Application of phellinus igniarius exosome in glioma treatment, extraction method and composition

Abstract

The invention particularly relates to application of phellinus igniarius exosomes in glioma treatment, an extraction method and a composition. The phellinus igniarius exosome is provided by taking phellinus igniarius as a raw material through operations such as soaking, crushing and centrifuging, and the phellinus igniarius exosome is verified to have anti-glioma activity in vitro. Based on the research result, the invention provides a new application of the phellinus igniarius-derived plant exosome in glioma treatment, and provides a potential active component with low cost for glioma drug development.

Description

Technical Field

[0001] The present invention relates to the technical field of plant exosomes, and particularly to the application of sanghuang exosomes and a composition containing the sanghuang exosomes in the treatment of glioma. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and is not necessarily to be regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] As a traditional Chinese medicine with a long medicinal history, the extraction and application of the active ingredients of sanghuang have always been a research hotspot. Sanghuang exosomes, as important bioactive substances in sanghuang, are small vesicles secreted by cells, and their diameters are usually between 30 - 150 nm. Research shows that exosomes can carry various specific substances such as proteins, RNAs, and DNAs, and exosomes play an important role in regulating physiological processes such as the balance of the intracellular and extracellular environments, signal transduction, and immune responses. And through humoral circulation or intercellular transmission, cell - to - cell communication and regulation are achieved. Therefore, exosomes are one of the hotspots in the research of contemporary diseases.

[0004] Numerous studies have proved that: Sanghuang contains polysaccharides, flavonoid compounds and other bioactive components, and these components show inhibitory effects on various cancer cells under laboratory conditions. For example, the polysaccharides in sanghuang can stimulate the immune system and increase the activity of natural killer cells in the body, thereby counteracting the proliferation of tumor cells. Flavonoid compounds have antioxidant and anti - inflammatory effects, which help reduce cell damage and prevent the occurrence of cancer. In addition to directly inhibiting cancer cells, sanghuang can also enhance the body's anti - cancer ability by regulating the internal environment of the human body. Sanghuang can clear heat and dry dampness, promote blood circulation to remove blood stasis, thereby improving the internal environment and reducing the risk of cancer occurrence. However, no relevant research on the anti - cancer effect of sanghuang exosomes has been found, and whether sanghuang exosomes have anti - tumor biological effects and their specific mechanisms of action have not been elucidated and remain to be further explored. Summary of the Invention

[0005] Taking sanghuang exosomes as the research object, the present invention provides a method for extracting sanghuang exosomes and first confirms the in vitro anti - glioma activity of sanghuang exosomes. Based on this research result, the present invention provides a new application of plant exosomes derived from sanghuang in the treatment of glioma: In the first aspect, there is provided an application of a sanghuang exosome, or a composition containing a sanghuang exosome, in the preparation of an anti - glioma product.

[0006] The sanghuang described in the above - mentioned first aspect is Phellinus igniarius (L. ex Fr.) Quel. Phellinus igniarius (L.ex Fr.) Quel.Fomes igniarius (L.) Fr.; Boletus igniarius L.; Polyporus igniarius The fruiting body of [Fr.]; the Sanghuang exosomes are extracellular vesicles or exosome-like nanoparticles secreted by plant cells with a particle size of 30 - 150 nm, and the aforementioned exosomes can be extracted using fresh Sanghuang as the raw material.

[0007] In one embodiment of the present invention, a method for extracting the above-mentioned Sanghuang exosomes is provided. After chopping the Sanghuang, it is added to a buffer salt solution for soaking, and then filtered to remove impurities after high-speed grinding to obtain a crude filtrate. Based on the principle of size separation, extracellular vesicles or nanoparticles with appropriate particle sizes in the crude filtrate are screened; in a preferred scheme, a complex enzyme can also be introduced into the above-mentioned crude filtrate for enzymatic hydrolysis to improve the release of intracellular contents. The specific steps are as follows: (1) Grind and juice the Sanghuang soaked in PBS, filter through gauze to obtain a crude filtrate, and centrifuge the crude filtrate at a high speed to retain the supernatant; (2) Add a complex enzyme preparation to the supernatant obtained in step (1) for enzymatic hydrolysis to obtain an enzymatic hydrolysate; freeze-centrifuge the enzymatic hydrolysate and then filter through a 100-mesh sieve to obtain a filtrate, and subject the filtrate to tangential flow filtration and concentration to obtain the product.

[0008] In step (1): Furthermore, the soaking time of the PBS is 5 - 7 h; the centrifugation speed of the crude filtrate is 800 - 1200 rpm, and the centrifugation time is 18 - 22 min.

[0009] In step (2): Furthermore, the dosage ratio of the supernatant to the complex enzyme preparation is 0.1 - 0.3%.

[0010] Furthermore, the complex enzyme preparation is composed of enzymes in the following proportions: 20%wt. pectinase, 40%wt. cellulase, 10%wt. β-1,3-glucanase, 30%wt. ligninase.

[0011] Furthermore, the filtration diameter of the tangential flow filtration is 30 - 600 nm.

[0012] The "composition containing Sanghuang exosomes" described in the above first aspect includes the Sanghuang exosomes prepared by the above extraction method, and should be in an active dose. The purposes of administration include, but are not limited to, having the effects of preventing, improving, treating, adjuvant treating or improving the prognosis of glioma. The present invention has confirmed the in vitro anti-glioma activity of Sanghuang exosomes. On the premise of clear biological activity, knowing the specific value of this "active dose" is conventional technical content in the art.

[0013] In some embodiments, in addition to the active dose of Sanghuang exosomes, the above composition further comprises other active ingredients, and the "other active ingredients" are ingredients with anti-glioma activity, and the specific types include but are not limited to small molecule entities, natural plant / animal extracts, macromolecular compounds or antibodies, and specific examples include temozolomide, carmustine, lomustine, gambogic amide, sesquiterpene lactones, bevacizumab, nimotuzumab, etc.

[0014] In some other embodiments, the "other active ingredients" are ingredients that assist anti-glioma activity, and classified by activity include but are not limited to liver-protecting ingredients, immunomodulatory ingredients, side effect-reducing ingredients, pain-relieving ingredients, etc.

[0015] In some other embodiments, the composition further comprises a pharmaceutically acceptable carrier. According to the general understanding in the art, "pharmaceutically acceptable" means that the carrier is chemically and / or physically compatible with other components in the composition, and even has a synergistic effect, and is physiologically compatible with the subjects of this composition or has the effect of preventing, improving or even treating diseases.

[0016] Furthermore, the above carrier includes but is not limited to pharmaceutically acceptable liquids, gels or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, suspension / dispersing agents, chelating agents, emulsifiers, diluents, adjuvants, excipients, auxiliary reagents or a combination of one or more of the above components.

[0017] Furthermore, the aqueous medium is selected from sodium chloride injection, Ringer's injection, isotonic glucose injection, sterile water injection or Ringer's glucose or lactate injection.

[0018] Furthermore, the non-aqueous medium is selected from non-volatile oils of plant origin, cottonseed oil, corn oil, sesame oil or peanut oil.

[0019] Furthermore, the isotonic agent is selected from sodium chloride or dextrose.

[0020] Furthermore, the buffer is selected from phosphate or citrate buffer.

[0021] Furthermore, the antioxidant is sodium bisulfate.

[0022] Furthermore, the suspension / dispersing agent is selected from sodium carboxymethylcellulose, hydroxypropyl methylcellulose or polyvinylpyrrolidone.

[0023] Furthermore, the chelating agent is selected from EDTA, EGTA.

[0024] Furthermore, the emulsifier is polysorbate 80.

[0025] Further, examples of the auxiliary reagent include a filler, a binder, a disintegrant, a buffer, a preservative, a lubricant, a flavoring agent, a thickening agent, a coloring agent, or an emulsifying agent.

[0026] The subjects of the above compositions include mammals or non-mammals. The mammals include mice, livestock, humans, dogs, rabbits, etc., and non-mammals such as fish. The dosage of the composition varies according to the type, age, sex, weight of the subject, the specific disease or pathological condition to be treated, the severity of the disease or pathological condition, the route of administration, and the judgment of the prescribing personnel, and also belongs to the technical content that can be routinely determined by those skilled in the art.

[0027] In the first aspect above, the anti-glioma product includes, but is not limited to, a drug, a modeling drug, or a kit having the function of detecting, diagnosing, preventing, improving, or treating glioma.

[0028] The dosage forms of the drug include, but are not limited to, oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, etc., external agents, suppositories, or sterilized injection solutions, and more preferably injections; feasible administration methods such as oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal. Oral or non-oral administration is preferred. Among them, the non-oral methods include subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.

[0029] In a second aspect, there is provided the use of Phellinus linteus exosomes, or a composition comprising the Phellinus linteus exosomes, in the preparation of a food, a beverage, a health product, or a cosmetic.

[0030] Examples of the food include biscuits, pastries, powder pastes, brewing granules, etc., examples of the beverage include compound fruit juices, concentrates, carbonated beverages, or tea beverages, etc.; examples of the health product include tablets, capsules, oral liquids, powders, granules, etc.; the dosage forms of the cosmetic include aqueous solutions, emulsions, creams, films, powders, oils, or suspensions, etc. The above examples are not specific limitations, and any product forms acceptable in the fields of food, beverage, health product, and cosmetic are within the protection scope of the present invention.

[0031] Compared with the prior art, the beneficial effects of the present invention are: Compared with current research, the present invention provides exosomes derived from the Chinese herbal medicine Phellinus igniarius, which are plant-derived, natural, can effectively act on glioblastoma to inhibit its progression, and have low toxicity to microglia, and their applications. The extraction cost of Phellinus igniarius exosomes is low, the extraction amount is large, and they can effectively inhibit the progression of glioma and have low toxicity to normal cells. Phellinus igniarius exosomes can effectively inhibit the autophagy of glioma cells and then induce the death of glioma cells, and at the corresponding concentration, they have no obvious cytotoxicity to normal microglia.

[0032] The research conclusion of the present invention has developed new clinical applications for Phellinus igniarius and also provided new potential active ingredients for the treatment of glioma, which is of great significance for the development of anti-glioma drugs. Brief Description of the Drawings

[0033] The accompanying drawings forming a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention.

[0034] Figure 1 It is a schematic diagram of the preparation process of Phellinus igniarius exosomes in the present invention; Figure 2 It is the particle size characterization result of Phellinus igniarius exosomes in Example 1; Among them, Figure 2 A in it is the NTA result diagram of Phellinus igniarius exosomes in Example 1, Figure 2 B in it is the TEM result diagram of Phellinus igniarius exosomes in Example 1; Figure 3 It is the cell proliferation result diagram after Phellinus igniarius exosomes in Example 2 are used to treat glioma (U87) and microglia (HMC3); Among them, Figure 3 A in it is the cell state photos of the control group (NC) and the Phellinus igniarius exosome group (PELNs) of glioma (U87), Figure 3 B in it is the relative activity results of the control group (NC-U87) and the Phellinus igniarius exosome group (PELNs-U87) of glioma, and the control group (NC-HMC3) and the Phellinus igniarius exosome group (PELNs-HMC3) of microglia; Figure 4 It is the scratch experiment display diagram of the control group and the Phellinus igniarius exosome treatment group in Example 2; Figure 5 It is the Western Blotting result display diagram of the regulation of AKT-MTOR related pathways by PELNs described in Example 2. Detailed Embodiments

[0035] It should be noted that the following detailed description is illustrative and aims to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0036] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0037] In order to enable those skilled in the art to more clearly understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below in conjunction with specific embodiments.

[0038] Example 1 In this embodiment, a sanghuang exosome is provided, and its extraction method steps are as follows: (1) Take fresh sanghuang, wash it, select γ-ray with a radiation dose of 3 - 5 kGy for irradiation and disinfection for 12 - 14 h, cut it into sanghuang pieces smaller than 1 cm 3 , add PBS solution and soak for 6 h.

[0039] (2) Transfer the soaked sanghuang pieces and PBS solution in step (1) to a food processor for shearing, grinding and juicing, with a rotation speed of 3000 rpm / min and a grinding time of 5 - 15 min; the obtained grinding liquid is filtered through three layers of sterile gauze to obtain a crude filtrate, which is transferred to a 50 ml sterile centrifuge tube for centrifugation to remove impurities, with a centrifugation speed of 1000 rpm and a centrifugation time of 20 min, and the supernatant is retained.

[0040] (3) Add a complex enzyme preparation to the supernatant in step (2) for enzymatic hydrolysis to obtain an enzymatic hydrolysate; the enzymatic hydrolysis temperature is 40 - 50 °C, and the enzymatic hydrolysis time is 4 - 8 h; the dosage ratio of the supernatant to the complex enzyme preparation is 0.1 - 0.3%. The above complex enzyme preparation is composed of enzymes in the following proportions: 20%wt. pectinase, 40%wt. cellulase, 10%wt. β-1,3-glucanase, 30%wt. ligninase.

[0041] (4) Transfer the enzymatic hydrolysate in step (3) to a freezing impurity removal device, perform freezing centrifugation filtration to remove impurities, and the filtration screen is 100 mesh. The obtained filtrate is subjected to tangential flow filtration (filter diameter is 30 - 600 nm) and concentrated to obtain plant exosome-like vesicles, which are irradiated and sterilized to obtain the sanghuang exosome.

[0042] The particle size and electron microscopy analysis results of the above sanghuang exosome are as follows: (1)Particle size analysis Wash the sample cell with pure water 3 times; after cleaning, calibrate the instrument with a standard (100 nm PS beads, polystyrene microspheres), and perform subsequent detection after calibration; wash the sample cell with 1×PBS; dilute the sample with 1×PBS and add it to the sample cell, observe the real-time dynamic image of exosome particles through the computer display screen; collect information and generate a corresponding detection report; after detection, wash the injection cell with 1×PBS and perform the detection of the next sample, draw a particle size spectrum, and obtain a particle size distribution map as shown in Figure 2 Figure A below. The average diameter of Phellinus igniarius exosomes is 137 nm.

[0043] Table 1 Particle size distribution of Phellinus igniarius exosomes (2)Transmission electron microscopy (TEM) analysis For TEM imaging, add 10 μL of the Phellinus igniarius exosome suspension to a pre-prepared Parafilm sealing film (the back of the sealing film is adhered to the table), place the copper grid with the film facing down, and let it naturally adsorb the suspension droplet for 10 - 15 minutes. Then, use a filter paper strip to absorb the excess liquid droplets and let it dry slightly. Pipette 10 μL of 2% phosphotungstic acid solution and drop it on the sealing film. Place the adsorbed copper grid with the film facing the staining solution and let it stand upside down for 3 - 5 min. Carefully use a filter paper strip to absorb the excess liquid droplets and dry it under an incandescent lamp. After drying for 30 min, send the sample for analysis, observe and take pictures with TEM, and the obtained transmission electron microscopy micrograph is as shown in Figure 2 Figure B below.

[0044] Example 2 In this example, the anti-glioma activity of the Phellinus igniarius exosomes described in Example 1 was verified.

[0045] 1. Detection of anti-glioma cell proliferation of Phellinus igniarius exosomes Take the glioma cell line U87 cells and the microglia cell line HMC3 in the logarithmic growth phase, inoculate the cell suspension into a 96-well cell culture plate at 6000 cells / well, and let it adhere overnight for 12 h; set up a blank control group (NC) and a PELNs test group (2.2E+11 particles / mL) respectively; set 9 replicates, with 3 wells in a group for parallel control, and continue to culture at 37 °C. Perform cell viability detection at 0 h, 24 h, and 48 h respectively; the detection method is CCK8 (10 μL / well), incubate at 37 °C for 4 h, place it in the dark for 30 min, and measure the OD450 absorbance value with an enzyme-linked immunosorbent assay (ELISA) reader to calculate the cell viability. As shown in Figure 3 Figure below.

[0046] 2. Detection of the anti-glioma cell repair effect of Phellinus igniarius exosomes Take breast cancer cell line U87 cells in logarithmic growth phase, and set up a blank control group and a Phellinus linteus exosome test group respectively; inoculate cell suspension in a 6-well plate, add complete medium to the blank control group, and inoculate the Phellinus linteus exosome solution in the test group at a concentration of 2.2E+11 particles / mL; set 2 replicates, with 2 wells in each group as parallel controls, and continue to culture in an incubator at 37°C for 24 h; take out the 6-well plate and place it in a live cell imager. Replace the blank control group with basal medium, make a scratch mark, and still add the Phellinus linteus exosome solution (2.2E+11 particles / mL) to the test group. After 48 h, count the data of the live cell imager and analyze the repair ability of glioma cells.

[0047] The results are as Figure 4 shown. The scratch boundary of tumor cells in the control group was no longer clear after 48 h, and the tumor cells began to migrate to the scratch area. However, obvious aggregation and shrinkage occurred in the glioma cells of the exosome group, and the number of migrating cells in the scratch area was significantly less than that in the control group. The above results indicate that Phellinus linteus exosomes effectively inhibit the repair of glioma cells.

[0048] 3. Detection of the expression of related proteins in which Phellinus linteus exosomes inhibit the apoptosis of glioma by AKT / mTOR signaling pathway Take glioma cell line U87 cells in logarithmic growth phase, inoculate 2 ml of cell suspension at 200,000 cells / well in a 6-well cell culture plate, and incubate overnight for 12 h to adhere to the wall; set up a blank control group and a PELNs test group (2.2E+11 particles / mL) respectively, and continue to culture for 24 h; add protein lysate to lyse the cells, centrifuge at 12,000 rpm for 15 min, and take the supernatant; measure the protein concentration by BCA assay kit: the total protein loading amount per well is 30 μg, add an appropriate amount of 6× protein loading buffer, and boil in water bath for 10 min; use the PAGE gel rapid kit to prepare the separating gel and the stacking gel: add an appropriate volume of protein sample, and add 6 μL / well of protein marker; first adjust the voltage to 90 V for constant voltage, and when the bromophenol blue indicator of the electrophoresis sample crosses the separating gel, change to 110 V for constant voltage electrophoresis until the bromophenol blue indicator reaches the bottom of the separating gel; cut out a PVDF membrane of appropriate size according to the number of samples and the size of the target protein, and soak it in methanol for about 5 s. Soak all the materials in the transfer membrane instrument in the electrotransfer solution, sponge pad - filter paper - PAGE gel - PVDF membrane - filter paper - sponge pad, drive out the air bubbles, and transfer the membrane at a constant current of 400 mA for 45 min; after the transfer is completed, take out the PVDF membrane, place it in a 53% skimmed milk powder solution, and incubate and block it on a shaker at room temperature for 1 h; Prepare the primary antibody with 5% skimmed milk powder and add it to the antibody incubation box. Place the membrane face up and incubate at 4°C overnight. After the incubation with the primary antibody, wash the membrane 3 times with TBST for 5 min each; incubate the membrane in the secondary antibody at 37°C for 30 min, and wash the membrane 3 times with TBST for 5 min each; prepare the ECL luminescent solution, drop it on the front of the PVDF membrane, and develop and fix the film. AsFigure 5 as shown

[0049] According to Figure 5 It is shown that in glioma cells cultured with the addition of Phellinus linteus exosomes, the expression levels of MTOR, phosphorylated p-MTOR, and p-AKT all decreased significantly, effectively inhibiting the PI3K / AKT / mTOR signaling pathway of glioma, indicating that Phellinus linteus exosomes have a therapeutic effect on glioma.

[0050] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. Use of Phellinus linteus exosomes, or a composition containing Phellinus linteus exosomes, in the preparation of an anti-glioma product.

2. The application according to claim 1, characterized in that The extraction method of the Phellinus linteus exosomes is as follows: (1) Grind and juice Phellinus linteus soaked in PBS, filter through gauze to obtain a crude filtrate, and centrifuge the crude filtrate at a high speed to retain the supernatant; the soaking time of PBS is 5 - 7 h; the centrifugation speed of the crude filtrate is 800 - 1200 rpm, and the centrifugation time is 18 - 22 min; (2) Add a composite enzyme preparation to the supernatant obtained in step (1) for enzymatic hydrolysis to obtain an enzymatic hydrolysate; freeze-centrifuge the enzymatic hydrolysate and filter through a 100-mesh sieve to obtain a filtrate, and subject the filtrate to tangential flow filtration and concentration to obtain the product; the dosage ratio of the supernatant to the composite enzyme preparation is 0.1 - 0.3%; the composite enzyme preparation is composed of enzymes in the following proportions: 20%wt. pectinase, 40%wt. cellulase, 10%wt. β-1,3-glucanase, 30%wt. ligninase.

3. The application according to claim 1, characterized in that, In the composition, the Phellinus linteus exosomes are in an active dosage; In the composition, in addition to the active dosage of Phellinus linteus exosomes, other active ingredients are also included, and the other active ingredients are ingredients with anti-glioma activity, including but not limited to small molecule entities, natural plant / animal extracts, high molecular compounds or antibodies. Further, they are selected from temozolomide, carmustine, lomustine, gambogic amide, sesquiterpene lactones, bevacizumab or nimotuzumab; Or, the other active ingredients are ingredients that assist anti-glioma activity, and according to the activity classification, they include but not limited to liver protection ingredients, immunomodulatory ingredients, side effect mitigation ingredients or analgesic ingredients.

4. The application according to claim 1, characterized in that, The composition also includes a pharmaceutically acceptable carrier, including but not limited to a pharmaceutically acceptable liquid, gel or solid carrier, aqueous medium, non-aqueous medium, antimicrobial agent, isotonic agent, buffer, antioxidant, suspension / dispersant, chelating agent, emulsifier, diluent, adjuvant, excipient, auxiliary reagent or a combination of one or several of the above ingredients.

5. The application according to claim 4, characterized in that, The aqueous medium is selected from sodium chloride injection, Ringer's injection, isotonic glucose injection, sterile water injection or Ringer's glucose or lactate injection; The non-aqueous medium is selected from non-volatile oils of plant origin, cottonseed oil, corn oil, sesame oil or peanut oil; The isotonic agent is selected from sodium chloride or dextrose; The buffer is selected from phosphate or citrate buffer; The antioxidant is sodium bisulfate; The suspension / dispersant is selected from sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose or polyvinylpyrrolidone; The chelating agent is selected from EDTA, EGTA; The emulsifier is polysorbate 80.

6. The application according to claim 4, characterized in that The auxiliary reagent is selected from filler, binder, disintegrant, buffer, preservative, lubricant, flavoring agent, thickening agent, coloring agent or emulsifier.

7. The application according to claim 2, wherein The anti-glioma product is one of, including but not limited to, a drug, a modeling drug or a kit having the function of detecting, diagnosing, preventing, improving or treating glioma.

8. The application according to claim 7, wherein The dosage forms of the drug include, but are not limited to, powder, granule, tablet, capsule, suspension, emulsion, syrup, spray, oral dosage form, external preparation, suppository or sterilized injection solution, and more preferably injection; The administration route is selected from oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal; preferably oral or parenteral administration; wherein, the parenteral administration includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.

9. Application of Phellinus linteus exosomes, or a composition comprising the Phellinus linteus exosomes, in the preparation of food, beverage, health product or cosmetic.

10. The application according to claim 9, characterized in that, The food is selected from biscuits, pastries, pastes or instant granules; The beverage is selected from compound fruit juice, concentrate, carbonated beverage or tea beverage; The health product is selected from tablets, capsules, oral liquids, powders or granules; The dosage form of the cosmetic is selected from aqueous solution, emulsion, cream, film, powder, oil or suspension.

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