug was started after 3 weeks of adaptive feeding.

[0076] 2. Weight monitoring

[0077] The body weights of the two groups of mice were recorded at the beginning of the experiment (0 d) and on d 3, 6, 9, 12, and 15 after administration.

[0078] 3. Survival status monitoring

[0079] The status of the mice was observed every day, and the number of dead mice was recorded daily. The observation was continued for 15 days, and the survival curve was drawn.

[0080] 4. Specimen Collection

[0081] At the end of the 15-day observation period, mice in each group were weighed, killed, and whole blood, thymus, spleen, liver, and testis were collected. All whole blood samples of mice were collected by enucleation, and 5 blood samples were taken from each group for routine blood tests after anticoagulation with EDTA-2K; the remaining 5 blood samples were centrifuged at 4°C, 3000 r / min for 15 min, and serum was separated for liver and kidney function tests. All organs were weighed separately, and the organ index was calculated according to the formula [organ index = (organ weight / body weight) × 100%].

[0082] 5. Data processing

[0083] SPSS 22.0 software was used for statistical analysis, and independent sample T test was used to analyze the significance of each group of data.

[0084] 2. Test indicators and results

[0085] (1) Body weight after administration

[0086] Monitor the body weight of animals in each group after administration ( Figure 4 ).

[0087] Results: Before administration (0 d), there was no statistical difference in the body weight between the high-dose group and the control group. After administration, the high-dose group had shiny fur, was active, gradually gained weight, and was in good general condition. From 3 to 15 d of administration, the body weight of the high-dose group was significantly higher than that of the control group ( * P < 0.05 or ** P < 0.01), indicating that oral administration of TSE can promote animal growth without obvious adverse reactions.

[0088] (2) Organ index after drug administration

[0089] 15 days after administration, the weight of thymus, spleen, liver and testis of each group of animals was measured, and the index of each organ was calculated ( Figure 5 ).

[0090] Results: After 15 days of administration, there was no statistical difference in the organ indexes of mice in each group, indicating that oral administration of TSE had no effect on the weights of the thymus, spleen, liver and testicles of mice, and no obvious organ toxicity was observed.

[0091] (3) Peripheral blood count after administration

[0092] Whole blood samples were collected from mice 15 days after administration to detect white blood cell count (WBC), lymphocyte count (Lymph), monocyte count (Mon), neutrophil count (Gran), red blood cell count (RBC), hemoglobin concentration (HGB), platelet count (PLT) and other indicators ( Figure 6 ).

[0093] Results: After 15 days of administration, there was no statistical difference in the peripheral blood indexes of mice in each group, indicating that oral administration of TSE had no effect on the peripheral blood of mice and no obvious blood system toxicity was observed.

[0094] (4) Liver and kidney function after administration

[0095] Serum samples were collected from mice 15 days after administration to detect liver function [serum alanine aminotransferase (ALT), serum aspartate aminotransferase (AST), serum albumin (ALB)] and renal function [serum urea nitrogen (BUN), serum creatinine (CR)] indicators ( Figure 7 ).

[0096] Results: After 15 days of administration, there was no statistical difference in the liver and kidney function indicators of mice in each group, indicating that oral administration of TSE had no effect on the liver and kidney function of mice and no obvious liver and kidney toxicity was observed.

[0097] According to the above experimental statistical data, the extract of the mycelium of the genus Toxoplasma fungus of the present invention has a significant inhibitory effect on tumor growth. At the same time, when the extract is taken for a long time, the mice grow faster and their weight gradually increases. It has no toxic effect on the organs of the mice and is suitable for long-term use. The extracted mycelium of the present invention can be used to prepare drugs for preventing and treating tumor growth and reducing the expression of tumor-related genes, and for preparing immunomodulatory drugs for patients with immunosuppression caused by diseases such as tumors. It is low-priced, easy to produce, safe and reliable.

[0098] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. If these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Use of Tolycopersicon fungi in the preparation of anti-tumor drugs, characterized in that: Toxoplasma spp Genus ( Tolypocladium sp.) strain F56, deposited in China Center for Type Culture Collection, accession number It is CCTCC NO:M2023568, and the deposit date is April 20, 2023; The efficacy of the anti-tumor drug is derived from the mycelium of the Toxoplasma fungus; The Tolylcoceras fungus is used to inhibit tumor growth; The tumor is colon cancer.

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