An antioxidant and immunoregulatory medicine and its preparation method

Abstract

This invention relates to the field of drug development technology, and more particularly to an antioxidant and immunomodulatory drug and its preparation method. This invention is the first to use termite nests containing individual termites and their symbiotic fungal gardens as raw materials to prepare an antioxidant and immunomodulatory drug that simultaneously targets two key pathological aspects of chemotherapy toxicity, effectively reducing the toxic side effects of chemotherapy. The drug prepared by this invention directly scavenges excess free radicals generated by chemotherapy drugs, increases SOD activity and the levels of antioxidants such as GSH, and can also act as an immunomodulator, activating natural killer cells, promoting T lymphocyte proliferation and differentiation, helping the damaged immune system recover rapidly, helping to maintain the patient's immune function during chemotherapy, reducing the risk of infection, and potentially having a positive impact on improving the tumor microenvironment.

Description

Technical Field

[0001] This invention relates to the field of drug development technology, and in particular to an antioxidant and immunomodulatory drug and its preparation method. Background Technology

[0002] Chemotherapy is one of the main clinical treatments for malignant tumors. However, while killing tumor cells, chemotherapy drugs often indiscriminately attack normal tissues, leading to a series of serious side effects. Oxidative damage and immunosuppression are two core pathological mechanisms that are interconnected, forming a vicious cycle. Chemotherapy drugs (such as platinum-based, anthracyclines, and alkylating agents) generate a large number of reactive oxygen species during metabolism, causing a sharp increase in the body's oxidative stress levels. This damages rapidly proliferating normal cells, such as intestinal mucosal epithelial cells and bone marrow hematopoietic stem cells, leading to oral mucositis, enteritis, and bone marrow suppression (mainly manifested as a significant decrease in white blood cells and platelets). Bone marrow suppression not only increases the risk of infection and bleeding but also severely limits the dosage and treatment progress of chemotherapy. Simultaneously, chemotherapy drugs directly or indirectly damage immune organs (such as bone marrow and spleen) and immune cells (such as T lymphocytes and natural killer cells), resulting in a comprehensive decline in the patient's immune function. This not only weakens the body's own anti-tumor immune surveillance ability but also makes patients more susceptible to opportunistic infections, seriously affecting their quality of life and treatment outcomes.

[0003] Termites are recognized as one of the world's five major pests. Despite this, termites themselves have edible and medicinal value. Termite nests and fungal gardens, formed by termites cultivating fungi and other microorganisms using special methods, are also considered delicacies. Termites belong to the order Isoptera in the class Insecta and contain various amino acids and trace elements essential for the human body. Termites live underground, inhabiting moist ecological environments, and possess a tremendous capacity for survival and reproduction. Even when termites die in their nests, they do not mold or rot. A termite nest is the dried body of the termite colony and its fungal garden. The termites are mainly *Odontotermes formosanus* Shiraki or *Macrotermes barneyi* Light, and the fungal garden is primarily composed of *Termitomyces albuminosus* (Berk) Heim.

[0004] The medicinal value of termites and their fungal gardens has been studied and reported in traditional Chinese medicine, and finished drugs are already in use, as shown in the applicant's earlier application (publication number CN102138940A). Furthermore, research on the activity and medicinal value of termites and their fungal gardens continues, such as the study "Efficacy Observation of Termite Nest Capsules Combined with Conventional Western Medicine in the Treatment of Liver and Kidney Yin Deficiency Type SLE," and the study "The Inhibitory Effect of Termite Fungal Garden Aqueous Extract on the Growth of Tumor Cells in Vitro." However, currently, termites and their fungal gardens have not been used as an adjunct to chemotherapy to reduce its toxic side effects. Summary of the Invention

[0005] Therefore, based on the above background, this invention further studies the application value of termites and their fungal gardens, and attempts to apply them to chemotherapy adjuvant therapy. It was found that they can simultaneously target two key pathological links of chemotherapy toxicity, protect the body from oxidative damage, and positively regulate immune function. Based on this, an antioxidant and immunomodulatory drug that can be used to prepare chemotherapy adjuvant preparations and its preparation method were developed.

[0006] The technical solution provided by this invention is as follows: The application of termite nests in the preparation of antioxidant and immunomodulatory drugs, wherein the termite nests are a mixture of termites and termite nurseries.

[0007] Preferably, the termites are selected from black-winged subterranean termites or yellow-winged macrotermites.

[0008] Preferably, the termite nursery is selected from Termitomyces albuminosus.

[0009] Based on the same inventive concept, the present invention also provides a method for preparing an antioxidant and immunomodulatory drug, comprising the following steps: (1) After removing impurities from the termite nest, dry it until the moisture content is controlled below 3.0% to obtain the termite nest clean medicinal material; the termite nest is a mixture of termites and termite nursery. (2) The termite nest clean medicine was crushed to obtain termite nest powder; (3) After irradiating and sterilizing the termite nest powder, it is ready.

[0010] Preferably, the termites are selected from black-winged subterranean termites or yellow-winged macrotermites.

[0011] Preferably, the termite nursery is selected from Termitomyces albuminosus.

[0012] Preferably, the dry weight percentage of termites in the termite nest raw material is not less than 35%, and the dry weight percentage of the termite nursery is not less than 60%.

[0013] Based on the same inventive concept, this invention also provides the use of the drug prepared by the method for preparing an antioxidant and immunomodulatory drug in the preparation of a chemotherapy adjuvant having at least one of the following functions A1) to A2), wherein the function includes: A1) Resisting oxidative damage caused by chemotherapy; A2) Regulates the body's immune function.

[0014] Based on the same inventive concept, the present invention also provides a chemotherapy adjuvant preparation comprising an effective amount of the drug prepared by the method described above for preparing an antioxidant and immunomodulatory drug.

[0015] Preferably, the drug prepared by the method for preparing an antioxidant and immunomodulatory drug is the only active ingredient.

[0016] The beneficial effects achieved by this invention are as follows: This invention is the first to use termite nests containing individual termites and their symbiotic fungal gardens as raw materials to prepare an antioxidant and immunomodulatory drug that can simultaneously target two key pathological aspects of chemotherapy toxicity, effectively reducing the toxic side effects of chemotherapy. In vitro cell experiments using intestinal epithelial cells and mouse bone marrow mononuclear cells have shown that the drug prepared in this invention directly eliminates excess free radicals produced by chemotherapy drugs, increases SOD activity and the level of antioxidants such as GSH, thereby protecting intestinal mucosal cells and bone marrow hematopoietic stem cells from oxidative damage and reducing bone marrow suppression and mucositis from the source.

[0017] Experiments conducted in a mouse model of immunosuppression induced by chemotherapy drugs have shown that the drug prepared in this invention can act as an immunomodulator, activating natural killer cells, promoting the proliferation and differentiation of T lymphocytes, helping the damaged immune system to recover rapidly, helping to maintain the immune function of patients during chemotherapy, reducing the risk of infection, and potentially having a positive impact on improving the tumor microenvironment. Detailed Implementation

[0018] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0019] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the experiments used in the following embodiments... Unless otherwise specified, all materials were purchased from commercial channels.

[0020] Example 1: A method for preparing an antioxidant and immunomodulatory drug, comprising the following steps: (1) After removing impurities from the termite nest, dry it until the moisture content is controlled below 3.0% to obtain the termite nest clean medicinal material; the termite nest is a mixture of termites and termite nursery. In this step, the termite fungus is collected directly from the breeding base along with black-winged subterranean termites to obtain the termite nest. Non-medicinal parts such as mud film and fragments in the termite nest fungus garden are removed. Then, the termite nest is placed in a container (with mesh to prevent the termites from escaping) and dried in a vacuum drying oven at 50℃ until the moisture content is ≤2.5%. During the drying process, the black-winged subterranean termites and the termite fungus dehydrate, and the black-winged subterranean termites die from dehydration.

[0021] The dry weight of termites in the termite nest clean medicine obtained in this step is about 40%, and the dry weight of termite fungus is about 60%.

[0022] The dry weight determination was performed as follows: Before drying the termite nest, a portion of the termite nest samples were randomly selected. Non-medicinal parts such as mud film and fragments in the fungal garden were removed. The live termites / termites in the samples were manually and completely separated from the fungal garden (using tools such as tweezers and brushes). The separated termites and fungal garden were placed separately into pre-weighed desiccant dishes and dried under the same conditions (e.g., a 50°C vacuum drying oven) until constant weight (moisture content ≤ 2.5%). After cooling in the desiccator, the dry weight of the termites and the dry weight of the fungal garden were accurately weighed separately. The dry weight percentages were then calculated.

[0023] (2) After pulverizing the termite nest clean medicinal material, pass it through an 80-mesh sieve to obtain termite nest powder; (3) The termite nest powder was packaged in a polyethylene bag and sterilized by irradiation with 60Co-γ rays at a dose of 8kGy.

[0024] In practical applications, the sterilized termite nest powder is filled into capsules and taken orally. It can also be prepared into other oral preparations by adding commonly used medical excipients, such as tablets, granules, or pills.

[0025] (1) In vitro cell antioxidant damage experiment 1) HIEC-6 (human intestinal epithelial cells) were purchased from Ausys Biotech.

[0026] Culture conditions: DMEM high glucose medium + 10% fetal bovine serum + 1% penicillin / streptomycin, cultured in a humid incubator at 37°C and 5% CO2.

[0027] Cells were digested and counted at a density of 5 × 10³ - 1 × 10³ per well. 4 Seeds were placed at a density of 1,000 cells into 96-well plates (for CCK-8) or specific culture plates / dishes. Once the cells adhered, the culture medium was replaced with fresh medium.

[0028] Cells from each cell culture plate were randomly grouped into blank control group, control group, model group, low-dose group, medium-dose group, and high-dose group. Then, the low-dose group, medium-dose group, and high-dose group were incubated with termite nest powder prepared in Example 1 at concentrations of 25, 50, and 100 μg / mL, respectively, for 2 hours. After incubation, 0.5 mM cyclophosphamide was added, and the incubation continued for 24 hours.

[0029] The control group was incubated for 26 hours after being fed with termite nest powder prepared in Example 1 at a concentration of 100 μg / mL. After adding physiological saline of the same volume as 50 μg / mL termite nest powder to the model group for 2 hours, 0.5 mM cyclophosphamide was added, and the group was cultured for 24 hours.

[0030] The blank control group was incubated with physiological saline containing the same amount of 50 μg / mL termite nest powder for 2 hours, followed by the addition of physiological saline containing the same amount of 0.5 mM cyclophosphamide for 24 hours.

[0031] Then, relevant tests were performed on each group of cells.

[0032] ① Add 10% volume of CCK-8 solution to each well and continue culturing for 1-4 hours. Measure the absorbance at 450 nm using a microplate reader. Then calculate the cell viability (%) = (OD administered - OD blank) / (OD control - OD blank) × 100%.

[0033] ② Measurement of intracellular reactive oxygen species levels (DCFH-DA probe method) After treatment, cells were resuspended in serum-free medium, and DCFH-DA was added to a final concentration of 10 μM. Cells were incubated at 37°C in the dark for 20-30 minutes. Cells were washed 2-3 times with PBS. Immediately, fluorescence intensity was detected using a fluorescence microplate reader at excitation light of 485 nm and emission light of 525 nm. ROS levels were directly proportional to fluorescence intensity.

[0034] ③ Intracellular SOD and GSH activity assay Cells were lysed, and the supernatant (cytoplasmic proteins) was collected. Total protein concentration was determined using the BCA method to standardize subsequent enzyme activity.

[0035] SOD activity was determined using the WST-8 assay kit (absorbance measured at 450 nm). GSH content was determined using the DTNB assay kit (absorbance measured at 412 nm).

[0036] The results are shown in Table 1.

[0037] Table 1. Effects of termite nest powder on the protection against chemotherapeutic drugs against oxidative damage in HIEC-6 cells.

[0038] * indicates that compared with the blank control group, P < 0.01. # indicates that compared with the model group, P < 0.05.

[0039] 2) Mouse bone marrow mononuclear cells were purchased from Shanghai Kanglang Biotechnology Co., Ltd.

[0040] Cultured using a dedicated hematopoietic stem cell culture medium, such as StemSpan™ SFEM.

[0041] CD34⁺ cells were obtained by immunomagnetic bead sorting at a density of 1 × 10⁶ cells per well. 4 Cells were seeded in 96-well plates.

[0042] The cells were grouped and processed in the same way as in the HIEC-6 experiment, and then tested.

[0043] The results are shown in Table 2.

[0044] Table 2: Effects of termite nest powder on the protection against oxidative damage in mouse bone marrow mononuclear cells from chemotherapy drugs.

[0045] * indicates that compared with the blank control group, P < 0.01. # indicates that compared with the model group, P < 0.05.

[0046] As shown in Tables 1 and 2, the survival rate, SOD, and GSH activities of HIEC-6 cells and bone marrow cells in the model group decreased sharply under the treatment of cyclophosphamide, indicating that chemotherapy induced severe cytotoxicity and oxidative stress. After treatment with termite nest powder (low, medium, and high doses), the survival rate and antioxidant indicators of both cell types significantly rebounded in a dose-dependent manner. In particular, in the high-dose group, all indicators approached or even reached normal levels. It is evident that the termite nest powder prepared in this invention can effectively counteract the cytotoxicity and oxidative stress induced by chemotherapy drugs (cyclophosphamide), significantly improve the survival rate and endogenous antioxidant enzyme levels of the two key target cells in a dose-dependent manner.

[0047] (2) Immunosuppressed mouse experiment Purchase C57BL / 6 mice, 6-8 weeks old, weighing 18-22g.

[0048] SPF-rated environment, free access to water and food, allow for 1 week of acclimatization.

[0049] The mice were randomly divided into a blank control group, a model group, a low-dose group, a medium-dose group, and a high-dose group, with 15 mice in each group.

[0050] The low-dose, medium-dose, and high-dose groups were administered the drug once daily by gavage, starting from day 1 of the experiment, for 10-14 consecutive days. During administration, the termite nest powder prepared in Example 1 was added to water to prepare a suspension. The low-dose group received 100 mg / kg / day of termite nest powder via gavage, the medium-dose group received 200 mg / kg / day, and the high-dose group received 400 mg / kg / day, for a total of 14 consecutive days. The daily volume of suspension administered by gavage was the same for all three groups.

[0051] Starting from day 4 of administration, except for the normal control group, mice in the other groups were injected intraperitoneally with cyclophosphamide (80 mg / kg) once a day for 3 consecutive days.

[0052] The normal control group was given the same volume of physiological saline as the drug group by gavage starting from day 1 of the experiment.

[0053] The model group was given the same volume of physiological saline as the drug group by gavage from day 1 of the experiment, and the mice were injected intraperitoneally with cyclophosphamide (80 mg / kg) once a day for 3 consecutive days starting from day 4.

[0054] Relevant tests were performed 24 hours after the last administration.

[0055] Peripheral blood was collected, all mice were anesthetized and euthanized, the bilateral femurs and tibias were separated and the spleens were removed, weighed, and the spleen index and thymus index were calculated.

[0056] Thymus index = Thymus weight (mg) / Body weight (g) Spleen index = Spleen weight (mg) / Body weight (g) ① Use a fully automated blood cell analyzer to analyze peripheral blood counts and detect white blood cell count, lymphocyte count, neutrophil count and platelet count.

[0057] ②Based on the isolated bilateral femurs and tibias, bone marrow cell counts were determined: 1 mL of pre-cooled PBS was drawn into a 1 mL syringe and used to rinse the bone marrow cavities of the mouse femurs and tibias. The rinsing fluid was collected; filtered through a 200-mesh filter to prepare a single-cell suspension. Viable cells were counted using the step blue staining method, and the total number of nucleated cells in each femur was calculated (×10⁻⁶). 6 ).

[0058] ③ Grind the spleen, lyse the red blood cells, resuspend them in PBS, count them, and take approximately 1×10⁻⁶ cells. 6 Cells were incubated with the following fluorescent antibody combinations (in the dark, 4°C, 30 minutes), and then the CD4+ / CD8+ cell ratio and the proportion of NK cells (CD3-) were detected by flow cytometry.

[0059] The results are shown in Table 3.

[0060] All data are expressed as mean ± standard deviation. GraphPad Prism software was used. One-way ANOVA was used for comparisons among multiple groups, and Tukey's post-hoc test was used for pairwise comparisons between groups. P < 0.05 was considered statistically significant.

[0061] Table 3: Effects of termite nest powder on resistance to chemotherapy-induced immunosuppression

[0062] * indicates that compared with the normal control group, P < 0.01. # indicates that compared with the model group, P < 0.05.

[0063] As shown in Table 3, the thymus / spleen index, peripheral blood count, bone marrow nucleated cell count, T cell ratio, and NK cell ratio were all significantly and comprehensively reduced in the model group mice. After gavage treatment with termite nest powder, a comprehensive dose-dependent recovery was observed: the thymus and spleen indices rebounded, indicating that the drug promoted the repair and proliferation of immune organs; the white blood cell, platelet, and bone marrow nucleated cell counts increased significantly, directly demonstrating that the drug could alleviate bone marrow suppression and promote blood cell regeneration; the CD4+ / CD8+ ratio recovered, indicating that the drug helped correct the T cell subset imbalance caused by chemotherapy and rebuild immune homeostasis. The rebound in the NK cell ratio signifies the restoration of the body's inherent tumor cell killing ability. It is evident that the termite nest powder prepared in this invention can significantly improve the immunosuppressive state in mice induced by chemotherapy (cyclophosphamide). It can increase the indices of atrophied thymus and spleen, promoting the recovery of immune organs; it can significantly increase the counts of white blood cells, lymphocytes, and platelets, increase the total number of nucleated cells in the bone marrow, help protect bone marrow hematopoietic function, and is beneficial in increasing the CD4+ / CD8+ cell ratio and the proportion of NK cells. It is evident that the termite nest powder drug prepared in this invention can effectively reduce chemotherapy-induced damage through a dual pathway of anti-oxidation and immunomodulation, providing strong support for its use as an adjuvant in chemotherapy.

[0064] The present invention and its embodiments have been described above. This description is not restrictive, and the embodiments shown are only one of the embodiments of the present invention. The actual structure is not limited to this. In conclusion, if those skilled in the art are inspired by this description and design similar structures and embodiments without departing from the spirit of the present invention, they should all fall within the protection scope of the present invention.

Claims

1. The use of termite nests for the preparation of an antioxidant, immunomodulatory drug, characterized in that, The termite nest is a mixture of termites and termite nurseries.

2. Use according to claim 1, characterized in that, The termites were selected from black-winged subterranean termites or yellow-winged macrotermites.

3. Use according to claim 1, characterized in that, The termite nursery was selected from Termitomyces albuminosus.

4. A method for the preparation of an antioxidant, immunomodulatory medicament, characterized by, Includes the following steps: (1) After removing impurities from the termite nest, dry it until the moisture content is controlled below 3.0% to obtain the termite nest clean medicinal material; the termite nest is a mixture of termites and termite nursery. (2) The termite nest clean medicine was crushed to obtain termite nest powder; (3) After irradiating and sterilizing the termite nest powder, it is ready.

5. A process for the preparation of an antioxidant, immunomodulatory medicament as claimed in claim 4, wherein, The termites were selected from black-winged subterranean termites or yellow-winged macrotermites.

6. A process for the preparation of an antioxidant, immunomodulatory medicament as claimed in claim 5, wherein, The termite nursery was selected from Termitomyces albuminosus.

7. A process for the preparation of an antioxidant, immunomodulatory medicament as claimed in claim 6, wherein, The dry weight percentage of termites in the raw material for the termite nest shall not be less than 35%, and the dry weight percentage of the termite nursery shall not be less than 60%.

8. The use of the drug prepared by the method for preparing an antioxidant and immunomodulatory drug according to any one of claims 4 to 7 in the preparation of a chemotherapy adjuvant having at least one of the following functions A1) to A2), characterized in that, The functions include: A1) Resisting oxidative damage caused by chemotherapy; A2) Regulates the body's immune function.

9. A chemotherapy adjuvant preparation, characterized in that, The effective amount includes the drug prepared by the method of preparing an antioxidant and immunomodulatory drug according to any one of claims 4 to 7.

10. A chemotherapy adjuvant preparation according to claim 9, characterized in that, The drug prepared by the method of any one of claims 4 to 7, which is an antioxidant and immunomodulatory drug, has a single active ingredient.

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