Application of composition in radiation protective agent

Abstract

Application of a composition in a radiation protective agent belongs to the technical field of radiation protective agents, and the specific scheme includes the following steps that the composition is composed of a Shengshiitake mushroom source exosome-like nano-vesicle solution and a shiitake mushroom polysaccharide solution, the concentration ratio of Shengshiitake mushroom source exosome-like nano-vesicles to shiitake mushroom polysaccharide in the composition is 1: 10-1: 40, and the concentration ratio of the Shengshiitake mushroom source exosome-like nano-vesicles to the shiitake mushroom polysaccharide is 1: 10-1: 40. The preparation method of the yang-secreting shiitake mushroom source exosome-like nano-vesicles comprises the following steps: step 1, cutting off roots of shiitake mushroom sporocarp, cutting the remaining part into blocks, and adding an extraction solvent; and step 2, after wall-breaking and crushing, carrying out suction filtration to obtain filtrate, carrying out differential centrifugation to obtain supernate, and carrying out ultra-high-speed centrifugation to obtain precipitate, namely the shiitake mushroom source exosome-like nano-vesicles. It is proved for the first time that after being compounded with shiitake mushroom polysaccharides, the shiitake mushroom source exosome-like nano-vesicles have the effect of cooperatively relieving cell damage induced by ionizing radiation.

Description

[0001] This application is a divisional application of the application date of February 1, 2024, application number 202410144597.0, and invention name "Application of a kind of exosome-like nanovesicles derived from shiitake mushroom in radiation protection". Technical Field

[0002] The present invention belongs to the technical field of radiation protection, and in particular relates to an application of a composition in the preparation of a radiation protection agent. Background Art

[0003] Ionizing radiation, a general term for radiation that can ionize matter, is a high-energy physical agent that poses a serious threat to human health. It can cause excessive production of reactive oxygen species (ROS) that act on biomolecules such as nucleic acids and proteins, leading to varying degrees of damage to the hematopoietic, reproductive, and immune systems, and even cancer. Radioprotectants are an important means of preventing oxidative damage from ionizing radiation. Currently, developed radiation protection agents primarily consist of chemical drugs, including sulfur-containing compounds, cytokines, and hormones. However, these agents are relatively expensive and have certain toxic side effects, making them unsuitable for long-term use. Naturally derived radiation protection factors, however, can enhance radiation tolerance and offer significant advantages in efficacy, cost, and safety, making them suitable for use in pharmaceuticals and health foods. Currently, selected natural radiation protection factors include small-molecule polyphenols, such as resveratrol, curcumin, and tea polyphenols, and large-molecule polysaccharides, such as poria cocos polysaccharides and yam polysaccharides. However, these two types of natural radiation protection functional factors currently have certain limitations in practical application: low bioavailability, high cost, and limited application process. Therefore, it is still necessary to find and develop new natural source radiation protection functional factors.

[0004] Extracellular vesicles (EVs) are extracellular structures enclosed by a lipid bilayer membrane. They contain bioactive substances such as proteins, lipids, and nucleic acids and play a vital role in intercellular communication. EVs can be released by all cell types (including animals, plants, and microorganisms) and come in a variety of sizes (100-1000 nm in diameter). In published literature, they are often referred to as microvesicles, exosomes, or microparticles. EVs have attracted widespread attention due to their desirable characteristics, such as small size, high biocompatibility, and high stability. Dietary exosome-like nanovesicles (DELNs), derived from food and typically measuring less than 300 nm, also contain a variety of active substances, such as proteins, lipids, and nucleic acids, and exhibit diverse biological functions, including anti-inflammatory, antioxidant, and anti-cancer properties. DELNs offer advantages such as low safety risks, simple preparation and processing, a stable nanostructure, and high bioavailability for improved absorption, providing a promising foundation for their development and application. Flower mushrooms are a variant of the common edible mushroom, Lentinus edodes, cultivated and produced by controlling growth conditions and altering the development of the fruiting body. Flower mushroom-derived exosomes and nanovesicles also fall into the category of DELNs, but it is currently unclear whether they possess radioprotective properties. Studying the radioprotective effects of flower mushroom-derived exosomes and nanovesicles is crucial for developing new natural radioprotectants. Furthermore, DELNs exhibit excellent biocompatibility and inherent stability. Therefore, it is also important to investigate whether flower mushroom-derived exosomes and nanovesicles have synergistic effects with other substances. Summary of the Invention

[0005] In order to solve the problems existing in the background technology, the present invention provides an application of shiitake mushroom-derived exosome-like nanovesicles in radiation protection, and designs a synergistic and synergistic combination formula of shiitake mushroom-derived exosome-like nanovesicles and shiitake mushroom polysaccharides in radiation protection.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] An application of shiitake mushroom-derived exosome-like nanovesicles in radiation protection comprises the following steps:

[0008] Step 1: After cutting off the roots of the fresh shiitake mushroom fruiting bodies, cut the remaining parts into pieces and add extraction solvent;

[0009] Step 2: After the cell wall is broken and crushed, the filtrate is filtered to obtain a filtrate, the supernatant is obtained by differential centrifugation, and the precipitate, i.e., the shiitake mushroom-derived exosome-like nanovesicles, is obtained by ultrahigh-speed centrifugation;

[0010] Step 3: Using the shiitake mushroom-derived exosome-like nanovesicles as a radiation protector to resist radiation damage.

[0011] Furthermore, in step 1, the extraction solvent is 1×PBS buffer.

[0012] Furthermore, in step 2, the differential centrifugation is performed at a centrifugal force of 800-1200×g for 20-30 min; 2000-3000×g for 20-30 min; 4000-5000×g for 30-50 min; and 8000-10000×g for 60-120 min, and the precipitate is removed and the supernatant is collected after each centrifugation; the centrifugal force in the ultrahigh-speed centrifugation is 120000-180000×g, the centrifugation time is 90-150 min, and the supernatant is discarded to obtain the precipitate.

[0013] Furthermore, in step three, the shiitake mushroom-derived exosome-like nanovesicle solution and the shiitake mushroom polysaccharide solution are mixed to form a mixed solution, which is used as a radiation protector to resist radiation damage.

[0014] Furthermore, the concentration ratio of the shiitake mushroom-derived exosome-like nanovesicles to the shiitake mushroom polysaccharide in the mixed solution is 1:10-1:40.

[0015] Furthermore, the preparation method of the shiitake mushroom polysaccharide comprises the following steps:

[0016] S1. Crush the dried shiitake mushrooms, mix with ultrapure water, and extract in a constant temperature water bath at 85-95°C. Centrifuge the extract, collect the supernatant, and filter it to obtain a filtrate.

[0017] S2. Rotate and evaporate the filtrate to 1 / 3 of its volume, add 95% ethanol or anhydrous ethanol and place at 2-4°C. After precipitation, centrifuge and separate. Take the precipitate and add ultrapure water to redissolve the polysaccharide. Then dialyze it through a 7000Da dialysis bag for 36 hours. Finally, freeze-dry to obtain the shiitake mushroom polysaccharide.

[0018] Furthermore, in step S1, the material-liquid ratio of shiitake mushroom powder to ultrapure water is 1:20-1:40 mg / mL; and the constant temperature water bath extraction time is 1-4 hours.

[0019] Furthermore, in step S2, the volume of 95% ethanol or anhydrous ethanol is 4 times the volume of the concentrated solution after rotary evaporation, and the storage time is 10-15 hours.

[0020] Furthermore, radiation damage includes decreased in vitro cell viability or varying degrees of damage to multiple organs in animals caused by direct or indirect exposure to ionizing radiation. Examples include hematopoietic damage and liver damage, which are primarily manifested by abnormal blood cell counts, decreased spleen index, oxidative stress, and abnormal liver function.

[0021] Furthermore, the radiation protectant is used to prepare medicines or health foods; types include oral solutions, lozenges, chewable tablets, hard capsules, soft capsules, and powders.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] 1. This invention demonstrates for the first time that shiitake mushroom-derived exosome-like nanovesicles have good gastrointestinal digestion stability and good cellular absorption properties;

[0024] 2. This study demonstrates for the first time that oral ingestion of Biyang floral mushroom exosome-like nanovesicles (BFMELNs) protects animals from ionizing radiation-induced damage.

[0025] 3. The present invention demonstrates for the first time that the exosome-like nanovesicles derived from Biyang floral mushroom combined with crude Biyang floral mushroom polysaccharide (CBFMP) have the effect of synergistically reducing ionizing radiation-induced cell damage. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Flowchart for the extraction of BFMELNs;

[0027] Figure 2 Transmission electron microscopy images of BFMELNs;

[0028] Figure 3 are the particle size distribution, polydispersity index (PDI) and zeta potential of BFMELNs;

[0029] Figure 4 The material identification diagrams of BFMELNs, including (A) protein gel Coomassie blue staining of BFMELNs, (B) agarose gel electrophoresis of BFMELNs, and (C) thin layer chromatography of BFMELNs;

[0030] Figure 5 Figures show the results of cellular uptake of BFMELNs, including (A) representative fluorescence images of HL-7702 cells treated with Dio-labeled BFMELNs, and (B) representative fluorescence images of Caco-2 cells treated with Dio-labeled BFMELNs;

[0031] Figure 6The results of gastrointestinal digestion stability of BFMELNs are shown in Figure 1, including (A) the change in particle size of BFMELNs in different simulated digestive solutions, and (B) the change in Zeta potential of BFMELNs in different simulated digestive solutions.

[0032] Figure 7 The radioprotective effect of BFMELNs on HL-7702 cells;

[0033] Figure 8 Effects of BFMELNs on (A) body weight and (B) spleen index of irradiated mice;

[0034] Figure 9 Effects of BFMELNs on (A) white blood cell (WBC) and (B) platelet (PLT) counts in the peripheral blood of irradiated mice;

[0035] Figure 10 Effects of BFMELNs on (A) superoxide dismutase (SOD) activity and (B) malondialdehyde (MDA) content in the serum of irradiated mice;

[0036] Figure 11 Effects of BFMELNs on (A) alanine aminotransferase (ALT) activity and (B) aspartate aminotransferase (AST) activity in the serum of irradiated mice;

[0037] Figure 12 The results of the radioprotective synergistic effect of BFMELNs and CBFMP are shown, including (A) relative cell viability of cells treated with BFMELNs and CBFMP alone and with the combination of BFMELNs + CBFMP and then irradiated; (B) index of the combined use of BFMELNs and CBFMP. DETAILED DESCRIPTION

[0038] The technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings and embodiments. Obviously, the described embodiments are only part of the embodiments of the invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0039] Example 1: Preparation and characterization of BFMELNs

[0040] Experimental methods: (1) Preparation of BFMELNs: Fresh raw materials of shiitake mushrooms were purchased from Biyang County, Henan Province. The extraction process of shiitake mushroom-derived exosome-like nanovesicles was as follows: Figure 1 As shown in the following example, fresh fruiting bodies of Pleurotus ostreatus were gently washed with deionized water, and the roots were removed. The remaining fruiting bodies were cut into pieces and extracted with 1× PBS buffer (3 times the volume of the fruiting bodies). The fruiting bodies were then crushed with a juicer or broken with a wall breaker. The filtrate was then filtered through qualitative filter paper with the assistance of a vacuum pump to obtain a filtrate. The filtrate was further subjected to differential centrifugation at 1000 × g for 20 min; 2000 × g for 20 min; 4000 × g for 40 min; and 10,000 × g for 60 min. After each centrifugation, the precipitate was removed and the supernatant was collected. The filtrate obtained from the final centrifugation was filtered through 0.2 μm pore size filter paper with the assistance of a vacuum pump to obtain a filtrate. The filtrate was then centrifuged at 150,000 × g for 120 min, and the supernatant was discarded to obtain the precipitate, which is the Pleurotus ostreatus-derived exosome-like nanovesicles (BFMELNs) and stored at -80°C.

[0041] (2) Physicochemical parameters and component identification of BFMELNs: The morphology of BFMELNs was observed using transmission electron microscopy. The zeta potential, PDI, and particle size distribution of BFMELNs were determined using a laser particle size analyzer. Polyacrylamide gel electrophoresis (PAGE), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and silica gel thin-layer chromatography (TLC) were used to detect proteins, RNA, and lipids in BFMELNs, respectively.

[0042] Experimental results: Figure 1 The preparation process of BFMELNs is demonstrated. Figure 2 Transmission electron microscopy images of BFMELNs show that BFMELNs have exosome-like spherical or cup-shaped structures. Figure 3 The hydrated particle size of BFMELNs was shown to be 111.94±10.48 nm, the PDI was 0.251±0.019, and the Zeta potential was -3.77±0.52 mV. Figure 4 A shows that most proteins in BFMELNs are concentrated in the molecular weight range of 25-50 kDa. Figure 4 B shows that most of the RNA in BFMELNs are small RNAs. Figure 4 C shows that BFMELNs are rich in lipids.

[0043] This example demonstrates that BFMELNs possess a nanoscale, cup-shaped structure similar to that of exosomes, leading to their designation as exosome-like nanovesicles derived from Miyang Shiitake Mushroom (BFMELNs). Furthermore, this example demonstrates that BFMELNs contain abundant active proteins, RNA, and lipids, which are the core substances that exert their functional activity. Because their morphology and contents are distinct from traditional active components of natural products (polyphenols, polysaccharides, terpenes, etc.), they can be used as a new class of active ingredients for functional research and corresponding development and application.

[0044] Example 2: Cellular uptake and stability of BFMELNs

[0045] Experimental methods: (1) Cellular uptake of BFMELNs: 30 μmol / L DiO fluorescent dye was added to the BFMELNs solution (BFMELNs dissolved in 1× PBS buffer) and incubated at 37°C for 30 min. The free fluorescent dye was then centrifuged at 150,000×g for 2 h and discarded, and the pellet was resuspended in PBS. DiO-labeled BFMELNs were incubated with human hepatocytes (HL-7702) and human colorectal adenocarcinoma cells (Caco-2) for 12 h, respectively. Images were collected using an inverted microscope at 3, 6, and 12 h of incubation. The cells were washed twice with PBS and the nuclei were stained with Hoechst 33342.

[0046] (2) Stability of BFMELNs: 1.34 μL of 18.5% w / v HCl and 24 μL of pepsin solution (80 mg / mL, dissolved in 0.1 M HCl, pH 2.0) were added to 1 mL of BFMELNs and incubated at 37°C on a shaker for 1 h for simulated gastric digestion. Then, 80 μL of a mixture of 0.1 M NaHCO3, 24 mg / mL bile extract, and 4 mg / mL pancreatic enzyme was added, and the pH was adjusted to 6.5 with 1 M NaHCO3 to form simulated intestinal fluid. The mixture was incubated at 37°C on a shaker for 1 h for simulated intestinal digestion. The stability of the BFMELNs was evaluated by measuring the particle size and zeta potential of the BFMELNs after the simulated gastric and intestinal digestions, respectively.

[0047] Experimental results: Good cellular absorption and uptake help improve the bioavailability of exosomes and further enhance their biological activity in the body. Figure 5 AB shows HL-7702 cells ( Figure 5 A) and Caco-2 cells ( Figure 5B) The uptake capacity of Dio-labeled BFMELNs increased in a time-dependent manner. At 6 h of incubation, BFMELNs aggregated in HL-7702 and Caco-2 cells, and the green fluorescence intensity increased. As the culture time extended to 12 h, the green fluorescence intensity gradually increased without significant attenuation. The in vivo stability results of BFMELNs are shown in Figure 2. Figure 6 AB showed that BFMELNs showed a slight increase in particle size and surface charge reversal after digestion in simulated gastric fluid for 1 h, which was due to the presence of a large amount of H + , thereby affecting the surface charge of some BFMELNs, shifting it from negative to positive, and causing nanovesicle aggregation. In simulated intestinal fluid, the particle size remained below 200 nm and the potential remained negative. These results demonstrate that BFMELNs can remain stable under various temperatures and simulated gastrointestinal digestion. This example demonstrates that the BFMELNs prepared by the present invention are readily absorbed by cells and exhibit good digestive stability, making them suitable for use in pharmaceuticals or health food applications.

[0048] Example 3: Radiation protection capability of BFMELNs

[0049] Experimental methods: (1) In vitro radiation protection ability of BFMELNs: HL-7702 cell suspension was incubated at 4×10 3 90 μL / well was added to a 96-well plate and cultured at 37°C, 5% CO2 for 12 hours. The cells were then divided into a blank control group (NC group), an irradiation control group (IR group), and a BFMELNs irradiation group (BFMELNs group). 10 μL of 1× PBS buffer was added to each well of the NC and IR groups. The BFMELNs group was divided into five concentration gradients and 10 μL of sample solution was added to each well: the final concentrations of the samples were 0.625, 1.25, 2.5, 5, and 10 μg / mL, respectively. After 12 hours of culture, the cells were cultured using 60 Cells were irradiated with Co-γ rays at a dose rate of 2 Gy / min for a total dose of 10 Gy. After irradiation, the cells were cultured in an incubator for 24 hours. The supernatant was removed and 110 μL of CCK-8 working solution (CCK-8: serum: culture medium = 1:1:9) was added. After a further 2 hours of incubation, the absorbance of each well was measured at 450 nm using a microplate reader.

[0050] (2) In vivo radiation protection ability of BFMELNs: Specific pathogen free (SPF) male Kunming mice were randomly divided into 6 groups. The experiment set up a blank control group (NC group, gavage with normal saline), an irradiated control group (IR group, gavage with normal saline), a positive control group (PC group, Licojun tablets), and BFMELNs low-, medium-, and high-dose groups (1 mg / (kg·bw) / d, BFMELNs-L group; 2 mg / (kg·bw) / d, BFMELNs-M group; 4 mg / (kg·bw) / d, BFMELNs-H group). Each mouse was gavaged for 30 consecutive days, and on the 31st day, all mice except the NC group received the same dose of ionizing radiation. The irradiation conditions were as follows: 60 Mice received a single whole-body irradiation with Co-γ rays at a dose rate of 2 Gy / min, for a total radiation dose of 6 Gy. Mice were fasted for one day before being sacrificed and various parameters collected. Mice were weighed daily during oral administration, and spleen weight was measured after sacrifice. The spleen index was calculated by dividing spleen weight by mouse body weight. The number of white blood cells and plasminogen activator cells (PLTs) in the mouse peripheral blood was measured using a hematology analyzer. Serum MDA content and the activities of superoxide dismutase (SOD), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) were measured using kits from the Nanjing Jiancheng Research Institute.

[0051] Experimental results: Relative cell viability can reflect the proliferation and growth status of cells, such as Figure 7 As shown, HL-7702 cells received 10 Gy 60 24 hours after Co-γ ray irradiation, the relative cell viability decreased significantly (P<0.001), proving that IR caused serious damage to the cells, while pre-treatment of cells with 0.625-10 μg / mL of BFMELNs significantly increased cell viability (P<0.05), proving that BFMELNs had a protective effect on IR-induced cell damage. Mouse weight is an important indicator of mouse growth status. Changes in weight can be used to preliminarily determine whether the experimental treatment has a negative impact on the growth of mice. Figure 8 As shown in A, as the number of days of gavage increased, the mice in each group grew well, their weight continued to increase steadily, and there was no significant difference between the groups (P>0.05), indicating that the maximum gavage dose of BFMELNs had no acute toxic side effects on mice. Figure 8 B shows that compared with the NC group, the organ index of the spleen of the IR group mice was significantly decreased (P<0.001). The BFMELNs-M and BFMELNs-H groups had a significant recovery effect on the spleen of the mice after irradiation (P<0.05), indicating that after 6Gy 60 Co-γ ray irradiation caused damage to the spleen of mice, and BFMELNs showed a relatively obvious radiation protective effect on the spleen of mice. Figure 9A. Compared with the NC group, the number of WBC in the peripheral blood of the IR group mice increased from 4.53±0.76×10 9 / L decreased significantly to 1.27±0.28×10 9 / L (P<0.001), while the WBC in the peripheral blood of mice pretreated with BFMELNs was higher than that in the IR group; similarly, Figure 9 B shows that radiation caused a significant decrease in the number of PLTs in mice (P<0.05), compared with 780.83±81.19×10 9 / L, low, medium and high doses of BFMELNs treatments all had significant improvement effects, and the corresponding numbers were (976.83±154.83×10 9 / L, P < 0.05), (1039.83 ± 166.30 × 10 9 / L, P < 0.01), (1024.50 ± 102.06 × 10 9 / L, P<0.01). Figure 10 A shows that the activity of SOD in the serum of mice was significantly reduced after irradiation (P<0.05). Compared with the SOD enzyme activity of the IR group, the SOD enzyme activity of each BFMELNs pretreatment group had a significant improvement effect, among which the BFMELNs-M (P<0.001) and BFMELNs-H (P<0.001) groups had the best improvement effect. Figure 10 B shows that the MDA content in the serum of mice in the IR group was significantly higher than that in the NC group (P<0.001). The increase in serum MDA proves that radiation aggravates the degree of peroxidation in the mice and causes damage. However, BFMELNs pretreatment can significantly reduce the MDA content in irradiated mice (P<0.01), showing a good radiation protection effect in vivo. The excessive increase in serum ALT and AST activity indicates that the liver is damaged. Figure 11 As shown in AB, compared with the NC group, the ALT activity (P<0.01) and AST (P<0.001) activities in the serum of mice in the IR group were significantly increased, indicating that ionizing radiation can cause significant damage to the liver of mice, and different doses of BFMELNs groups have the effect of preventing liver damage to a certain extent (P<0.05).

[0052] This example shows that BFMELNs have a protective effect on IR-induced cell damage in vitro and in vivo. 60 Co-γ ray-induced hematopoietic function and liver damage and the effect of alleviating oxidative stress, and no toxic side effects are shown by oral administration within the effective range.

[0053] Example 4: Radiation protection combination formula of BFMELNs and CBFMP

[0054] Experimental method: (1) After the dried shiitake mushrooms were crushed, they were sieved through a 40-mesh sieve to obtain shiitake mushroom powder. The powder was mixed with ultrapure water at a material-liquid ratio of 1:30 (g / mL) and then extracted in a constant temperature water bath at 90°C for 2 hours. The extract was centrifuged at 4000 r / min for 10 minutes and the filtrate was filtered and rotary evaporated to 1 / 3 of the volume. Then, 95% ethanol or anhydrous ethanol with a volume of 4 times that of the concentrated solution after rotary evaporation was added and placed at 4°C for 12 hours. After the precipitate was precipitated, it was centrifuged at 4000 r / min for 10 minutes. The precipitate was then taken and added to ultrapure water to redissolve the polysaccharide, and dialyzed through a 7000Da dialysis bag for 36 hours. Finally, it was freeze-dried to obtain the crude polysaccharide of Miyang shiitake mushroom. CBFMP was dissolved in 1×PBS and filtered through a 0.22μm filter to prepare a CBFMP solution. The radiation protection combination formula of BFMELNs and CBFMP is named BFMELNs+CBFMP. The preparation method is as follows: BFMELNs solution and CBFMP solution are mixed so that the concentration ratio of the two is 1:30. For example, 200 μg / mL BFMELNs solution and 6000 μg / mL CBFMP solution are mixed in a 1:1 ratio to obtain 100+3000 μg / mL BFMELNs+CBFMP solution.

[0055] (2) The method used was consistent with that in the in vitro radiation protection example of BFMELNs in Example 3, and the specific groups were NC group, IR group, BFMELNs group, CBFMP group, and BFMELNs+CBFMP group. 10 μL of 1×PBS buffer was added to the NC group and the IR group. The sample groups were divided into 5 concentration gradients, and 10 μL of sample solution was added to each well: the final concentrations of the samples in the BFMELNs group were 0.625, 1.25, 2.5, 5, and 10 μg / mL, respectively; the final concentrations of the samples in the CBFMP group were 18.75, 37.5, 75, 150, and 300 μg / mL, respectively; and the final concentrations of the samples in the BFMELNs+CBFMP group were 0.625+18.75, 1.25+37.5, 2.5+75, 5+150, and 10+300 μg / mL, respectively.

[0056] Experimental results: Combination Index (CI) is a method for evaluating the combined effects of drugs, which was first proposed in 1984. This method is based on the dose-effect curve of the sample. By calculating the ratio of the actual effect to the theoretical effect when the sample is used in combination, it is determined whether the combined sample has a synergistic effect, an additive effect or an antagonistic effect. A CI value less than 1 indicates a synergistic effect, equal to 1 indicates an additive effect, and greater than 1 indicates an antagonistic effect. Figure 12 A shows that HL-7702 cells were exposed to 10 Gy 60After Co-γ ray irradiation, the relative cell viability decreased significantly to about 75% (P<0.01). CBFMP and BFMELNs can effectively improve the decreased viability of HL-7702 cells caused by ionizing radiation (P<0.05). BFMELNs alone, CBFMP alone, and BFMELNs + CBFMP combined treatment of HL-7702 cells all have a certain recovery effect on the decreased cell viability induced by IR. The combined index (CI value) of the BFMELNs + CBFMP combination was calculated using CompuSyn software based on the recovery rate of IR cell viability by each treatment. Figure 12 B shows the BFMELNs+CBFMP at the final concentrations of 0.625+18.75, 1.25+37.5, 2.5+75, 5+150 μg / mL after treatment of HL-7702 cells with 10 Gy 60 After Co-γ irradiation, CI values ​​calculated based on relative cell viability at each concentration were 0.12663, 0.10497, 0.22040, and 0.79311, respectively. All CI values ​​were less than 1, indicating that BFMELNs and CBFMP exhibited a synergistic protective effect against ionizing radiation-induced cell damage within the above concentration range. This synergistic effect may be due to the fact that the exosome-like nanovesicles derived from the shiitake mushroom act as carriers for the polysaccharides, effectively delivering them to their target sites. Furthermore, interactions between the exosome-like nanovesicles and the polysaccharides may alter their properties and enhance their radiation protection. This example demonstrates that BFMELNs and CBFMP exhibit synergistic protective effects against IR-induced damage at certain concentrations.

[0057] The results of the four examples above demonstrate that the BFMELNs prepared using the present invention are stable nanoparticles that are well taken up by cells and exhibit good radiation damage prevention (radioprotection) effects both in vivo and in vitro. They also have the potential to synergize with other types of radioprotectants, such as polysaccharides. BFMELNs are derived from the edible mushroom, shiitake mushrooms, have natural ingredients, and have been proven safe in animal studies. Therefore, BFMELNs can be used in radiation protection (anti-radiation) drugs or health foods.

[0058] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. Use of a composition in the preparation of a drug for ionizing radiation protection, characterized in that: The composition is composed of a Miyang shiitake mushroom-derived exosome-like nanovesicle solution and a shiitake mushroom polysaccharide solution, wherein the concentration ratio of the Miyang shiitake mushroom-derived exosome-like nanovesicle to the shiitake mushroom polysaccharide in the composition is 1:10-1:

40. The preparation method of the Miyang shiitake mushroom-derived exosome-like nanovesicles comprises the following steps: Step 1: After cutting off the roots of the fresh shiitake mushroom fruiting bodies, cut the remaining parts into pieces and add an extraction solvent; the extraction solvent is 1×PBS buffer; Step 2: After the cell wall is broken and crushed, the filtrate is filtered to obtain a filtrate, the supernatant is obtained by differential centrifugation, and the precipitate is obtained by ultrahigh-speed centrifugation, namely, the Miyang Flower Oyster Mushroom-derived exosome-like nanovesicles; The differential centrifugation is carried out at centrifugal forces of 800-1200×g for 20-30 min; 2000-3000×g for 20-30 min; The centrifugation is performed at 4000-5000×g for 30-50 min; or 8000-10000×g for 60-120 min, and the precipitate is removed and the supernatant is collected after each centrifugation. The centrifugal force in the ultrahigh-speed centrifugation is 120000-180000×g, and the centrifugation time is 90-150 min.

2. Use of a composition according to claim 1 in the preparation of a drug for protecting against ionizing radiation, characterized in that: The preparation method of the shiitake mushroom polysaccharide comprises the following steps: S1. Crush the dried shiitake mushrooms, mix with ultrapure water, and extract in a constant temperature water bath at 85-95°C. Centrifuge the extract, collect the supernatant, and filter it to obtain a filtrate. S2. Rotate and evaporate the filtrate to 1 / 3 of its volume, add 95% ethanol or anhydrous ethanol and place at 2-4°C. After precipitation, centrifuge and separate. Take the precipitate and add ultrapure water to redissolve the polysaccharide. Then dialyze it through a 7000Da dialysis bag for 36 hours. Finally, freeze-dry to obtain the shiitake mushroom polysaccharide.

3. Use of a composition according to claim 2 in the preparation of a drug for protecting against ionizing radiation, characterized in that: In step S1, the material-liquid ratio of shiitake mushroom powder to ultrapure water is 1:20-1:40 mg / mL; and the constant temperature water bath extraction time is 1-4 hours.

4. Use of a composition according to claim 2 in the preparation of a drug for protecting against ionizing radiation, characterized in that: In step S2, the volume of 95% ethanol or anhydrous ethanol is 4 times the volume of the concentrated solution after rotary evaporation, and the storage time is 10-15 hours.

5. Use of the composition according to claim 1 in preparing a health food having an auxiliary protective effect against the hazards of ionizing radiation.

Images