A strain of bat lepidoptera cordyceps and its application in preparing anti-neutron radiation medicine

Abstract

The application discloses a bat moth lepidoptera cordyceps strain and application thereof in preparation of anti-neutron radiation medicines. Samsoniella hepiali The strain is the bat moth lepidoptera cordyceps strain P969 (Pleurotus spodoleucus), which is preserved in the China Center for Type Culture Collection and has a preservation number of CCTCC NO: M 20251172 and a preservation date of May 26, 2025. Mycelium extracts of the strain can protect biological tissues against neutron radiation and repair organ damage caused by neutron radiation, so that the strain has a wide development space and a good development and application prospect.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a strain of Cordyceps militaris from the bat moth and its application in the preparation of anti-neutron radiation drugs. Background Technology

[0002] Cordyceps militaris (a type of insect) Samsoniella hepiali It is an important medicinal entomopathogenic fungus, belonging to the Cordyceps family (Cordyceps). Cordycipitaceae ) genus Cordyceps ( Samsoniella Cordyceps militaris (also known as the ghost moth lepidopterus) is believed to enhance immunity, have antibacterial and anti-inflammatory properties, protect organs such as the lungs, liver, kidneys, and testes, inhibit tumors, and prevent and treat cardiovascular and cerebrovascular diseases, diabetes, and depression. Samsoniella hepiali Phylogenetic Analysis of the Mitochondrial Genome of a Model Strain (Sun Tao, Li Tianhao, et al.) This article, through second-generation genome sequencing of the model strain of *Cordyceps militaris* (ICMM 82-2), assembled and annotated its mitochondrial genome. The results showed that the mitochondrial genome of the model strain is a closed circular structure, measuring 24246 bp. Its gene regions account for 85.10% of the genome, encoding a total of 42 genes, including 15 PCGs, 2 rRNA genes, and 25 tRNA genes.

[0003] Neutron radiation is a type of high linear energy transfer (LET) ray. Compared with gamma rays, neutron radiation can cause damage to the body's tissue structure and even loss of physiological function in a small dose and in a short period of time. It can cause deterministic effects such as acute radiation sickness, radiation pneumonia, radiation thyroid disease, and radiation skin damage, as well as stochastic effects such as cancer and genetic effects. Its potential harm is enormous.

[0004] Currently, the protection and treatment of neutron damage remains very difficult. There is no existing technology that can prevent the harm caused by neutron radiation through fungi. The search and screening of anti-neutron radiation drugs is of great significance for radiation medicine protection. Summary of the Invention

[0005] To address the shortcomings of existing technologies, the present invention aims to provide a strain of Cordyceps militaris from the bat moth and its application in the preparation of anti-neutron radiation drugs, and to verify the anti-neutron radiation effect of the strain through mouse efficacy experiments.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0007] This invention provides a strain of Cordyceps militaris from the ghost moth, specifically strain P969 of Cordyceps militaris. Samsoniella hepialiThis strain is deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M20251172 and deposit date of 2025-05-26.

[0008] The present invention also provides mycelial extracts of Cordyceps militaris strains.

[0009] The present invention also provides a composition containing mycelial extracts of Cordyceps militaris strains.

[0010] The above composition is added with conventional excipients and processed using conventional processes to produce capsules, freeze-dried powders, or bacterial liquid preparations, etc.

[0011] The application of the *Cordyceps militaris* strain, mycelial extract, or composition containing *Cordyceps militaris* mycelial extract in the preparation of anti-neutron radiation drugs.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] 1. The extract of Cordyceps militaris mycelium from the bat moth of the present invention has a protective effect on mice exposed to neutron radiation;

[0014] 2. The extract of Cordyceps militaris mycelium from the bat moth of the present invention has a protective effect on damaged organs. Attached Figure Description

[0015] Figure 1 strain P969 ( Samsoniella hepiali The morphology of a morphologist under an electron microscope;

[0016] Figure 2 A phylogenetic tree of strain P969 based on the 18S rRNA gene sequences of each strain;

[0017] Figure 3 Analysis of changes in body weight in mice after drug administration and irradiation;

[0018] Figure 4 Figure showing the survival changes in mice 28 days after drug administration and irradiation;

[0019] Figure 5 The effects of Cordyceps militaris mycelium extract on damaged organs in mice. Detailed Implementation

[0020] The technical solution of this patent will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments below are the preferred embodiments of this patent, but this patent is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the scope of this patent technology.

[0021] A strain of Cordyceps militaris (L.ex Fr.) Link var. hepiali Chen strain, which is Cordyceps militaris (L.ex Fr.) Link var. hepiali Chen strain P969 ( Samsoniella hepiali ), and the strain is deposited in the China Center for Type Culture Collection, with the deposit number of CCTCC NO: M 20251172, and the deposit date is May 26, 2025.

[0022] The present invention also provides a mycelial extract of Cordyceps militaris (L.ex Fr.) Link var. hepiali Chen strain.

[0023] The present invention also provides a composition containing the mycelial extract of Cordyceps militaris (L.ex Fr.) Link var. hepiali Chen strain.

[0024] The above composition is added with conventional excipients and made into capsules, lyophilized powder or bacterial liquid preparations according to conventional processes.

[0025] Application of the Cordyceps militaris (L.ex Fr.) Link var. hepiali Chen strain, or the mycelial extract, or the composition containing the mycelial extract of Cordyceps militaris (L.ex Fr.) Link var. hepiali Chen strain in the preparation of anti-neutron radiation drugs.

[0026] After culturing the strain, it is sent to the China Center for Type Culture Collection for strain identification. Figure 1 Figure 0000070 shows the morphological characteristics of the strain under an electron microscope. The strain grows slowly on PDA medium. After about 15 days of culture, the colony diameter is 2 cm. The center of the colony is slightly convex, white, villous, and the reverse side of the colony is light yellow. Microscopic examination shows that the aerial hyphae are flexuous.

[0027] The 18S sequence of the strain is determined and analyzed, and the 18S rRNA gene sequence of strain P969 is shown in SEQ ID No.1. Figure 2 Figure 0000075 is the phylogenetic tree of strain P969 based on the 18S rRNA gene sequences of each strain. After sequence analysis and alignment, the strain is identified as Samsoniella hepiali Cordyceps militaris (L.ex Fr.) Link var. hepiali Chen Examples

[0028] I. Experimental materials and equipment

[0029] Experimental animals

[0030] 30 SPF-grade C57BL / 6 male mice, with a body weight of 21.6 - 26.0 g, and an average of 23.3 ± 1.0 g. The production license number of the experimental animals is SCXK (Shanghai) 2023 - 0009. During the animal feeding period, good ventilation is ensured, the room temperature is maintained at 24 ± 2°C, the relative humidity is maintained at 60 ± 10%, and the lighting time is 12 h / d (from 6:00 A.M. to 18:00 P.M.).

[0031] Experimental materials

[0032] Universal tissue fixative (neutral): Composed of 4% paraformaldehyde + PBS (500 ml), store at room temperature. Catalog number: G1101, Lot: GP24043042934.

[0033] EDTA decalcification solution (slow decalcification): 500 ml, store at room temperature. Catalog number: G1105, Lot: GP24043061121.

[0034] Instruments and equipment

[0035] Neutron radiation source: High-current deuterium-tritium neutron source scientific facility.

[0036] Preparation of Cordyceps militaris mycelium extract from the ghost moth

[0037] A mycelial powder suspension of *Cordyceps militaris* strain was prepared to a concentration of 75 mg / mL. Water was added, and the suspension was ultrasonically disrupted for 30 min. Extraction was performed at 100℃ for 30 min, and the suspension was then set aside. The suspension was diluted halfway with ddH2O, dispensed into 5 mL vials, sterilized at 121℃ for 30 min, and stored at room temperature after cooling.

[0038] The dosage was 375 mg / kg body weight, and the administration volume was 200 μL, administered once daily by gavage for 7 consecutive days.

[0039] II. Animal grouping methods and dosing regimens

[0040] Thirty C57BL / 6 mice were divided into three groups according to their initial body weight: the model group (n=10), the Cordyceps militaris mycelium extract group (n=10), and the normal group (n=10).

[0041] The model group and the group receiving the extract of Cordyceps militaris mycelium from the bat moth were given prophylactic oral administration once daily for 7 days before neutron irradiation.

[0042] III. Experimental Methods

[0043] Neutron whole-body irradiation

[0044] Thirty minutes before irradiation, mice were mixed together in a plastic irradiation box (10 mice / box) and given a single whole-body irradiation.

[0045] Irradiation conditions: source distance 10 cm, total dose 8 Gy, irradiation time 2 h.

[0046] Weight monitoring

[0047] The body weight of the four groups of mice was recorded on the irradiation day (0 d) and on the 7th, 14th, 21st and 28th days after irradiation.

[0048] Survival status monitoring

[0049] The mice were observed daily, and the number of dead mice was recorded daily. The observation was continued for 28 days, and a survival curve was plotted.

[0050] Specimen Collection

[0051] Mice in each group were sacrificed on day 28 after irradiation. Each group of mice was weighed, and then sacrificed to collect whole blood (EDTA-2K anticoagulated), heart, liver, lungs, both kidneys, both testes, small intestine, thymus, spleen, and femur. The heart, liver, lungs, both kidneys, both testes, small intestine, thymus, and spleen were weighed separately, and organ indices were calculated using formulas for complete blood count and histopathological examination.

[0052] The left femur was harvested, the muscle was dissected, and the bone was fixed in 4% paraformaldehyde-PBS at room temperature for 24 h. After rinsing with ddH2O, the bone was transferred to EDTA decalcification solution and decalcified at 4°C. The solution was changed weekly. Decalcification was completed when the syringe needle could penetrate the bone. The process lasted for 2 weeks. The bone was then removed and embedded in paraffin for histopathological studies.

[0053] IV. Experimental Results

[0054] (a) Analysis of mouse body weight after drug administration and irradiation

[0055] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. If these modifications and variations fall within the scope of the claims of this invention and their equivalents, then this invention is also intended to include these modifications and variations. Figure 3 Analysis of changes in body weight in mice after drug administration and irradiation. Figure 4 This figure shows the changes in survival of mice 28 days after drug administration and irradiation. From... Figure 3 and Figure 4 As can be seen, there was no significant difference in body weight among the groups at the start of administration (-6 days) (P = 0.884). At day 4 of administration (-3 days), there was no significant difference in body weight among the groups (P = 0.380). At the end of administration / irradiation day (0 days), there was no significant difference in body weight among the groups (P = 0.474).

[0056] 7 days after irradiation

[0057] The study included a model group (6 birds), a group treated with Cordyceps militaris mycelium extract (10 birds), and a control group (10 birds). Significant differences were found between the model group and the group treated with Cordyceps militaris mycelium extract (P = 0.010), between the model group and the control group (P = 0.000), and between the group treated with Cordyceps militaris mycelium extract and the control group (P = 0.011).

[0058] 14 days after irradiation

[0059] The model group (5 birds), the *Cordyceps militaris* mycelium extract group (9 birds), and the normal group (10 birds) showed significant differences between the model group and the *Cordyceps militaris* mycelium extract group (P = 0.016), and between the model group and the normal group (P = 0.010).

[0060] 21 days after irradiation

[0061] The model group (n=5), the group receiving Cordyceps militaris mycelium extract (n=9), and the normal group (n=10) were divided into three groups. There were no significant differences among the groups.

[0062] 28 days after irradiation

[0063] The model group (n=5), the Cordyceps militaris mycelium extract group (n=9), and the normal group (n=10). There was a significant difference between the Cordyceps militaris mycelium extract group and the normal group (P = 0.022).

[0064] The above results indicate that the mycelial extract of Cordyceps militaris has a certain protective effect against neutron radiation in mice and has the potential to prepare anti-neutron radiation drugs.

[0065] Figure 5 Effects of Cordyceps militaris mycelium extract on damaged organs in mice:

[0066] thymus

[0067] In the normal group, the thymus tissue structure was intact and regular, with a clear distinction between the cortex and medulla. The cortex contained densely distributed lymphocytes, which were plump and relatively uniform in size, indicating a healthy and active state. The medulla contained numerous epithelial cells, and the entire thymus exhibited an active immune function.

[0068] The thymus structure in the model group was significantly disrupted, with the boundary between the cortex and medulla becoming blurred. The number of lymphocytes was significantly reduced, and the intercellular spaces increased, making the entire tissue appear loose. These changes severely affect the immune function of the thymus, reducing the body's immune defense capabilities.

[0069] The thymus tissue in the treatment group showed some signs of recovery. The boundary between the cortex and medulla was clearer than that in the model group, although it may not have fully recovered to the level of the normal group, but there was a significant improvement. The number of lymphocytes increased, and the arrangement between cells gradually became more compact, indicating that the production and survival of lymphocytes had improved to some extent. This may be because the drug promoted lymphocyte proliferation or inhibited their apoptosis, thereby enhancing the immune function of the thymus and helping the body restore its immune defense capabilities.

[0070] spleen

[0071] In a normal spleen, the white and red pulp are clearly defined and have a well-ordered structure. The lymphoid nodules in the white pulp are prominently displayed, serving as an important area for lymphocyte aggregation. The red pulp contains abundant blood cells, including erythrocytes, leukocytes, and platelets. Macrophages are also widely distributed within the red pulp.

[0072] In the model group, the boundary between the white and red pulp of the spleen became blurred, and the structure became disordered. The morphology of the lymph nodes changed significantly, with possible reduction in size, cell number, and lymphocyte activity. The blood cell components in the red pulp decreased, which could lead to impaired immune and hematopoietic functions of the spleen, causing an imbalance in the body's immune status and making it susceptible to various diseases.

[0073] In the treatment group, the boundary between the white and red pulp of the spleen gradually became clearer, and the structure began to return to normal. The morphology of the lymph nodes was repaired to some extent, their volume increased, and the number and activity of lymphocytes increased. The blood cell component in the red pulp gradually increased. This indicates that the drug has a certain improving effect on the spleen's immune and hematopoietic functions, and helps to restore the body's immune balance.

[0074] Small intestine

[0075] In the normal group, the small intestinal villi exhibit a regular and orderly arrangement, resembling a dense and well-organized "forest." The villi are of moderate length, and their surface is covered with a layer of intact and healthy epithelial cells. These epithelial cells are regularly shaped and tightly connected. Simultaneously, the intestinal gland structure is normal.

[0076] In the model group, the intestinal villi were severely damaged, becoming short and sparse, resembling a destroyed "forest" that had lost its original neat arrangement and full shape. Epithelial cells shed, disrupting the integrity of the villus surface and creating many defective areas. This significantly reduced the absorptive surface area of ​​the small intestine, resulting in a marked decrease in the absorption capacity of nutrients.

[0077] The damage to the small intestinal villi in the treatment group was significantly improved. The villi length increased, became denser, and gradually returned to a near-normal arrangement. The integrity of the epithelial cells was repaired to some extent, the number of shed cells decreased, new epithelial cells began to grow and replenish, and the absorptive surface area of ​​the small intestine increased.

[0078] marrow

[0079] Normal bone marrow tissue is rich in hematopoietic cells, including various blood cell precursors at different developmental stages. These hematopoietic cells are densely packed and exhibit active proliferation and differentiation. Adipocytes constitute a small proportion of the bone marrow and do not interfere with the growth and development of hematopoietic cells. The stable bone marrow microenvironment provides suitable conditions for the survival and differentiation of hematopoietic stem cells, thus ensuring the body's continuous and stable hematopoietic function and meeting the body's needs for various blood cells.

[0080] Significant changes occurred in the bone marrow tissue of the model group, with a sharp decrease in the number of hematopoietic cells, and previously dense areas of hematopoietic cells becoming sparse. Conversely, there was a significant increase in fat cells, occupying a large amount of bone marrow space, resulting in a pronounced fatty bone marrow structure. This change can severely affect the hematopoietic function of the bone marrow, preventing the body from producing enough blood cells, such as red blood cells, white blood cells, and platelets, which may lead to a series of health problems such as anemia, infection, and bleeding.

[0081] The number of hematopoietic cells in the bone marrow tissue of the treatment group was significantly increased compared to the model group, while the number of adipocytes decreased accordingly. This indicates that the drug may have a positive impact on the hematopoietic microenvironment of the bone marrow, promoting the proliferation and differentiation of hematopoietic stem cells and inhibiting the growth and accumulation of adipocytes. With the increase in hematopoietic cells, the hematopoietic function of the bone marrow gradually recovered, enabling it to provide the body with sufficient blood cells, maintain normal physiological functions, and improve various health problems caused by bone marrow damage.

[0082] Overall, the model group showed obvious pathological changes in all tissue sites, while the tissue morphology of the drug-treated group was improved compared with that of the model group, showing a certain therapeutic effect.

[0083] In summary, compared with the model group, the mycelial extract of *Cordyceps militaris* increased the body weight of mice on days 7 and 14 after irradiation and reduced the damage caused by irradiation. The mycelial extract of *Cordyceps militaris* significantly improved the 28-day survival rate of neutron-irradiated mice, indicating that it has a certain protective effect against neutron irradiation damage. Pathological examination results showed that the mycelial extract of *Cordyceps militaris* had a significant protective effect on the thymus, spleen, small intestine, and femur of irradiated mice.

[0084] The above results indicate that the mycelial extract of Cordyceps militaris has a certain protective effect against neutron radiation in mice and has the potential to prepare anti-neutron radiation drugs.

Claims

1. A strain of Cordyceps militaris, characterized in that, This strain is *Cordyceps militaris* strain (*Hepiali*). Samsoniella hepiali P969, this strain is deposited at the China Center for Type Culture Collection, with accession number CCTCCNO: M 20251172, and the deposit date is May 26, 2025.

2. The mycelium extract of the bat moth cordyceps mothworm strain of claim 1, wherein, The preparation method of the mycelial extract is as follows: prepare a mycelial powder suspension of Cordyceps militaris strain to a concentration of 75 mg / mL, add water and sonicate for 30 min, extract at 100℃ for 30 min and set aside, dilute half with ddH2O, dispense into 5 mL vials, sterilize at 121℃ for 30 min, cool and store at room temperature.

3. A composition comprising the mycelial extract of claim 2.

4. The composition of claim 3, wherein, The composition is prepared as capsules, lyophilized powder, or bacterial liquid preparation.

5. The use of the mycelial extract as described in claim 2 or the composition as described in claim 3 in the preparation of anti-neutron radiation drugs.

Images