CN118576592A - Application of ergothioneine or its composition in preparing radiation protection agent - Google Patents

Abstract

The present invention relates to the application of ergothioneine or its composition in the preparation of a radiation protective agent. Ergothioneine or its composition according to one embodiment of the present invention can effectively remove radiation-induced DNA damage and reactive oxygen species, protect normal cells from radiation-induced death, can be used as a radiation protective agent, and can effectively prevent or alleviate radiation diseases.

Description

Application of ergothioneine or its composition in preparing radiation protection agent

Technical Field

The invention relates to ergothioneine or a composition thereof, and in particular to application of ergothioneine or a composition thereof in radiation protection.

Background Art

Radiotherapy that relies on high-energy ionizing radiation (such as X-rays and gamma rays) is one of the three traditional therapies used in clinical treatment of tumors. More than 60% of cancer patients need to receive radiotherapy as a primary or adjuvant treatment. High-energy ionizing radiation damages biological macromolecules such as DNA, proteins and lipids by direct ionization or indirect generation of reactive oxygen species (ROS), thereby killing tumor cells. However, this non-specific killing mechanism will inevitably damage healthy tissues around the lesions and cause serious side effects. From the perspective of the disease progression, early radiation damage manifests as oxidative stress, cell senescence and apoptosis in fast-turnover cells, which in turn induces tissue inflammation. Late radiation damage manifests as tissue fibrosis, vascular damage, nerve damage and organ failure caused by excessive repair of inflammation.

According to the different parts of radiotherapy, the corresponding radiation diseases can be roughly divided into: 1) Whole body or large area (>60%) radiation exposure is easy to induce bone marrow radiation syndrome; 2) Head and neck radiotherapy can damage oral mucosa, salivary glands, eyes and nervous system; 3) Chest radiation is easy to induce radiation damage to organs such as esophagus, lungs, heart, liver, etc.; 4) Pelvic and abdominal radiotherapy usually causes radiation damage to gastrointestinal tract, kidneys, bladder, ovaries, vagina, etc. These radiation diseases not only seriously reduce the quality of life of patients, but also limit the increase of radiation dose and hinder the effect of radiotherapy.

However, the clinical treatment of these radiation diseases only stays at the stage of symptomatic treatment to alleviate symptoms. Currently, only one radioprotectant, amifostine, has been approved clinically to relieve xerostomia and oral mucositis induced by radiotherapy for head and neck cancer. However, the disadvantages of amifostine, such as its extremely short half-life in vivo and narrow medication safety window, severely limit its application in high-dose ionizing radiation protection. Therefore, there is an urgent need to develop a broad-spectrum, efficient and safe radiotherapy protective agent.

Summary of the invention

To overcome at least one defect of the above-mentioned prior art, in a first aspect, one embodiment of the present invention provides the use of ergothioneine or a composition thereof in the preparation of a radiation protective agent.

According to one embodiment of the present invention, the ergothioneine composition comprises ergothioneine, sodium hyaluronate and water.

According to one embodiment of the present invention, in the ergothioneine composition, the mass percentage of the sodium hyaluronate is 0.3-2%.

According to one embodiment of the present invention, the ergothioneine composition is a hydrogel.

In a second aspect, an embodiment of the present invention provides a radiation protectant, comprising ergothioneine or a combination thereof.

In a third aspect, one embodiment of the present invention provides the use of ergothioneine or a composition thereof in the preparation of a medicament for preventing or treating radiation sickness.

According to one embodiment of the present invention, the ergothioneine composition comprises ergothioneine, sodium hyaluronate and water; and/or,

The radiation sickness includes radiation gastroenteritis.

According to one embodiment of the present invention, the radiation disease includes one or more of radiation oral injury, radiation esophageal injury, radiation gastric injury, radiation intestinal injury, radiation lung injury, bone marrow radiation syndrome, radiation liver injury, radiation kidney injury, radiation bladder injury, radiation ovarian injury, radiation vaginal mucosal injury, radiation eye injury, radiation skin injury, radiation vascular injury, radiation nerve injury, and radiation muscle injury.

According to one embodiment of the present invention, the radiation disease is damage induced by exposure to ionizing radiation, and the radiation source of the ionizing radiation includes one or more of X-ray radiation, gamma-ray radiation, radionuclide radiation, electron radiation, neutron radiation, and proton radiation.

In a fourth aspect, one embodiment of the present invention provides a drug for preventing or treating radiation sickness, comprising ergothioneine or a combination thereof.

The ergothioneine or the composition thereof according to one embodiment of the present invention can effectively remove radiation-induced DNA damage and reactive oxygen species, protect normal cells from radiation-induced death, can be used as a radiation protector, and can effectively prevent or alleviate radiation sickness.

In the present invention, the above-mentioned technical solutions can also be combined with each other to achieve more preferred combination solutions. Other features and advantages of the present invention will be described in the subsequent description, and some advantages can become obvious from the description, or can be understood by practicing the present invention. The purpose and other advantages of the present invention can be realized and obtained through the contents particularly pointed out in the description and the drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are only for the purpose of illustrating particular embodiments and are not to be construed as limiting the invention.

in:

Fig. 1 is the clearance rate diagram of ergothioneine of different concentrations to 1,1-diphenyl-2-trinitrophenylhydrazine free radical (DPPH) of embodiment 1-1;

Fig. 2 is the clearance rate diagram of ergothioneine of different concentrations to 2,2-azino-bis(3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt free radical (ABTS) of Example 1-2;

Fig ^. 3 is the clearance rate diagram of superoxide anion free radical (O _2.- ) of ergothioneine of different concentrations of Example 1-3;

Fig. 4 is the scavenging diagram of hydroxyl radical of thioneine of different concentrations of embodiment 1-4;

Fig. 5 is the scavenging situation diagram of the intracellular free radical of X-ray induction by ergothioneine measured by laser scanning confocal microscope of embodiment 1-5;

Fig. 6 is the mitigation diagram of ergothioneine to X-ray-induced cellular DNA damage measured by laser scanning confocal microscope of Example 1-6;

Fig. 7 is the relief situation diagram of the cell viability decline caused by X-ray of ergothioneine recorded by CCK method of Example 1-7;

FIG8 is a graph showing the intestinal adhesion performance of the sodium hyaluronate hydrogel measured by a small animal in vivo imaging device in Example 2;

Fig. 9 is the UV spectrogram of the thioneine-sodium hyaluronate hydrogel of Example 2;

Fig. 10 is the infrared spectrogram of thioneine-sodium hyaluronate freeze-dried gel of Example 2;

Figure 11 is the drug release curve of thioneine-sodium hyaluronate hydrogel of Example 2;

Figure 12 is a H&E histopathological staining image of the ergothioneine-sodium hyaluronate hydrogel intervention for radiation gastroenteritis of Example 2;

Figure 13 is a Ly6G immunofluorescence staining diagram of ergothioneine-sodium hyaluronate hydrogel intervention radiation-induced inflammatory infiltration in Example 2;

Figures 14A and 14B are diagrams showing changes in α-diversity of intestinal flora induced by radiation intervention of ergothioneine-sodium hyaluronate hydrogel in Example 2;

Figure 15 is a diagram showing the disordered structure of intestinal flora community induced by radiation intervention of ergothioneine-sodium hyaluronate hydrogel in Example 2;

Figure 16 is a H&E histopathological staining image of the ergothioneine-sodium hyaluronate hydrogel intervention radiation dermatitis of Example 2;

Figure 17 is an optical microscope image of the ergothioneine calcium alginate microspheres of Comparative Example 1;

Figure 18 is a scanning electron microscope image of the ergothioneine calcium alginate microspheres of Comparative Example 1;

Figure 19 is the thioneine standard curve measured by ultraviolet spectrophotometer of Comparative Example 1;

Figure 20 is an H&E histopathological staining image of ergothioneine calcium alginate microspheres of Comparative Example 1 intervening in radiation enteritis.

DETAILED DESCRIPTION

The preferred embodiments of the present invention are described in detail below, wherein the accompanying drawings constitute a part of the present invention and are used together with the embodiments of the present invention to illustrate the principles of the present invention, but are not used to limit the scope of the present invention.

One embodiment of the present invention provides the use of L-ergothioneine (EGT) or a composition thereof in the preparation of a radiation protective agent.

In one embodiment, ergothioneine is a 2-thioimidazole amino acid, which is a levorotatory isomer and has the following chemical formula:

In one embodiment, ergothioneine can be produced by biosynthetic fermentation, and its purity can be 99.8%.

One embodiment of the present invention provides a radiation protectant, including ergothioneine or a combination thereof.

One embodiment of the present invention provides use of ergothioneine or a composition thereof in the preparation of a medicament for preventing or treating radiation disease.

In one embodiment, in the ergothioneine composition, the mass percentage of ergothioneine can be 0.01-90%, further can be 0.1-70%, and further can be 0.5-50%, for example 0.02%, 0.05%, 0.08%, 0.1%, 0.2%, 0.5%, 1%, 2%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 60%, 80%.

In one embodiment, the ergothioneine composition includes ergothioneine, sodium hyaluronate and water. Wherein, the mass percentage of sodium hyaluronate can be 0.3-2%, further can be 0.5-1%, for example 0.6%, 0.8%, 1.2%, 1.5%, 1.6%, 1.8%.

In one embodiment, the ergothioneine composition may be a hydrogel, and may further be a hydrogel comprising ergothioneine and sodium hyaluronate.

In one embodiment, the ergothioneine composition includes ergothioneine and other types of drugs, and other types of drugs include anti-inflammatory drugs, antibiotics, antiviral drugs, hormone drugs, immunosuppressants, probiotics, prebiotics, short-chain fatty acids, proteins, polypeptides, exosomes, and one or more of nucleic acid drugs.

In one embodiment, radiation sickness is damage induced by exposure to ionizing radiation, and the radiation source of the ionizing radiation may include one or more of X-ray radiation, gamma-ray radiation, radionuclide radiation, electron radiation, neutron radiation, and proton radiation.

In one embodiment, the radiation disease comprises one or more of radiation oral injury, radiation esophageal injury, radiation gastric injury, radiation intestinal injury, radiation lung injury, bone marrow radiation syndrome, radiation liver injury, radiation kidney injury, radiation bladder injury, radiation ovarian injury, radiation vaginal mucosal injury, radiation eye injury, radiation skin injury, radiation vascular injury, radiation nerve injury, and radiation muscle injury.

In one embodiment, the dosage form of the drug includes one or more of a liquid preparation, a semisolid preparation, a solid preparation, a gas preparation, and a micro-nano drug delivery system.

In one embodiment, the liquid preparation includes one or more of a solution, a suspension, an emulsion, an injection, an eye drop, a lotion, and an ointment; the injection may be one or more of a water injection, a powder injection, and an infusion. The emulsion may be one or more of an oil-in-water emulsion, a water-in-oil emulsion, and a double emulsion.

In one embodiment, the semisolid preparation includes one or more of a gel, an ointment, a cream, a suppository, and a paste.

In one embodiment, the solid preparation includes one or more of capsules, tablets, granules, micropills, and films. Tablets can be one or more of ordinary tablets, chewable tablets, effervescent tablets, orally disintegrating tablets, dispersible tablets, lozenges, sustained-release tablets, controlled-release tablets, and enteric-coated tablets. Capsules can be one or more of hard capsules, soft capsules, sustained-release capsules, controlled-release capsules, and enteric-coated capsules.

In one embodiment, the gaseous preparation includes one or more of a powder spray, an aerosol and a spray.

In one embodiment, the propellant of the aerosol comprises one or more of fluorochloroalkanes, hydrofluoroalkanes, propane, n-butane, isobutane, compressed carbon dioxide, nitrogen, and nitric oxide.

In one embodiment, the carrier of the powder aerosol comprises one or more of lactose, xylitol, mannitol, amino acids, and phospholipids.

In one embodiment, the micro-nano drug delivery system includes one or more of microspheres, microcapsules, microemulsions, liposomes, nanospheres, nanoparticles, nanocapsules, and nanoemulsions.

One embodiment of the present invention provides a pharmaceutical composition for preventing or treating radiation diseases, comprising an active ingredient ergothioneine and a pharmaceutically acceptable excipient.

In one embodiment, the pharmaceutical excipients of solid preparations can be divided into one or more of fillers, adhesives, wetting agents, disintegrants, lubricants, glidants, capsule materials, plasticizers, coatings, colorants, flavoring agents, retardants, pore-forming agents, and suppository bases according to their functions; the pharmaceutical excipients of liquid preparations or semisolid preparations can be divided into one or more of solvents, cosolvents, solubilizers, pH regulators, osmotic pressure regulators, suspending agents, dispersants, gelling agents, emulsifiers, oil phase bases, preservatives, antibacterial agents, and transdermal absorption enhancers according to their functions; the pharmaceutical excipients of gaseous preparations can be divided into one or more of solvents, cosolvents, antioxidants, antibacterial agents, propellants, lubricants, glidants, antistatic agents, and carriers according to their functions.

In one embodiment, the pharmaceutical excipients of the drug include one or more of lactose, sucrose, mannitol, sorbitol, calcium sulfate, calcium carbonate, calcium hydrogen phosphate, magnesium oxide, talc, micro-powdered silica gel, starch and its derivatives, cellulose and its derivatives, gum arabic, agar, sodium alginate, guar gum, hyaluronic acid, chitosan, tragacanth gum, pectin, gelatin, albumin, shellac, polyethylene hydrocarbons, polyacrylic acids, polyesters, polyethers, polyamino acids, silicones, hydrocarbons, higher fatty acids and their alcohol esters, hydrogenated vegetable oils, polydimethylsiloxanes, sodium fatty alcohol sulfates, stearates, oleates, fatty acid sorbitans, polyoxyethylene fatty alcohol ethers, polysorbates, fatty acid glycerides, stearic acid, cocoa butter, phospholipids, and cholesterol.

In one embodiment, starch and its derivatives include one or more of starch, pregelatinized starch, dextrin, dry starch, sodium carboxymethyl starch, and hydroxyethyl starch. Cellulose and its derivatives include one or more of powdered cellulose, microcrystalline cellulose, sodium carboxymethyl cellulose, cross-linked sodium carboxymethyl cellulose, methyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, cellulose acetate, cellulose acetate phthalate, hydroxypropyl methyl cellulose phthalate, and hydroxypropyl methyl cellulose acetate succinate. Polyethylene hydrocarbon compounds include one or more of povidone, cross-linked povidone, polyvinyl alcohol, polyvinyl alcohol phthalate, ethylene-vinyl acetate copolymer, polyvinyl acetate phthalate, and polyisobutylene pressure-sensitive adhesive. Polyacrylic acid compounds include one or more of carbomer, acrylic resins, sodium polyacrylate, cross-linked sodium polyacrylate, and polyacrylic acid pressure-sensitive. Polyester compounds include one or more of polylactic acid, lactic acid-glycolic acid copolymer, polycaprolactone, and polydinonyl sebacate. Polyether compounds include one or more of polyethylene glycols, poloxamers, and polyoxyethylene fatty acid esters. The hydrocarbon compound includes one or more of vaseline, paraffin, and liquid paraffin. The higher fatty acid and its alcohol ester include one or more of lanolin, beeswax, and spermaceti.

In one embodiment, the osmotic pressure regulator includes one or more of sodium chloride, mannitol, glucose, phosphate, and acetate.

In one embodiment, the transdermal absorption enhancer includes one or more of ethanol, propylene glycol, ethyl acetate, dimethyl sulfoxide, dimethylamide, oleic acid, linoleic acid, lauryl alcohol, laurocapron and its homologues, lecithin, urea, salicylic acid, pyrrolidines, menthol, and camphor.

In one embodiment, the antioxidant includes one or more of vitamin E, butylated hydroxytoluene, ascorbic acid, sulfite, citric acid, and tartaric acid.

In one embodiment, the preservative includes one or more of methylparaben, ethylparaben, phenol, benzoic acid, sorbic acid, benzalkonium chloride, alkyltrimethylammonium bromide, and chlorobutanol.

In one embodiment, the drug is classified by function and includes one or more of ordinary preparations, sustained-release preparations, controlled-release preparations, and targeted preparations.

In one embodiment, the administration route of the drug includes one or more of oral administration, buccal administration, sublingual administration, inhalation administration, nasal administration, transdermal administration, injection, ocular administration, and cavity administration.

In one embodiment, the dosage of the drug can be 0.001-300 mg/kg body weight, further can be 0.1-250 mg/Kg, and further can be 5-150 mg/Kg. For example, 1 mg/Kg, 5 mg/Kg, 10 mg/Kg, 20 mg/Kg, 50 mg/Kg, 100 mg/Kg, 150 mg/Kg, 200 mg/Kg, 250 mg/Kg, 300 mg/Kg, 350 mg/Kg, 400 mg/Kg, 450 mg/Kg.

In one embodiment, the drug is administered in a reasonable manner according to the dosage form, and the drug can be administered before and/or after irradiation, and the optimal administration time is from three days before irradiation to a period of time after irradiation. The specific course of treatment depends on the relief of the patient's symptoms.

In one embodiment, the radiation gastroenteritis model is established by intraperitoneal irradiation of male Balb/C mice (6-8 weeks old). The radiation source includes one or more of gamma rays, X-rays, and various particle radiation exposures. The X-ray irradiation dose can be 0.1-50 Gy, and further can be 3-28 Gy, for example 5 Gy, 10 Gy, 15 Gy, 20 Gy, 25 Gy, 30 Gy, 35 Gy, 40 Gy, 45 Gy.

In one embodiment, for the prevention or treatment of radiation gastroenteritis, the dosage form of the drug is preferably an oral gel, which can effectively prolong the retention time of water-soluble small molecule ergothioneine in the gastrointestinal tract to prolong its efficacy. The excipients included in the oral gel may include one or more of a thickener, an antioxidant, and a pH regulator. The gel matrix may include one or more of natural polymers, semi-synthetic and synthetic polymers; preferably, the gel matrix includes one or more of alginate, pectin, gelatin, agar, gum arabic, hyaluronate, chitosan and derivatives, starch and its derivatives.

In one embodiment, the radiation dermatitis model is established by local back irradiation of male Balb/C mice (6-8 weeks old). The radiation source includes one or more of gamma rays, X-rays and various particle beam exposures. The X-ray irradiation dose can be 0.1-90Gy, and further can be 15-50Gy, such as 5Gy, 10Gy, 20Gy, 25Gy, 30Gy, 35Gy, 40Gy, 45Gy, 55Gy, 60Gy, 65Gy, 70Gy, 75Gy, 80Gy, 85Gy.

The ergothioneine or the composition thereof according to one embodiment of the present invention can effectively alleviate the cell damage and tissue damage caused by radiation, and play a therapeutic role on radiation diseases.

The ergothioneine or a composition thereof according to one embodiment of the present invention can effectively remove radiation-induced DNA damage and reactive oxygen species, protect normal cells from radiation-induced death, thereby effectively preventing or alleviating radiation sickness, and improving the quality of life of radiotherapy patients and the radiotherapy dose threshold.

The radiation protection agent or medicine containing ergothioneine according to one embodiment of the present invention can be administered before radiation occurs to prevent radiation diseases; it can also be administered after radiation occurs to treat radiation diseases; it can also be administered both before and after radiation occurs.

The radiation protection agent or medicine containing ergothioneine according to one embodiment of the present invention is a hydrogel (or oral gel) containing ergothioneine and sodium hyaluronate, which has a good protective effect on radiation gastroenteritis and radiation dermatitis.

Below, in conjunction with accompanying drawing and specific embodiment, ergothioneine or its composition of one embodiment of the present invention is further described.

Example 1-1

First, 100mM DPPH free radical solution is prepared with absolute ethanol as solvent. Subsequently, the thioneine aqueous solution (EGT solution) of 2 μg/mL, 7 μg/mL, 15 μg/mL, 30 μg/mL is prepared respectively. The DPPH free radical solution and the EGT solution of each concentration are mixed in equal volumes and reacted in dark for 30min, and then the absorption value of each mixture at 517nm is detected using an ultraviolet spectrophotometer. The removal results of DPPH by different concentrations of EGT are as shown in Figure 1. As can be seen from Figure 1, thioneine presents a dose-dependent increase in the scavenging ability of DPPH free radicals, and the scavenging rate of DPPH free radicals by low-content thioneine (15 μg/mL) can exceed 75%.

Example 1-2

First, 7mM ABTS aqueous solution and 2.45mM potassium persulfate were reacted under room temperature lucifuge for 16 hours to obtain ABTS free radical solution, and then the above mixture was diluted 40 to 50 times with appropriate volume of PBS to prepare ABTS free radical working solution. Subsequently, 1 μg/mL, 2 μg/mL, 5 μg/mL, 10 μg/mL of ergothioneine aqueous solution (EGT solution) were prepared respectively. DPPH free radical working solution and the EGT solution of each concentration were mixed in equal volumes and reacted in lucifuge for 10 min, and then the absorption value of each mixture at 734 nm was detected using an ultraviolet spectrophotometer. The removal results of different concentrations of EGT to ABTS free radicals are shown in Figure 2. As can be seen from Figure 2, ergothioneine presents a dose-dependent increase in the scavenging ability of ABTS free radicals, and the scavenging rate of low-content ergothioneine (5 μg/mL) to ABTS free radicals can reach nearly 100%.

Examples 1-3

First, 1mM NADH, 0.25mM NBT, 15μM PMS aqueous solution and 60μg/mL, 300μg/mL thioneine aqueous solution were prepared. 200μL PBS, 100μL NADH, 100μL NBT and 100μL thioneine aqueous solution were mixed, and then 100μL PMS was added to start O ₂ · ^-generation reaction. After the reaction was started, the absorption value of each mixture at 560nm was measured immediately, and 300s was recorded by UV-vis spectrophotometer. The scavenging ability of thioneine at different working concentrations to superoxide anions, the results are shown in Figure 3. As can be seen from Figure 3, the thioneine solution with a concentration of 10μg/mL showed a strong scavenging ability to superoxide anion free radicals.

Examples 1-4

First, 4mM FeSO ₄ solution and 40mM H ₂ O ₂ aqueous solution with pH 4 acetate buffer as solvent, 1mM TMB solution and 100μg/mL, 600μg/mL thioneine aqueous solution with DMSO as solvent were prepared. Then, 150μL TMB, 150μL H ₂ O ₂ and 150μL thioneine aqueous solution were mixed, and 150μL FeSO ₄ solution was added to start the reaction and the reaction was protected from light for 5min. The absorption curve of each mixed solution in the range of 550-750nm was measured by UV-visible spectrophotometer. The scavenging ability of thioneine at different working concentrations for hydroxyl radicals is shown in Figure 4. As can be seen from Figure 4, the thioneine solution with a concentration of 150μg/mL showed a strong scavenging ability for hydroxyl radicals.

Examples 1-5

The removal of intracellular reactive oxygen species induced by irradiation by ergothioneine was investigated by reactive oxygen probe (DCFH-DA). Rat small intestinal crypt epithelial cells (IEC-6) were cultured in confocal culture dishes at a density of 2×10 ⁵ per dish. After the cells adhered to the wall, complete medium containing 50 μg/mL ergothioneine was added and incubated for 3 h. Then, DCFH-DA probes and Hoechst 33342 dyes at appropriate concentrations were added to pure DMEM, and 1 mL of the above dye solution was added to each dish to treat the cells at 37 ° C for 30 min. After 6 Gy irradiation was completed, fluorescence images were collected immediately with a laser scanning confocal microscope, and specific results were shown in Figure 5. In Figure 5, "Ctrl" represents untreated cells, "EGT" represents cells pre-incubated with ergothioneine, "irradiation" represents irradiated cells, "irradiation+EGT" represents cells pre-incubated with ergothioneine+irradiated, and "positive control" represents cells treated with reactive oxygen inducing agents.

As shown in Figure 5, a large amount of reactive oxygen species is produced in IEC-6 cells after X-ray irradiation, which is manifested as a large range of DCF positive signals. However, there is no obvious DCF positive signal in cells incubated with ergothioneine in advance, indicating that ergothioneine can effectively remove reactive oxygen species in cells induced by irradiation.

Examples 1-6

The mitigation of irradiation-induced DNA double-strand breaks by ergothioneine was investigated by immunofluorescence experiments. IEC-6 cells were cultured in 24-well plates with cell slides at a density of 5×10 ⁴ cells per well. After the cells adhered, complete medium containing 50 μg/mL ergothioneine was added, incubated for 3 hours, and then irradiated with 6 Gy. After irradiation for 3 hours, the cells were fixed with 4% paraformaldehyde, and then 200 μL of punching agent (0.2% Triton 100×, 99.8% PBS) was added for 10 minutes, and then blocked with blocking agent (1% Triton 100×, 5% FBS, 94% PBS) for 1 hour. After that, the cells were treated with phosphorylated histone H ₂ AX antibody and incubated overnight at 4°C. After recovering the primary antibody, the cells were washed and treated with fluorescent secondary antibodies at 37°C for 1 hour. After incubation with Hoechst 33342 dye to stain the nucleus, the slides were sealed. Finally, fluorescence images were taken using a laser scanning confocal microscope, and the specific results are shown in Figure 6. In Figure 6, "Ctrl" represents untreated cells, "EGT" represents cells pre-incubated with ergothioneine, "irradiation" represents irradiated cells, and "irradiation+EGT" represents cells pre-incubated with ergothioneine+irradiation.

As shown in Figure 6, obvious DNA damage appeared in the nucleus of IEC-6 cells after X-ray irradiation, which was manifested by the appearance of a large number of γ-H ₂ AX foci. The positive signal of γ-H ₂ AX in cells pre-treated with ergothioneine was significantly reduced, indicating that ergothioneine can alleviate the DNA damage caused by irradiation.

Examples 1-7

The CCK method was used to investigate the mitigation of the decrease in IEC-6 cell viability caused by radiation by ergothioneine. IEC-6 cells were cultured in 96-well plates at a density of 4×10 ³ per well. After cell attachment, they were incubated for 3 h in a complete medium containing 50 μg/mL ergothioneine, and then irradiated with 0, 2, 6, 8, and 10 Gy, respectively. After irradiation for 40 h, 100 μL of 10% CCK-8 solution was quickly added to each well, incubated for 1 h, and finally the absorbance of each well at 450 nm was detected by an ELISA instrument. For specific results, see Figure 7. In Figure 7, "Ctrl" represents irradiated cells, and "EGT" represents irradiated cells after pre-incubation of ergothioneine.

As shown in Figure 7, with the increase of irradiation dose, the viability of cells in the control group (Ctrl) and the group (EGT) incubated with ergothioneine before irradiation gradually decreased. However, incubation of ergothioneine before irradiation can significantly improve the viability of IEC-6 cells under the same radiation dose, which shows that ergothioneine shows a favorable radiation protection effect at the cellular level.

Example 2

Study on the intestinal adhesion properties of sodium hyaluronate (HA) oral gel

First, prepare a 0.5 mg/mL cyanine fluorescein (Cy5) aqueous solution. Secondly, use an appropriate volume of Cy5 solution to dissolve an appropriate amount of HA, and swell it sufficiently to form a 1% HA-Cy5 gel. Then, 7 BALB/c mice were randomly divided into 3 groups and gavaged with 200 μL of distilled water (1 mouse in the blank group), Cy5 aqueous solution, and Cy5-HA hydrogel, respectively. The mice were killed 2h, 6h, and 12h after gavage, and the gastrointestinal tissues and other organs of the mice were collected for in vivo fluorescence imaging analysis.

As shown in Figure 8, 2 hours after intragastric administration, Cy5 fluorescence was distributed almost throughout the gastrointestinal tract of the mice in the Cy5 aqueous solution group and the Cy5-HA group. The gastrointestinal fluorescence signal of the Cy5 aqueous solution group was significantly weaker than that of the Cy5-HA group, which may be due to the fact that free Cy5 is more easily absorbed into the blood and redistributed to other organs or excreted from the body. In addition, 2 hours after intragastric administration, the fluorescence signal in the stomach of the Cy5-HA group was the strongest, while the fluorescence signal in the lower jejunum of the Cy5 aqueous solution group was the strongest, indicating that HA has bioadhesive properties in the gastrointestinal tract.

After 6 hours, there was almost no Cy5 distribution in the upper gastrointestinal tract of the Cy5 aqueous solution group, and the colon became the main accumulation site of Cy5. In contrast, the Cy5-HA group showed obvious fluorescence signals in the stomach, duodenum and jejunum, further reflecting the improvement of HA on the retention of small molecules in the gastrointestinal tract. In addition, when the time was extended to 12 hours, the Cy5-HA group showed obvious fluorescence signals in the gastrointestinal tract compared with the Cy5 aqueous solution group. The above results show that gastrointestinal adhesive sodium hyaluronate gel has the ability to enhance the retention of water-soluble small molecules in the gastrointestinal tract, and is expected to further enhance its intestinal protective effect by promoting the intestinal retention of ergothioneine.

Preparation and characterization of ergothioneine-sodium hyaluronate hydrogel

1. Preparation: Prepare 5mg/mL of thioneine aqueous solution, ultrasonically dissolve it fully; add appropriate amount of sodium hyaluronate to the aqueous solution after ultrasonication, stir, stand for 4-5 hours to fully swell and deaerate, and obtain final thioneine-sodium hyaluronate hydrogel. Wherein, the mass percentage of sodium hyaluronate is 1%.

2. UV detection: After diluting thioneine-sodium hyaluronate hydrogel, thioneine aqueous solution, and sodium hyaluronate hydrogel 500 times with deionized water, the UV absorption spectra of the three in the 200-300nm band were measured and compared. For specific results, see Figure 9. In the figure, "EGT-HA" represents thioneine-sodium hyaluronate hydrogel, "EGT" represents thioneine aqueous solution, and "HA" represents sodium hyaluronate hydrogel.

As shown in Figure 9, by comparing the UV spectra of EGT-HA and EGT solution of the same concentration, it can be seen that the introduction of HA did not change the peak position and absorption intensity of the characteristic absorption peak of EGT at 257nm. This indicates that EGT-HA gel is formed through physical interaction between EGT and HA.

3. Infrared detection: Take thioneine-sodium hyaluronate freeze-dried hydrogel, a powder mixture of thioneine and sodium hyaluronate, pure thioneine powder and pure sodium hyaluronate powder, grind them thoroughly, take appropriate amounts and grind and mix them with potassium bromide powder, press them into tablets, measure and compare the infrared absorption spectra of each sample, and see Figure 10 for specific results.

As shown in Figure 10, by comparing EGT-HA freeze-dried gel, mixed powder of EGT and HA, pure HA powder and pure EGT powder, it can be found that EGT-HA freeze-dried gel and the mixed powder of the two have almost the same infrared spectra. This shows that the process of HA fully swelling in EGT solution to form EGT-HA gel does not involve chemical reaction, which once again shows that there is only physical interaction between the two.

4. Drug release behavior: First, prepare EGT aqueous solutions with concentrations of 1, 2, 4, 6, 8, 10, and 12 μg/mL, measure the absorbance of each solution at 257nm, and establish a standard curve. Then, three equal volumes of the above EGT-HA gel were respectively placed in dialysis bags with a cutoff diameter of 1000Da and sealed with clips. The above dialysis bags were immersed in gastrointestinal simulation buffers containing 100mL of pH 1.2, pH 6.8, and pH 7.4 in sequence, and released for 2, 3, and 19h at 100rpm and 37°C, respectively. At the selected time point, 2mL of supernatant was drawn from the release medium, and 2mL of the corresponding buffer was immediately added. Finally, the absorbance value of each supernatant at 257nm was detected, the cumulative drug release rate of the sampling point was calculated, and the drug release curve of the EGT-HA gel was plotted. See Figure 11 for specific results.

The release of EGT from EGT-HA gel and maintaining its inherent chemical structure is the prerequisite for EGT to be absorbed by its specific receptors and play a protective role in radiotherapy. As shown in Figure 11, EGT-HA gel can cumulatively release about 50% of EGT in two hours in simulated gastric fluid, continue to release about 30% of EGT in three hours in simulated intestinal fluid, and continue to release in simulated colon fluid for about 19 hours to achieve complete release of EGT. The above results show that EGT-HA gel can release EGT in all segments of the gastrointestinal tract, which provides a basis for EGT-HA as a gastrointestinal radiation protector.

Protection test of ergothioneine-sodium hyaluronate hydrogel against radiation gastroenteritis

The protective effect of the optimal prescription of ergothioneine sodium hyaluronate oral gel on radiation gastroenteritis was evaluated by H&E histopathological staining. BALB/c male mice aged 6 to 8 weeks were randomly divided into: 1) normal group: gavage with water, no irradiation; 2) EGT-HA group: gavage with ergothioneine sodium hyaluronate oral gel, no irradiation; 3) irradiation group: gavage with water and whole abdomen irradiation; 4) irradiation + EGT-HA group, gavage with ergothioneine sodium hyaluronate oral gel and whole abdomen irradiation. The administration method was gavage three times every other day before irradiation, and gavage every other day after irradiation until the mice were sacrificed on the sixth day after irradiation. One hour before irradiation, mice were gavaged with water or EGT-HA, and then anesthetized with 1% sodium pentobarbital solution, followed by whole abdominal irradiation, and the irradiation parameters were 6Gy, 160KV, and 25mA. On the sixth day after irradiation, the mice were sacrificed, and the stomach and small intestine tissues were fixed in paraformaldehyde fixative for 48 hours and sent for H&E pathological examination. The H&E pathological sections were photographed using an inverted fluorescence microscope, and the specific results are shown in Figure 12.

As shown in Figure 12, on the sixth day after radiation, the epithelium of the mouse gastric pits was severely damaged, the gastric mucosa was severely eroded, and gastric cells, including mucus neck cells, parietal cells, and chief cells, were severely missing. In contrast, oral administration of ergothioneine sodium hyaluronate gel can greatly alleviate acute radiation gastritis, and the degree of epithelial defects and mucosal erosion is greatly reduced.

Compared with the intact and orderly intestinal villi and crypts in the non-irradiated group, the intestinal structure of the irradiated mice was severely damaged on the sixth day, characterized by a large number of broken and fallen villi, loss of crypts, and inflammatory infiltration. In contrast, oral ergothioneine sodium hyaluronate gel can significantly maintain the integrity and height of the villi of irradiated mice and alleviate inflammatory infiltration. Therefore, the oral ergothioneine preparation showed a good protective effect against radiation gastroenteritis.

Ergothioneine-sodium hyaluronate hydrogel alleviates radiation-induced inflammatory infiltration

The Ly6G immunofluorescence staining method was used to evaluate the relief of radiation-induced inflammatory infiltration by the superior prescription ergothioneine sodium hyaluronate oral gel. For details of the animal experiment process, please refer to the "Protection test of ergothioneine-sodium hyaluronate hydrogel against radiation gastroenteritis" section. On the sixth day after irradiation, the mice were sacrificed, and the small intestinal tissue of the mice was taken and immersed in paraformaldehyde fixative for 48 hours, and then sent to Seville Biotechnology Co., Ltd. for Ly6G immunofluorescence staining. The immunofluorescence stained sections were photographed using a laser scanning confocal microscope, and the specific results are shown in Figure 13.

As shown in Figure 13, on the 6th day after irradiation, obvious Ly6G signals (white bright spots) appeared in the mucosa and submucosa of the intestinal tissue after irradiation, while the Ly6G signal in the irradiation + EGT-HA group was much weaker. Ly6G is a specific marker for neutrophils. Neutrophils are an important type of white blood cell that can promote the progression of radiation enteritis by performing a respiratory burst. Radiation enteritis can essentially be considered as a tissue inflammation marked by leukocyte infiltration. Therefore, this result indicates that EGT-HA can alleviate the severity of radiation enteritis caused by radiation.

Ergothioneine-sodium hyaluronate hydrogel alleviates radiation-induced intestinal flora disturbance

The 16s rDNA sequencing technology was used to evaluate the relief of radiation-induced intestinal flora disturbance by the optimal prescription of ergothioneine sodium hyaluronate oral gel. For details of the animal experiment process, please refer to the "Protection test of ergothioneine-sodium hyaluronate hydrogel against radiation gastroenteritis" section. On the sixth day after irradiation, the mice were sacrificed, and fecal samples from the cecum and colon of each group of mice were taken under sterile conditions and placed in sterile EP tubes, and quickly frozen in a -80℃ refrigerator until sent for inspection. The fecal samples of each group were stored in dry ice and sent to Ouyi Biotechnology Co., Ltd. for 16s rDNA sequencing. The specific results and discussions are as follows.

Studies have shown that radiation-induced intestinal flora disturbance is one of the important mechanisms that promote the occurrence and development of radiation enteritis, which can aggravate radiation enteritis by destroying the permeability of the intestinal barrier, affecting the growth and proliferation of intestinal cells, and inducing the release of inflammatory factors. Radiation reduces the diversity of intestinal flora and significantly changes the community structure of intestinal microorganisms. The α-diversity parameters of intestinal flora can be used to evaluate the richness and distribution uniformity within species. As shown in Figures 14A and 14B, on the 6th day after radiation, the α-diversity parameters of the intestinal flora of mice in the irradiated group were low, including the number of species observed (left) and the ShannonWiener index (right), indicating that the diversity of the intestinal flora of mice decreased after radiation. Oral administration of EGT-HA alleviated this downward trend, suggesting that EGT-HA has the ability to maintain the diversity of intestinal flora. In addition, EGT-HA can positively regulate radiation-induced intestinal microbial structure disorder.

As shown in Figure 15, the abundance of Proteobacteria in the intestinal flora of mice in the irradiated group increased significantly, while the abundance of Bacteroidetes decreased significantly. Proteobacteria contains many common pathogenic Gram-negative bacteria, such as Escherichia coli, which are important causes of dysbiosis and intestinal inflammation. In contrast, intestinal Bacteroidetes ferment exogenous fiber to produce beneficial short-chain fatty acids, promote the development and balance of the immune system, and play anti-inflammatory and anti-infective effects, playing an important role in maintaining intestinal homeostasis. Oral administration of EGT-HA gel can increase the abundance of beneficial Bacteroidetes and reduce the abundance of harmful Proteobacteria, indicating that EGT-HA has a positive remodeling ability for irradiation-induced intestinal flora imbalance.

Protection test of ergothioneine-sodium hyaluronate hydrogel against radiation dermatitis

BALB/c male mice with hair loss on their backs aged 6-8 weeks were divided into 4 groups: 1) Normal group: no treatment; 2) Irradiation group: irradiation of the back of mice with an x-ray tube for 90s (50kV, 75μA), and upper body lead plate sealing; 3) Irradiation + superoxide dismutase (SOD) ointment group; 4) Irradiation + ergothioneine sodium hyaluronate hydrogel (EGT-HA) group. In groups 3 and 4, SOD ointment and EGT-HA hydrogel were evenly applied on the back skin of mice (covering an area of ^4cm2 , ≈0.2g) 1h before irradiation. The mice were photographed every day for 21 consecutive days. See Figure 16 for specific results.

As shown in Figure 16, on the 5th day of irradiation, obvious tissue erythema appeared in the irradiation group and the irradiation + SOD ointment group, while no obvious abnormality was observed in the irradiation + EGT-HA group; on the 15th day of irradiation, the simple irradiation group and the irradiation + SOD ointment group reached the peak of radiation dermatitis, with large areas of moist desquamation and skin bleeding and ulceration; in contrast, the irradiation + EGT-HA group was able to effectively delay the onset of inflammation, showing only mild moist desquamation. On the 21st day of irradiation, the simple irradiation group showed skin ulceration, bleeding and scab, while the irradiation + EGT-HA gel and irradiation + SOD ointment groups showed varying degrees of recovery, among which the irradiation + EGT-HA gel group had less damage and recovered faster. This shows that EGT-HA skin gel can effectively protect against radiation dermatitis.

Comparative Example 1

Preparation of calcium alginate microspheres loaded with ergothioneine

A 16 mg/mL aqueous solution of thioneine was prepared and fully dissolved by ultrasound; appropriate amounts of sodium alginate and pectin powder were added to the completely dissolved thioneine solution and stirred at 300 rpm for 1 h to prepare a completely swollen sol containing 4% sodium alginate and 1% pectin (hereinafter referred to as "A4P1@EGT").

The sol solution was allowed to stand for 1 hour for degassing; thereafter, the sol solution was transferred to a syringe and microspheres were prepared by gas shearing method. The peristaltic pump pushing speed was 1 mm/min, the pressure of the spraying gas was 0.1 MPa, and the crosslinking agent was a 400 mM CaCl ₂ solution. After the sol solution was sprayed, it was cured in the crosslinking agent for 20 minutes. Fresh microspheres were collected by centrifugation and lightly washed, and the fresh microspheres were freeze-dried to obtain A4P1@EGT freeze-dried microspheres. The fresh A4P1@EGT microspheres were characterized by optical microscopy, and the freeze-dried microspheres were characterized by scanning electron microscopy. The results are shown in Figures 17 and 18.

As can be seen from Figure 17, the A4P1 calcium-based microspheres are spherical with uniform size and regular shape, and the particle size of fresh microspheres is mostly between 5-20 μm. As can be seen from Figure 18, the A4P1 calcium-based microspheres are freeze-dried to form relatively regular spherical shapes with a size of about 500 nm.

Test on the radiation protection effect of calcium alginate microspheres loaded with ergothioneine

Prepare 1 μg/ml, 3.125 μg/ml, 6.25 μg/ml, 8 μg/ml, 10 μg/ml, and 12.5 μg/ml EGT aqueous solutions respectively, and use a UV spectrophotometer to measure the absorbance of each solution at the maximum UV absorption wavelength of ergothioneine, 256 nm, and draw a standard curve with concentration as the abscissa and absorbance as the ordinate. See Figure 19 for the results. Take the supernatant solution remaining after centrifugation and dilute it by an appropriate multiple, measure the absorbance of the supernatant dilution at 256 nm, and calculate the encapsulation efficiency of the microspheres. Encapsulation efficiency = (1-W _f /W _t ) × 100%, where W _f is the amount of free drug in the supernatant, and W _t is the total amount of drug input.

A4P1@EGT fresh microspheres were prepared according to the above method. The absorbance of the supernatant was measured using an ultraviolet spectrophotometer and the encapsulation efficiency of the microspheres was calculated. According to the encapsulation efficiency, an appropriate volume of distilled water was added to resuspend the fresh microspheres so that the concentration of ergothioneine was 5 mg/mL. BALB/c male mice aged 6 to 8 weeks were randomly divided into 1) normal group: gavage with water, no irradiation; 2) A4P1@EGT group: gavage with A4P1@EGT microspheres, no irradiation 3) irradiation group: gavage with water and whole abdomen irradiation; 4) irradiation + A4P1@EGT group: gavage with A4P1@EGT and whole abdomen irradiation. The administration method was gavage three times every other day before irradiation, and gavage every other day after irradiation until the mice were sacrificed on the sixth day after irradiation. One hour before irradiation, mice were gavaged with water or A4P1@EGT, and then anesthetized with 1% sodium pentobarbital solution, followed by whole abdominal irradiation, with irradiation parameters of 6Gy, 160KV, 25mA. On the sixth day after irradiation, mice were sacrificed, and small intestinal tissue was taken and fixed in paraformaldehyde fixative for 48h and sent for H&E pathological examination. H&E pathological sections were photographed with an inverted fluorescence microscope. See Figure 20 for specific results.

As shown in Figure 19, the standard curve of ergothioneine is y=0.0593x+0.0684R ² =0.9936, and the encapsulation efficiency of A4P1@EGT calculated according to this formula is about 17%. As shown in Figure 20, compared with the complete intestinal villi and orderly arranged crypts of the normal group, the intestinal structure of irradiated mice was severely damaged on the sixth day, characterized by a large amount of villi breaking and falling off, crypt loss and inflammatory infiltration. In contrast, oral ergothioneine calcium alginate microspheres only showed a relatively general radiation protection effect, which was manifested as a significant shortening and a large degree of shedding of small intestinal villi.

According to the above test results, the thioneine-sodium hyaluronate hydrogel of Example 2 has a good protective effect on radiation gastroenteritis and radiation dermatitis. And in the protection against radiation gastroenteritis, the thioneine-sodium hyaluronate hydrogel of Example 2 is better than the calcium alginate microspheres loaded with thioneine of Comparative Example 1.

The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by any technician familiar with the technical field within the technical scope disclosed by the present invention should be covered within the protection scope of the present invention.

Claims (10)

1. Application of ergothioneine or its composition in the preparation of a radiation protector. 2. application according to claim 1, wherein, described thioneine composition comprises thioneine, sodium hyaluronate and water. 3. application according to claim 2, wherein, in the ergothioneine composition, the mass percentage of the sodium hyaluronate is 0.3-2%. 4. application according to claim 1, wherein the ergothioneine composition is a hydrogel. 5. A radioprotectant comprising ergothioneine or a combination thereof. 6. Use of ergothioneine or its composition in the preparation of a medicine for preventing or treating radiation disease. 7. Application according to claim 6, wherein the ergothioneine composition comprises ergothioneine, sodium hyaluronate and water; and/or, The radiation sickness includes radiation gastroenteritis. 8. The use according to claim 6, wherein the radiation disease comprises one or more of radiation oral injury, radiation esophageal injury, radiation gastric injury, radiation intestinal injury, radiation lung injury, bone marrow radiation syndrome, radiation liver injury, radiation kidney injury, radiation bladder injury, radiation ovarian injury, radiation vaginal mucosal injury, radiation eye injury, radiation skin injury, radiation vascular injury, radiation nerve injury, and radiation muscle injury. 9. The use according to claim 6, wherein the radiation disease is damage induced by exposure to ionizing radiation, and the radiation source of the ionizing radiation includes one or more of X-ray radiation, gamma-ray radiation, radionuclide radiation, electron radiation, neutron radiation, and proton radiation. 10. A medicine for preventing or treating radiation sickness, comprising ergothioneine or a combination thereof.