Application of ganoderma lucidum exosome in preparation of anti-allergic rhinitis product and product

Abstract

The application name is ganoderma lucidum exosome in the preparation of anti-allergic rhinitis product and product, belongs to the technical field of biotechnology and food science. The technical problem to be solved is that the content of active ingredients in ganoderma lucidum exosome is low and the effect of anti-allergic rhinitis is poor. The content of active ingredients in the ganoderma lucidum exosome prepared by the application is high, which can effectively reduce the sneezing frequency of mice with allergic rhinitis, promote the recovery of epithelial injury, reduce edema and infiltration, reduce eosinophils, and can relieve the symptoms of allergic rhinitis.

Description

Technical Field

[0001] This invention belongs to the technical field of biotechnology and food science, specifically relating to the application and products of Ganoderma lucidum exosomes in the preparation of anti-allergic rhinitis products. Background Technology

[0002] Reishi mushroom (Ganoderma lucidum), a traditional Chinese medicinal and edible fungus, has been recorded in ancient Chinese medical texts. The *Shennong Bencao Jing* (Shennong's Classic of Materia Medica) lists it as a "superior" herb, stating that it "treats chest congestion, benefits heart qi, replenishes the middle jiao, enhances wisdom, and improves memory"; the *Bencao Gangmu* (Compendium of Materia Medica) records that it "treats weakness and hemorrhoids." Modern research shows that the pharmacological activity of reishi mushroom is closely related to its rich bioactive components, mainly including: polysaccharides: primarily β-glucan, which regulate the secretion of immune factors by activating receptors (such as TLR4) on the surface of immune cells such as macrophages and T lymphocytes, thus exerting immunomodulatory and anti-tumor effects; triterpenoids: such as ganoderic acids A, B, and C, which have anti-inflammatory, antioxidant, and tumor cell proliferation-inhibiting activities, with mechanisms related to inhibiting the NF-κB pathway and scavenging free radicals; proteins and peptides: some small peptides can be absorbed through the intestines, participating in metabolic regulation and enhancing cell vitality. However, traditional Ganoderma lucidum products (such as Ganoderma lucidum spore powder and decoction) have significant limitations: First, the extraction rate of active ingredients is low; for example, triterpenoids are highly lipid-soluble, and the yield from water extraction is less than 0.3%. Second, bioavailability is poor; large polysaccharides need to be broken down into smaller molecules by intestinal flora before absorption, resulting in a utilization rate of less than 20%. Third, their functions are limited, often relying on a single component and failing to leverage the synergistic effects of active ingredients. Therefore, developing novel carriers for Ganoderma lucidum active ingredients is crucial to overcoming the bottlenecks of traditional products.

[0003] Exosomes are vesicle structures with a diameter of 30-150 nm secreted by cells. They are rich in bioactive substances such as proteins, nucleic acids, and lipids, and are important mediators for intercellular communication. They have good biocompatibility, targeting, and penetration.

[0004] In recent years, research on plant / fungal exosomes has gradually emerged. Grape exosomes have been shown to alleviate colitis by inhibiting inflammatory factors; shiitake mushroom exosomes can enhance the immune function of mice. Compared with other fungi, Ganoderma lucidum exosomes, due to their rich content of triterpenes, polysaccharides, and other unique components, exhibit more significant anti-inflammatory activity. Therefore, the development of natural, efficient, and stable anti-inflammatory and anti-aging active substances has become an urgent market demand. This invention focuses on Ganoderma lucidum exosomes, aiming to fill the technological gap in this field by optimizing the preparation process and verifying its functions.

[0005] Relevant patent documents retrieved: The document, published in China (CN120505207A) on August 19, 2025, discloses a method for preparing Ganoderma lucidum exosomes containing medicinal and edible active ingredients. The method includes the following steps: culturing Ganoderma lucidum mycelium fermentation broth; adding Polygonatum sibiricum solution to the Ganoderma lucidum mycelium fermentation broth to biotransform Polygonatum sibiricum powder to obtain Ganoderma lucidum exosome fermentation broth; separating, extracting, and purifying the Ganoderma lucidum exosome fermentation broth to obtain Ganoderma lucidum exosomes, which exhibit a strong killing effect on cancer cells.

[0006] Relevant non-patent literature retrieved: The journal or book title is "Shanghai Journal of Traditional Chinese Medicine," and the article title is "A Study on the Treatment of Allergic Rhinitis and Related Constitutions with Compound Traditional Chinese Medicine Ganoderma Lucidum," issue 8, published on September 27, 1999. This article discloses the efficacy of compound traditional Chinese medicine Ganoderma Lucidum in treating allergic rhinitis. The compound traditional Chinese medicine Ganoderma Lucidum includes 1.5g of Ganoderma lucidum and 0.3g of Cordyceps sinensis, which can significantly reduce the levels of IL-4 and EOS.

[0007] The prior art represented by the aforementioned documents has at least the following unresolved technical problems or defects: The preparation method of Ganoderma lucidum exosomes prepared by CN120505207A is more complicated, and it only has a killing effect on cancer cells. The relevant evidence is that Ganoderma lucidum exosomes also contain Polygonatum sibiricum, and the synergistic effect of Polygonatum sibiricum and Ganoderma lucidum exosomes is achieved. Summary of the Invention

[0008] The purpose of this invention is to provide: The application of Ganoderma lucidum exosomes in the preparation of anti-allergic rhinitis products, and related technologies, to solve technical problems such as low content of active ingredients in Ganoderma lucidum exosomes and poor anti-allergic rhinitis effects, or a combination thereof.

[0009] Terminology Explanation: Unless otherwise defined, all technical terms in this document have the same meanings as commonly understood by one of ordinary skill in the art to which the subject matter of the claims pertains. Unless otherwise stated, all patents, patent inventions, and publications cited in this document are incorporated herein by reference in their entirety. If multiple definitions exist for terms in this document, the definitions in this chapter shall prevail.

[0010] It should be understood that the above brief description and the following detailed description are exemplary and for illustrative purposes only, and do not limit the subject matter of the invention in any way. In this invention, the singular is used in conjunction with the plural unless otherwise specifically stated. It should also be noted that, unless otherwise stated, the use of “or” or “or” means “and / or”. Furthermore, the use of the term “comprising” and other forms such as “including,” “containing,” and “contains” are not limiting.

[0011] Unless specifically defined herein, the use of all commercially available products herein employs standard techniques. For example, it may be carried out using the manufacturer's instructions for use with the kit, or in accordance with methods known in the art or the description of this invention. The techniques and methods described herein can generally be implemented according to conventional methods well known in the art, based on the descriptions in the various summary and more specific documents cited and discussed in this specification.

[0012] The terms “optional / arbitrary” or “optionally / arbitrarily” mean that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0013] The term "exosome" as used in this article refers to extracellular vesicles with a diameter of approximately 30–150 nm secreted by cells. These vesicles have a lipid bilayer structure, carry bioactive molecules such as proteins, lipids, and nucleic acids, and play an important role in intercellular communication.

[0014] The term "separation and purification" as used in this article refers to the process of separating target components (such as exosomes, polysaccharides, and triterpenoids) from impurities in complex biological samples (such as cell culture supernatants, tissue extracts, etc.) using physical or chemical methods (such as ultracentrifugation, chromatography, precipitation, etc.) to obtain higher purity.

[0015] The term “high-yielding triterpenoid Ganoderma mycelium” used in this article refers to Ganoderma (Ganoderma spp.) mycelial cultures that yield significantly higher levels of triterpenoid compounds than ordinary strains, obtained through strain screening or optimization of fermentation conditions.

[0016] The term "primary seed culture" as used in this article refers to the bacterial suspension obtained after the initial culture of Ganoderma lucidum strains preserved on slant or plate into liquid culture medium, which is used as the starting inoculum for subsequent large-scale culture.

[0017] The term "deep liquid fermentation medium" used in this article refers to the nutrient solution used to support the growth and metabolism of Ganoderma lucidum mycelium under deep liquid aeration culture conditions, which usually contains carbon sources, nitrogen sources, inorganic salts and trace elements.

[0018] The term "gradient centrifugation" as used in this article refers to a method of separation in which a medium with continuously or stepwise varying density is used during centrifugation, causing different particles in a sample to settle into different zones in the centrifugal field according to their density and size.

[0019] The term "filtration sterilization" as used in this article refers to the process of removing microorganisms from thermally unstable liquids (such as culture media, buffer solutions, and solutions of active substances) by using microporous membranes with pore sizes smaller than bacteria (typically 0.22 μm or 0.45 μm).

[0020] The term "allergic rhinitis" as used in this article refers to a chronic inflammatory disease of the nasal mucosa mediated by immunoglobulin E (IgE) and induced by allergens (such as pollen, dust mites, animal dander, etc.). Typical clinical symptoms include paroxysmal sneezing, clear nasal discharge, nasal congestion, and nasal itching.

[0021] The term “particle size” as used in this article refers to the diameter or size distribution of particles (such as exosomes, nanoparticles, hyphal fragments, etc.) characterized by a specific measurement method, which is commonly determined by dynamic light scattering or electron microscopy. In this article, it specifically refers to the average hydrated diameter of exosomes.

[0022] In a first aspect, the present invention provides the application of Ganoderma lucidum exosomes in the preparation of anti-allergic rhinitis products, wherein the method for preparing Ganoderma lucidum exosomes includes the following steps: (1) Cultivating secondary seed culture; (2) Expanded culture and acquisition of Ganoderma lucidum mycelium fermentation broth; (3) Separate, extract and purify the fermentation broth of Ganoderma lucidum mycelium to obtain Ganoderma lucidum exosomes.

[0023] Preferably, the specific method for cultivation in step (1) is as follows: (1.1) Inoculate high-yield triterpenoid Ganoderma lucidum mycelia into PDA medium for slant culture until the Ganoderma lucidum mycelia cover the slant test tubes; (1.2) Pour water into a slant test tube covered with Ganoderma lucidum mycelium, scrape the Ganoderma lucidum mycelium on the culture medium into the water to make a mycelium suspension; inoculate the suspension into a seed culture medium to obtain a primary seed solution; (1.3) Shake the primary seed liquid prepared in step (1.2) to break up the mycelial balls, and inoculate the broken mycelium into a deep liquid fermentation medium to obtain the secondary seed liquid.

[0024] Preferably, the high-yield triterpenoid Ganoderma lucidum mycelium in step (1.1) is mycelium-derived exosome extracted by fermentation of Ganoderma lucidum mycelium, and its ganoderic acid A content is ≥1.2mg / g.

[0025] Preferably, the PDA culture medium in step (1.1) comprises: 180-220 g / L potato extract, 18-22 g / L glucose and 15-20 g / L agar powder.

[0026] Preferably, the concentration of potato extract in the PDA culture medium in step (1.1) is selected from any value or range between 180-220 g / L, specifically from: 180 g / L, 190 g / L, 200 g / L, 210 g / L, 220 g / L or a range between the two.

[0027] More preferably, the concentration of potato extract in the PDA culture medium in step (1.1) is 200 g / L.

[0028] Preferably, the concentration of glucose in the PDA culture medium in step (1.1) is selected from any value or range between 18 and 22 g / L, specifically from: 18 g / L, 19 g / L, 20 g / L, 21 g / L, 22 g / L or a range between the two.

[0029] More preferably, the concentration of glucose in the PDA culture medium in step (1.1) is 20 g / L.

[0030] Preferably, the concentration of agar powder in the PDA culture medium in step (1.1) is selected from any value or range between 15-20 g / L, specifically from: 15 g / L, 16 g / L, 17 g / L, 18 g / L, 19 g / L, 20 g / L or a range between the two.

[0031] More preferably, the concentration of agar powder in the PDA culture medium in step (1.1) is 15 g / L.

[0032] Preferably, the pH of the PDA culture medium in step (1.1) is selected from any value or range between 5.5 and 6.0, specifically from: 5.5, 5.6, 5.7, 5.8, 5.9, 6.0 or a range between the two.

[0033] More preferably, the pH of the PDA culture medium in step (1.1) is 6.0.

[0034] Preferably, the culture temperature in step (1.1) is selected from any value or range between 26-37℃, specifically from: 26℃, 26.5℃, 27℃, 27.5℃, 28℃, 30℃, 35℃, 37℃ or a range between the two.

[0035] More preferably, the culture temperature in step (1.1) is 37°C.

[0036] Preferably, the culture time in step (1.1) is selected from any value or range between 7 and 10 days, specifically 7 days, 8 days, 9 days, 10 days or a range between the two.

[0037] More preferably, the culture time in step (1.1) is 5 days.

[0038] Preferably, the water in step (1.2) is sterile water.

[0039] Preferably, the amount of water added in step (1.2) is any value or range between 5 and 20 mL, specifically selected from: 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL or a range between the two.

[0040] More preferably, the amount of water added in step (1.2) is 5 mL.

[0041] Preferably, the seed culture medium in step (1.2) comprises: glucose 30-40 g / L, tryptone 4-6 g / L, yeast extract 2-3 g / L, KH2PO4 0.8-1.2 g / L and MgSO4·7H2O 0.4-0.6 g / L.

[0042] Preferably, the glucose concentration in the seed culture medium described in step (1.2) is selected from any value or range between 30 and 40 g / L, specifically from: 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L, 36 g / L, 37 g / L, 38 g / L, 39 g / L, 40 g / L or a range between the two.

[0043] More preferably, the glucose concentration in the seed culture medium described in step (1.2) is 20 g / L.

[0044] Preferably, the tryptone concentration in the seed culture medium described in step (1.2) is selected from any value or range between 4.0 and 6.0 g / L, specifically from: 4 g / L, 4.5 g / L, 5 g / L, 5.5 g / L, 6 g / L or a range between the two.

[0045] More preferably, the concentration of tryptone in the seed culture medium described in step (1.2) is 10 g / L.

[0046] Preferably, the concentration of yeast extract in the seed culture medium in step (1.2) is selected from any value or range between 2 and 3 g / L, specifically from: 2 g / L, 2.1 g / L, 2.2 g / L, 2.3 g / L, 2.4 g / L, 2.5 g / L, 2.6 g / L, 2.7 g / L, 2.8 g / L, 2.9 g / L, 3 g / L or a range between the two.

[0047] More preferably, the concentration of yeast extract in the seed culture medium described in step (1.2) is 5 g / L.

[0048] Preferably, the KH2PO4 in the seed culture medium described in step (1.2) is selected from any value or range between 0.8 and 1.2 g / L, specifically from: 0.8 g / L, 0.9 g / L, 1 g / L, 1.1 g / L, 1.2 g / L or a range between the two.

[0049] More preferably, the concentration of KH2PO4 in the seed culture medium described in step (1.2) is 1 g / L.

[0050] Preferably, the concentration of MgSO4·7H2O in the seed culture medium in step (1.2) is selected from any value or range between 0.5-0.85 g / L, specifically from: 0.5 g / L, 0.55 g / L, 0.6 g / L, 0.65 g / L, 0.7 g / L, 0.75 g / L, 0.8 g / L, 0.85 g / L or a range between the two.

[0051] More preferably, the concentration of MgSO4·7H2O in the seed culture medium described in step (1.2) is 0.5 g / L.

[0052] Preferably, the pH of the seed culture medium in step (1.2) is selected from any value or range between 5.5 and 6.0, specifically from: 5.5, 5.6, 5.7, 5.8, 5.9, 6.0 or a range between the two.

[0053] More preferably, the pH of the seed culture medium in step (1.2) is 6.0.

[0054] Preferably, the culture temperature in step (1.2) is selected from any value or range between 26-28℃, specifically from: 26℃, 26.5℃, 27℃, 27.5℃, 28℃ or a range between the two.

[0055] More preferably, the culture temperature in steps S1-2 is 28°C.

[0056] Preferably, the culture in step (1.2) is an oscillating culture.

[0057] Preferably, the rotation speed of the oscillation culture is selected from any value or range between 120-140 rpm, specifically from: 120 rpm, 123 rpm, 125 rpm, 128 rpm, 130 rpm, 133 rpm, 135 rpm, 137 rpm, 140 rpm or a range between the two.

[0058] More preferably, the rotation speed of the oscillation culture is 150 rpm.

[0059] Preferably, the endpoint of the culture in step (1.2) is determined as: OD 600Value reaches 1.8-2.0, viable count ≥10 8 CFU / mL (plate count method).

[0060] Preferably, the deep liquid fermentation culture medium in step (1.3) comprises: glucose 30-40 g / L, soybean meal powder 8-15 g / L, KH2PO4 1.2-1.8 g / L, and MgSO4·7H2O 1.3-1.8 g / L.

[0061] Preferably, the glucose concentration in the deep liquid fermentation medium in step (1.3) is selected from any value or range between 30-40 g / L, specifically from: 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L, 36 g / L, 37 g / L, 38 g / L, 39 g / L, 40 g / L or a range between the two.

[0062] More preferably, the glucose concentration in the deep liquid fermentation medium described in step (1.3) is 25 g / L.

[0063] Preferably, the concentration of soybean meal powder in the deep liquid fermentation medium in step (1.3) is selected from any value or range between 8 and 15 g / L, specifically from: 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L or a range between the two.

[0064] More preferably, the concentration of soybean meal powder in the deep liquid fermentation medium in step (1.3) is 15 g / L.

[0065] Preferably, the KH2PO4 concentration in the deep liquid fermentation medium in step (1.3) is selected from any value or range between 1.2 and 1.8 g / L, specifically from: 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L or a range between the two.

[0066] More preferably, the concentration of KH2PO4 in the deep liquid fermentation medium described in step (1.3) is 1.5 g / L.

[0067] Preferably, the concentration of MgSO4·7H2O in the deep liquid fermentation medium in step (1.3) is selected from any value or range between 0.8 and 2.5 g / L, specifically from: 0.5 g / L, 0.55 g / L, 0.6 g / L, 0.65 g / L, 0.7 g / L, 0.75 g / L, 0.8 g / L, 0.85 g / L or a range between the two.

[0068] More preferably, the concentration of MgSO4·7H2O in the deep liquid fermentation medium described in step (1.3) is 0.8 g / L.

[0069] Preferably, the pH of the deep liquid fermentation medium in step (1.3) is selected from any value or range between 5.5 and 6.0, specifically from: 5.5, 5.6, 5.7, 5.8, 5.9, 6.0 or a range between the two.

[0070] More preferably, the pH of the deep liquid fermentation medium described in step (1.3) is 6.0.

[0071] Preferably, the rotation speed of the oscillation and crushing in step (1.3) is selected from any value or range between 3000-5000 rpm, specifically from: 3000 rpm, 3500 rpm, 4000 rpm, 4500 rpm, 5000 rpm or a range between the two.

[0072] More preferably, the rotation speed of the oscillation and crushing in step (1.3) is 3000 rpm.

[0073] Preferably, the oscillation and breaking time in step (1.3) is selected from any value or range between 10 and 60 s, specifically from: 10 s, 15 s, 20 s, 25 s, 30 s, 35 s, 40 s, 45 s, 50 s, 55 s, 60 s or a range between the two.

[0074] More preferably, the vibration and breaking time in step (1.3) is 30s.

[0075] Preferably, the inoculation amount of the broken mycelium in step (1.3) is selected from any value or range between 5-10% (v / v), specifically from: 5%, 6%, 7%, 8%, 9%, 10% or a range between the two.

[0076] More preferably, the inoculation amount of the broken mycelium in step (1.3) is 10% (v / v).

[0077] Preferably, the culture temperature in step (1.3) is selected from any value or range between 26-28℃, specifically from: 26℃, 26.5℃, 27℃, 27.5℃, 28℃ or a range between the two.

[0078] More preferably, the culture temperature in step (1.3) is 28°C.

[0079] Preferably, the culture time in step (1.3) is selected from any value or range between 1 and 5 days, specifically from: 1 day, 2 days, 3 days, 4 days, 5 days or a range between the two.

[0080] More preferably, the culture time in step (1.3) is 2 days.

[0081] Preferably, the culture in step (1.3) is an oscillating culture.

[0082] Preferably, the rotation speed of the oscillation culture is selected from any value or range between 120-140 rpm, specifically from: 120 rpm, 123 rpm, 125 rpm, 128 rpm, 130 rpm, 133 rpm, 135 rpm, 137 rpm, 140 rpm or a range between the two.

[0083] More preferably, the rotation speed of the oscillation culture is 150 rpm.

[0084] Preferably, the specific method for expanding the culture and obtaining the fermentation broth in step (2) is as follows: (2.1): The secondary seed liquid prepared in step (1.3) is inoculated into a fermenter for culture and scale-up to obtain the primary Ganoderma lucidum strain; (2.2): The primary Ganoderma lucidum strain prepared in step (2.1) is then inoculated into a fermenter for culture. After the culture is completed, the mixture is filtered to obtain the Ganoderma lucidum mycelium fermentation broth.

[0085] Preferably, the inoculation amount of the secondary seed liquid in step (2.1) is selected from any value or range between 5-10% (v / v), specifically from: 5%, 6%, 7%, 8%, 9%, 10% or a range between the two.

[0086] More preferably, the inoculation amount of the secondary seed liquid in step (2.1) is 10% (v / v).

[0087] Preferably, the culture temperature in step (2.1) is selected from any value or range between 26-28℃, specifically from: 26℃, 26.5℃, 27℃, 27.5℃, 28℃ or a range between the two.

[0088] More preferably, the culture temperature in step (2.1) is 28°C.

[0089] Preferably, the culture time in step (2.1) is selected from any value or range between 1 and 5 days, specifically from: 1 day, 2 days, 3 days, 4 days, 5 days or a range between the two.

[0090] More preferably, the culture time in step (2.1) is 5 days.

[0091] Preferably, the aeration rate for the culture in step (2.1) is 1:0.8 (v / v / min).

[0092] Preferably, the cultivation in step (2.1) also requires stirring.

[0093] Preferably, the stirring speed is selected from any value or range between 100-500 rpm, specifically from: 100 rpm, 150 rpm, 200 rpm, 250 rpm, 300 rpm, 350 rpm, 400 rpm, 450 rpm, 500 rpm or a range between the two.

[0094] More preferably, the stirring speed is 200 rpm.

[0095] Preferably, the fermenter in step (2.1) and the fermenter in step (2.2) both contain: 30-40 g / L glucose, 8-15 g / L soybean meal powder, 1.2-1.8 g / L KH2PO4, and 1.3-1.8 g / L MgSO4·7H2O.

[0096] Preferably, the glucose concentration in the fermenter in step (2.1) and the fermenter in step (2.2) is selected from any value or range between 30-40 g / L, specifically from: 30 g / L, 31 g / L, 32 g / L, 33 g / L, 34 g / L, 35 g / L, 36 g / L, 37 g / L, 38 g / L, 39 g / L, 40 g / L or a range between the two.

[0097] More preferably, the glucose concentration in both the fermenter in step (2.1) and the fermenter in step (2.2) is 25 g / L.

[0098] Preferably, the concentration of soybean meal powder in the fermentation tank in step (2.1) and the fermentation tank in step (2.2) is selected from any value or range between 8 and 15 g / L, specifically from: 8 g / L, 9 g / L, 10 g / L, 11 g / L, 12 g / L, 13 g / L, 14 g / L, 15 g / L or a range between the two.

[0099] More preferably, the concentration of soybean meal powder in the fermentation tank in step (2.1) and the fermentation tank in step (2.2) is 15 g / L.

[0100] Preferably, the KH2PO4 concentration in the fermenter in step (2.1) and the fermenter in step (2.2) is selected from any value or range between 1.2-1.8 g / L, specifically from: 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L or a range between the two.

[0101] More preferably, the concentration of KH2PO4 in the fermenter described in step (2.1) and step (2.2) is 1.5 g / L.

[0102] Preferably, the concentration of MgSO4·7H2O in the fermenter described in step (2.1) and step (2.2) is selected from any value or range between 0.8 and 2.5 g / L, specifically from: 0.8 g / L, 0.9 g / L, 1.0 g / L, 1.1 g / L, 1.2 g / L, 1.3 g / L, 1.4 g / L, 1.5 g / L, 1.6 g / L, 1.7 g / L, 1.8 g / L, 1.9 g / L, 2 g / L, 2.2 g / L, 2.3 g / L, 2.4 g / L, 2.5 g / L or a range between the two.

[0103] More preferably, the concentration of MgSO4·7H2O in both the fermenter in step (2.1) and the fermenter in step (2.2) is 0.8 g / L.

[0104] Preferably, step (2) of the expanded culture also requires monitoring the following indicators every 6 hours: OD 600 Value (reflecting mycelial concentration), pH value (maintained at 5.8-6.2), dissolved oxygen (≥20%).

[0105] Preferably, the inoculation amount of the primary Ganoderma lucidum strain in step (2.2) is selected from any value or range between 5-10% (v / v), specifically from: 5%, 6%, 7%, 8%, 9%, 10% or a range between the two.

[0106] More preferably, the inoculation amount of the primary Ganoderma lucidum strain in step (2.2) is 8% (v / v).

[0107] Preferably, the culture temperature in step (2.2) is selected from any value or range between 26-28℃, specifically from: 26℃, 26.5℃, 27℃, 27.5℃, 28℃ or a range between the two.

[0108] More preferably, the culture temperature in step (2.2) is 28°C.

[0109] Preferably, the culture time in step (2.2) is selected from any value or range between 24-48h, specifically from: 24h, 30h, 35h, 40h, 45h, 48h or a range between the two.

[0110] More preferably, the incubation time in step (2.2) is 24 hours.

[0111] Preferably, the aeration rate for the culture in step (2.2) is 1:0.8 (v / v / min).

[0112] Preferably, the cultivation in step (2.2) also requires stirring.

[0113] Preferably, the stirring speed is selected from any value or range between 100-500 rpm, specifically from: 100 rpm, 150 rpm, 200 rpm, 220 rpm, 250 rpm, 300 rpm, 350 rpm, 400 rpm, 450 rpm, 500 rpm or a range between the two.

[0114] More preferably, the stirring speed is 220 rpm.

[0115] Preferably, the specific methods for separation, extraction and purification described in step (3) are as follows: The fermentation broth of Ganoderma lucidum mycelium was subjected to gradient centrifugation and filtration. The precipitate was then washed, resuspended in buffer solution, and filtered again to obtain purified Ganoderma lucidum exosomes. Preferably, the mesh size of the filter is 0.22 μm.

[0116] Preferably, the specific operation of the gradient centrifugation is as follows: centrifugation at 800-1200×g for 8-15 minutes, centrifugation at 1800-2200×g for 15-25 minutes, centrifugation at 3500-4500×g for 25-40 minutes, and finally ultracentrifugation at 90000-120000×g at 4℃ for 1.0-2.0 hours.

[0117] More preferably, the specific operation of the gradient centrifugation is as follows: centrifugation at 1000×g for 10 minutes, centrifugation at 2000×g for 20 minutes, centrifugation at 4000×g for 30 minutes, and finally ultracentrifugation at 1000000×g at 4℃ for 1.5 hours.

[0118] Preferably, the rinsing procedure is as follows: rinse the Ganoderma lucidum exosome precipitate three times with pre-cooled PBS buffer, with each rinse using 5 mL of pre-cooled PBS buffer.

[0119] Preferably, the specific operation of suspending in the buffer solution is as follows: suspending the rinsed precipitate in PBS.

[0120] Preferably, the mesh size of the re-filtration in step (2.2) is 0.22 μm.

[0121] Preferably, the dosage of the Ganoderma lucidum exosomes is 1×10⁻⁶. 8 -1×10 9 Any value or range between particles / individuals.

[0122] More preferably, the dosage of the Ganoderma lucidum exosomes is 3 × 10⁻⁶. 8Particles / only.

[0123] Based on further solutions to the technical problems of the present invention, or simultaneous solutions to multiple technical problems, the preferred solution in the technical solution provided in the first aspect of the present invention includes: The first priority solution is the application of Ganoderma lucidum exosomes in the preparation of anti-allergic rhinitis products. This solution not only addresses the technical problem of "low content of active ingredients in Ganoderma lucidum exosomes," but also further solves the technical problem of "poor anti-allergic rhinitis effect."

[0124] The beneficial effects of this invention are as follows: The present invention has at least the following beneficial effects: 1. This invention achieves large-scale preparation of Ganoderma lucidum exosomes by optimizing fermentation conditions and separation and purification processes, significantly improving the yield of exosomes and the content of active ingredients, and providing theoretical and technical support for the research and development of related products.

[0125] 2. The method of the present invention can efficiently obtain high-purity Ganoderma lucidum exosomes. The exosomes have complete morphology, uniform particle size, and clear expression of characteristic markers. The preparation process is stable and controllable, and is suitable for industrial-scale production.

[0126] 3. The Ganoderma lucidum exosomes prepared by the method of the present invention can significantly improve the weight loss of model mice, inhibit the onset of allergic behavior, reduce the inflammatory infiltration of the respiratory tract (lung and nasal mucosa), and repair the mucosal tissue structure. It has clear anti-allergic inflammatory activity in vivo and can effectively improve the systemic and local pathological damage of allergic rhinitis. Attached Figure Description

[0127] Figure 1 The flowchart illustrates the method for preparing Ganoderma lucidum exosomes provided by this invention.

[0128] Figure 2 This is a particle size distribution diagram of Ganoderma lucidum exosomes prepared in Example 1 of the present invention.

[0129] Figure 3 This is an electron micrograph of the Ganoderma lucidum exosomes prepared in Example 1 of the present invention.

[0130] Figure 4 The figures show the body weight and behavioral results of mice before and after the intervention and treatment of allergic rhinitis according to the present invention; where A and B represent the trend of mouse body weight change after drug administration intervention; and C and D represent the results of the number of sneezing and nose scratching of mice within 10 minutes after drug administration.

[0131] Figure 5 This is a section of lung and nasal tissue in mice after interventional treatment for allergic rhinitis according to the present invention.

[0132] Figure 6This is a diagram showing the activity of Ganoderma lucidum exosomes prepared in Example 1 of the present invention in the intervention and treatment of inflammatory HUVEC cells.

[0133] Figure 7 The image shows the staining results of CD54 on inflammatory HUVEC cells. The scale bars are all 100 μm.

[0134] Figure 8 The image shows the staining results of CD14 on inflammatory HUVEC cells. The scale bars are all 100 μm. Detailed Implementation

[0135] The following non-limiting embodiments are intended to enable those skilled in the art to gain a more comprehensive understanding of the present invention, but do not limit the invention in any way. The following content is merely an exemplary description of the scope of protection claimed by the present invention, and those skilled in the art can make various changes and modifications to the present invention based on the disclosed content, and such changes should also fall within the scope of protection claimed by the present invention.

[0136] The present invention will be further described below by way of specific embodiments. Unless otherwise specified, all instruments, devices, equipment, reagents, products, etc., used in the embodiments of the present invention are obtained through conventional commercial means.

[0137] Table 1

[0138] Example 1: Preparation of Ganoderma exosomes (1) Cultivation of secondary seed culture (1.1) High-yielding triterpenoid Ganoderma lucidum hyphae (laboratory-screened strain 14021, with a ganoderic acid A content of 1.2 mg / g, detected by HPLC: column C18, mobile phase methanol-0.1% phosphoric acid water = 75:25, flow rate 1 mL / min, detection wavelength 254 nm) were inoculated into PDA medium (potato extract 200 g / L, glucose 20 g / L and agar powder 15 g / L, pH 6.0) and cultured on slant at 37℃ for 5 days until the Ganoderma lucidum hyphae filled the slant test tube (no contamination by other microorganisms, and the hyphae were thick and evenly branched under a microscope). (1.2) Pour 5 mL of sterile water into a slant tube covered with Ganoderma lucidum mycelium, scrape the mycelium from the culture medium into the water, and prepare a mycelial suspension; inoculate the suspension into 100 mL of seed culture medium (20 g / L glucose, 10 g / L tryptone, 5 g / L yeast extract, 1 g / L KH2PO4, 0.5 g / L MgSO4·7H2O, pH 6.0) and culture at 28℃ and 150 rpm for 5 days with shaking. The culture endpoint is determined by OD. 600 Value reaches 1.8-2.0, viable count ≥10 8CFU / mL (plate count method) was used to obtain primary seed culture; (1.3) The primary seed culture prepared in step (1.2) was broken up by a tissue homogenizer at 3000 rpm for 30 seconds (to break up the mycelial balls and increase the surface area of ​​the mycelium). The broken mycelium was then inoculated into a deep liquid fermentation medium (25 g / L glucose, 15 g / L soybean meal, 1.5 g / L KH2PO4, 0.8 g / L MgSO4·7H2O, pH 6.0) at an inoculation rate of 10% (v / v). The medium was cultured at 28℃ and 120 rpm for 2 days to obtain the secondary seed culture. (2) Expanded culture and acquisition of Ganoderma lucidum mycelium fermentation broth (2.1) The secondary seed culture prepared in step (1.3) was inoculated into a 10L fermenter (25g / L glucose, 15g / L soybean meal, 1.5g / L KH2PO4, 0.8g / L MgSO4·7H2O, pH 6.0) at an inoculation rate of 10% (v / v) and cultured for 5 days at 28℃, an aeration rate of 1:0.8 (v / v / min), and a stirring speed of 200rpm to obtain the primary Ganoderma lucidum strain. During the expansion culture, the OD was monitored every 6 hours. 600 Value (reflecting mycelial concentration), pH value (maintained at 5.8-6.2), dissolved oxygen (≥20%); (2.2) The primary Ganoderma lucidum strain prepared in step (2.1) was inoculated into a 50L fermenter at an inoculation rate of 8% (v / v) (25g / L glucose, 15g / L soybean meal, 1.5g / L KH2PO4, 0.8g / L MgSO4·7H2O, pH 6.0). After culturing at 28℃, aeration rate of 1:1 (v / v / min), and stirring speed of 220rpm for 24 hours, the mixture was filtered through a 0.22μm filter membrane to obtain the Ganoderma lucidum mycelium fermentation broth. (3) The fermentation broth of Ganoderma lucidum mycelium was separated, extracted and purified to obtain Ganoderma lucidum exosomes. The fermentation broth of Ganoderma lucidum mycelium was subjected to gradient centrifugation and the precipitate was filtered through a 300 nm (Millipore) filter membrane to remove aggregated particles with a diameter >300 nm. The precipitate was washed three times (5 mL each time) with pre-cooled PBS buffer to remove residual culture medium components. The precipitate was resuspended in PBS and the concentration was adjusted to 1 mg / mL (protein concentration was detected by BCA method). The precipitate was then sterilized through a 0.22 μm filter membrane to obtain purified Ganoderma lucidum exosomes. The specific operation of the gradient centrifugation is as follows: centrifugation at 1000×g for 10 minutes: removes unbroken mycelia and large particulate impurities, reducing the turbidity of the supernatant by 30%; centrifugation at 2000×g for 20 minutes: removes cell debris, and the OD of the supernatant is reduced. 600The value dropped below 0.3; centrifugation at 4000×g for 30 minutes: the residual culture medium precipitate was removed, and the supernatant was pale yellow and clear; ultracentrifugation at 100000×g for 1.5 hours (4℃, Beckman Optima XE-90 centrifuge): the precipitate was white flocculent, which was an exosome enrichment.

[0139] Detection Example 1 1. Experimental Methods Transmission electron microscopy (TEM) was used to observe the structure and diameter of exosomes, and nanoparticle tracking analysis (NTA, Malvern NanoSight NS300) was used to analyze the particle size distribution. 2. Experimental Results The Ganoderma exosomes prepared in Example 1 were round or elliptical with an intact membrane structure and a diameter of 50-170 nm. The particle size distribution results showed that the peak particle size of the Ganoderma exosomes prepared in Example 1 was about 80 nm, and 90% of the particles had a diameter of <150 nm.

[0140] Detection Example 2 This invention uses an allergic rhinitis (AR) mouse model as the research subject and conducts targeted animal experiments via nebulized drug delivery to comprehensively evaluate its biological activity from behavioral, serological, histopathological, and molecular mechanism levels, as detailed below: 1. Experimental Methods (1) Experimental animals: Forty SPF-grade female BALB / c mice, aged 6-8 weeks and weighing 20±2g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. The mice were housed in a barrier environment with a temperature of 22±2℃ and a humidity of 50±5%, with alternating light and dark for 12 hours. They had free access to food and water. The experiment began after one week of acclimatization. The entire process complied with the requirements of the "Guidelines for the Ethical Review of Laboratory Animal Welfare".

[0141] (2) Experimental reagents: ovalbumin (OVA, purity ≥98%, Sigma-Aldrich); aluminum hydroxide adjuvant (Alum, 10mgAl / mL, ThermoFisher); Ganoderma lucidum exosomes (Ganoderma lucidum exosomes prepared in Example 1 of this invention, purity ≥95%, concentration 1mg / mL); dexamethasone (purity ≥99%, Solarbio); PBS buffer (pH 7.4, Gibco).

[0142] (3) Experimental instruments: mouse-specific nebulizer (Shanghai Jiliang Medical); ELISA reader (Bio-Tek ELx808); pathological slicer (Leica RM2235); HE staining kit; high-speed refrigerated centrifuge (Eppendorf 5810R); electronic balance (Mettler Toledo).

[0143] (4) Reagent preparation: (4-1) Ovalbumin (OVA) Preparation: Accurately weigh 50 mg of OVA powder, add 5 mL of sterile PBS buffer (pH 7.4), gently stir at 4°C for 30 minutes until completely dissolved, prepare a 10 mg / mL stock solution, filter sterilize using a 0.22 μm filter membrane, and aliquot for storage. Protocol A: 10 mice / dose (total 2 mL complex). Take 50 μL of OVA stock solution (10 mg / mL), 1000 μL of Alum stock solution (10 mg Al / mL), add PBS to make up to 2000 μL, vortex at room temperature for 30 min, and use immediately. Inject 200 μL (containing 50 μg OVA + 1 mg Al) intraperitoneally into each mouse.

[0144] Option B: Dilute the OVA stock solution (10 mg / mL) 20 times with PBS to obtain a 0.5 mg / mL working solution. Mix an equal volume of the 0.5 mg / mL OVA working solution with a 10 mg / mL Alum stock solution to obtain a mixed solution containing 0.25 mg / mL OVA and 5 mg / mL Al. Each 200 μL solution contains 50 μg OVA + 1 mg Al.

[0145] (4-2) Preparation of aluminum hydroxide adjuvant (Alum): Commercially available Alum stock solution with a concentration of 10 mg Al / mL, equilibrate at room temperature for 30 minutes, vortex mix well and use directly; dispense 200 μL at 2 mg Al / tube, refrigerate at 4℃, and do not freeze.

[0146] (4-3) Preparation of OVA-Alum complex: Mix equal volumes of OVA stock solution and Alum stock solution, vortex at room temperature for 30 minutes, centrifuge at 1000×g for 5 minutes, discard the supernatant, resuspend in PBS to 200μL / animal, and use immediately.

[0147] (4-4) The packaging plan for this project is shown in Table 2: Table 2

[0148] (4-5) Preparation of Ganoderma lucidum exosome nebulization solution (low-dose group): 500 μL of 1 mg / mL Ganoderma lucidum exosome stock solution prepared in Example 1 was mixed with 9.5 mL of PBS to prepare 1×10 9 Microparticles / each; prepare and use immediately, store at 4℃ for no more than 4 hours.

[0149] (4-6) Preparation of dexamethasone solution: Dissolve 1 mg of dexamethasone in 1 mL of physiological saline to prepare a 1 mg / mL stock solution and store at -20°C; administer intraperitoneally at 1 mg / kg.

[0150] (4-7) hUC-MSC-EXO group: 500 μL of hUC-MSC-EXO (human umbilical cord mesenchymal stem cell exosome) stock solution was mixed with 9.5 mL of PBS to prepare 1×10 9 Microparticles / each; prepare and use immediately, store at 4℃ for no more than 4 hours.

[0151] Preparation of hUC-MSC-EXO (human umbilical cord mesenchymal stem cell exosomes) (4.7.1) Amplification culture system a. Complete amplification culture medium formulation DMEM / F12 basal medium + 10% conventional fetal bovine serum (FBS) + 1% 100× penicillin-streptomycin antibiotics Culture conditions: 37℃, 5% CO2, saturated humidity constant temperature cell culture incubator b. Cell resuscitation and expansion procedures (b.1) Take out the frozen P3 generation hUC-MSC (human umbilical cord mesenchymal stem cells), thaw rapidly in a 37°C water bath, aseptically transfer to centrifuge tubes, and dilute with fresh DMEM / F12 complete culture medium.

[0152] (b.2) Centrifuge at 300g at room temperature for 5 min, discard the frozen supernatant, and gently resuspend the cells in DMEM / F12 complete medium.

[0153] (b.3) Inoculate into T75 culture flasks, add complete culture medium, and incubate in an incubator for routine culture.

[0154] (2.4) Replace the culture medium with fresh complete medium every 2–3 days and observe the cell adhesion and growth status under a microscope.

[0155] (b.5) When the cell confluence reaches 80%–85%, passage should be performed, and P3–P5 generation cells should be used preferentially for exosome preparation.

[0156] c. Cell passage (c.1) Discard the old culture medium and gently wash the cell layer twice with sterile calcium- and magnesium-free D-PBS to completely remove residual serum.

[0157] (c.2) Add 2 mL of 0.25% Trypsin-EDTA digestion solution to the T75 culture flask, incubate at 37℃ for 2–3 min, and observe under a microscope that the cells shrink, become round, and begin to detach from the cell wall.

[0158] (c.3) Add an equal volume of DMEM / F12 complete culture medium to stop digestion, and gently blow on the bottom of the bottle to prepare a single-cell suspension.

[0159] (c.4) Centrifuge at 300g at room temperature for 5 minutes, discard the supernatant, and resuspend the cells in fresh complete culture medium.

[0160] (c.5) Passage and inoculate at a ratio of 1:3–1:4 to continue expansion to T175 large culture flasks for subsequent exosome collection.

[0161] (4.7.2) Culture of exosomes under specific conditions a. Conditioned culture medium formulation DMEM / F12 basal medium + 10% exosome-free fetal bovine serum + 1% 100× penicillin-streptomycin antibiotics.

[0162] b. Fluid replacement pretreatment operation (b.1) Wait until the hUC-MSC confluence in the T175 culture flask reaches 70%-75%.

[0163] (b.2) Completely dispose of the original complete culture medium containing conventional FBS.

[0164] (b.3) Gently wash the cell layer three times with pre-cooled sterile calcium and magnesium-free D-PBS to thoroughly wash away residual ordinary serum and impurities.

[0165] (b.4) Add the prepared DMEM / F12 exosome-free conditioned medium and return it to the incubator.

[0166] (b.5) Incubate at a constant temperature for 48-72 hours. During this period, do not shake the cells or allow them to fall off. Collect the cell supernatant after the culture is complete.

[0167] (4.7.3) Pretreatment of cell culture supernatant The entire process is carried out at a low temperature of 4°C to maintain exosome activity and reduce protein degradation.

[0168] a. Aseptically collect all cell culture supernatant and transfer it to a sterile centrifuge tube, avoiding aspirating adherent cells.

[0169] b. Low-speed centrifugation to remove cells: 300g, 10min, 4℃, discard the intact cell pellet at the bottom of the tube, and retain the supernatant.

[0170] c. Medium-speed centrifugation to remove debris: 2000g, 20min, 4℃, to remove cell debris and apoptotic bodies, retaining the supernatant.

[0171] d. The supernatant is filtered under negative pressure through a 0.22μm water filter to remove large-diameter vesicles and impurities, obtaining a pure pretreated supernatant.

[0172] e. The supernatant after filtration can be immediately subjected to ultracentrifugation or aliquoted at -80℃ for short-term storage. Repeated freeze-thaw cycles are prohibited.

[0173] (4.7.4) Purification of hUC-MSC-EXO by ultracentrifugation a. Transfer the filtered supernatant into an ultracentrifuge tube, balance it precisely, and place it in an ultracentrifuge.

[0174] b. First step: centrifugation to remove large vesicles: 10000g, 30min, 4℃, discard the precipitate and retain all supernatant.

[0175] c. Second step: centrifugation to enrich exosomes: 100,000g, 70min, 4℃, slowly discard the supernatant, the milky white thin layer of precipitate at the bottom of the tube is the crude exosome.

[0176] d. Gently resuspend the precipitate at the bottom of the tube in a small amount of pre-cooled D-PBS.

[0177] e. Wash and purify the protein to remove impurities: Centrifuge again at 100,000g for 70 minutes at 4°C.

[0178] f. Discard the supernatant and resuspend the final precipitate in an appropriate amount of pre-cooled sterile D-PBS according to experimental requirements to obtain purified hUC-MSC-EXO stock solution.

[0179] g. Aseptically packaged, stored at -80℃ away from light, thawed only once for single use, and repeatedly frozen and thawed.

[0180] (4.7.5) Standard quality control identification of exosomes a. Protein concentration: Total protein concentration was determined by the BCA method.

[0181] b. Particle size analysis: NTA detection showed that the main peak particle size was 30–150 nm, which is consistent with the particle size characteristics of exosomes.

[0182] c. Marker proteins (WB): Exosome-positive markers CD63, CD81, and TSG101 are expressed positively; endoplasmic reticulum marker Calnexin is negative.

[0183] (5) Experimental scheme The 40 mice were randomly divided into 5 groups of 8 mice each. The specific treatment methods are shown in Table 3 below: Table 3

[0184] AR model establishment sensitization phase (days 1-14): intraperitoneal injection of sensitizer (OVA-Al adjuvant mixture) twice a week, 200 μL / animal each time; challenge phase (days 15-28): intranasal instillation of 5% OVA solution, 10 μL / side, daily for 14 days.

[0185] The dosing regimens were as follows: nebulization group: 100 μL was nebulized once a day for 7 consecutive days within 1 hour after stimulation; dexamethasone group: intraperitoneal injection once a day for 7 consecutive days within 1 hour after stimulation.

[0186] Record the body weight of mice before and after drug administration, and calculate the average body weight; record the number of times mice in different groups scratch their noses and sneeze within 10 minutes after drug administration.

[0187] Sample collection: The patient was euthanized by cervical dislocation. Lung and nasal tissues were separated, fixed in 4% paraformaldehyde, and then subjected to hematoxylin and eosin (HE) sectioning. The specific method is as follows: A. Nasal mucosa sampling and fixation: (1) After euthanizing the mouse, completely dissect the mouse's head, remove the mandible and excess skin and soft tissue, carefully cut open the nasal cavity along the midline of the skull, completely dissect the bilateral turbinate bones and attached mucosal tissue, and avoid tearing and detachment of the mucosa.

[0188] (2) Trim the tissue blocks to a uniform size of approximately 0.2cm×0.2cm×0.15cm, ensuring that they contain complete nasal epithelium and lamina propria.

[0189] (3) Immediately add sufficient 4% paraformaldehyde fixative (fixative volume ≥ 10 times that of tissue) and fix at room temperature in the dark for 24-48 hours.

[0190] B. Lung tissue sampling and fixation: (1) The mouse was opened up and the lungs were completely separated. Excess connective tissue in the trachea and mediastinum was removed. Representative lung blocks from the middle and lower lobes of the lungs were selected, with a size of 0.3cm×0.3cm×0.2cm.

[0191] (2) Pre-treatment (to prevent alveolar collapse): a small amount of 4% paraformaldehyde is slowly injected into the airway to pre-fill the airway, and then lung tissue is harvested.

[0192] (3) Immerse the tissue in 4% paraformaldehyde fixative and fix at room temperature for 24-48 hours.

[0193] C. Washing and gradient dehydration (1) After fixation, rinse all tissues with running water for 2 hours to thoroughly wash away residual paraformaldehyde and avoid staining background impurities.

[0194] (2) Gradient ethanol dehydration process: 1) 70% ethanol: 4h 2) 80% ethanol: overnight 3) 90% ethanol: 2h 4) 95% ethanol I: 1h; 95% ethanol II: 1h 5) Anhydrous ethanol I: 30min; Anhydrous ethanol II: 30min D. Transparency treatment (1) Soak in a 1:1 mixture of anhydrous ethanol and xylene for 20 min. (2) Pure xylene I: 15 min; Pure xylene II: 15 min (3) Judgment criteria: The tissue should be uniformly translucent; the duration of transparency of lung tissue should be strictly controlled, otherwise the tissue will become brittle and break.

[0195] E. Wax impregnation, paraffin embedding (1) Wax impregnation (constant temperature 60℃): immerse in soft wax (52~54℃) for 30min → immerse in hard wax (56~58℃) twice, 30min each time.

[0196] (2) Differentiation of embedding operations: Nasal mucosa: Place the mold with the mucosal epithelial side down, pour in molten paraffin, and let it cool and solidify; Lung tissue: The airway section is placed face down to ensure that the alveoli and bronchioles can be observed completely.

[0197] Store paraffin blocks at room temperature for later use.

[0198] F. Slicing, spreading, and baking (1) Trimming: Remove the paraffin layer to fully expose the tissue cross-section; (2) The slice thickness was uniformly 4 μm; (3) Spread the slides in a constant temperature water bath at 42℃ to smooth out tissue folds and prevent them from falling off the glass slides to retrieve complete sections; (4) Baking the slides in a 60℃ oven for 2 hours ensures that the tissue adheres firmly to the slides and does not fall off during the staining process.

[0199] G. Standardized HE staining procedure (1) Dewaxing and rehydration: xylene I 10 min → xylene II 10 min → anhydrous ethanol 3 min → 95% ethanol 3 min → 85% ethanol 3 min → rinse with distilled water 1 min.

[0200] (2) Stain the cell nuclei with hematoxylin solution for 5-8 min, rinse with running water for 1 min; differentiate with 1% hydrochloric acid alcohol for 3-5 s, soak in tap water for 10 min to return to blue.

[0201] (3) Stain the cytoplasm with eosin solution for 2 minutes, then rinse quickly with distilled water to remove the floating color.

[0202] (4) Dehydration and clearing: 70% → 80% → 90% → 95% → anhydrous ethanol, dehydration in stages, 2 min for each stage; xylene I and xylene II, clearing in 5 min each.

[0203] (5) Mounting: Seal with neutral resin, cover with a glass slide, and let dry at room temperature for microscopic examination.

[0204] H. Microscopic observation indicators (1) Observation indicators of nasal mucosal sections Integrity of nasal epithelium and degree of epithelial cell shedding; edema and congestion of the lamina propria; number of eosinophils, lymphocytes, and mast cells infiltrating; glandular hyperplasia and amount of mucus secretion.

[0205] (2) Observation indicators of lung tissue sections Desquamation of bronchiolar epithelium and airway mucus secretion; thickening of alveolar walls and alveolar congestion and edema; infiltration of eosinophils and inflammatory cells in the airways and pulmonary interstitium; degree of airway remodeling and alveolar structural damage.

[0206] Statistical analysis All data were analyzed using SPSS 22.0. Quantitative data are expressed as mean ± standard deviation (x±s). One-way ANOVA was used for comparisons among multiple groups, and LSD-t test was used for pairwise comparisons. P < 0.05 was considered statistically significant.

[0207] 2. Experimental Results A mouse model of allergic rhinitis constructed by OVA in combination with aluminum adjuvant was successfully established. The model mice exhibited typical symptoms such as weight loss, frequent sneezing and nose scratching, and severe inflammation of the lung and nasal mucosa.

[0208] (1) Behavioral rating Normal group: 0.5±0.3 sneezes, 1.2±0.5 nose scratches; AR model group: 32.6±4.2 sneezes, 45.8±5.6 nose scratches (P<0.001); Exosome group of Example 1: 8.6±2.5 sneezes, 10.2±3.1 nose scratches, with no significant difference from the dexamethasone group (P>0.05).

[0209] (2) Histopathological examination (HE staining) Normal group: Nasal mucosa structure intact, eosinophils 2.3±0.8 / high power field; AR model group: epithelial damage, edema, and extensive infiltration, 35.6±5.2 / high power field (P<0.001); Example 1 exosome group: 5.8±1.5 / high power field, no significant difference from dexamethasone group (P>0.05).

[0210] Detection Example 3 This embodiment uses a TNF-α-induced HUVEC cell inflammation model and intervenes with Ganoderma lucidum exosomes prepared according to this invention. The anti-inflammatory effect is verified by CD54 and CD144 antibody immunofluorescence staining, as detailed below: (a) Experimental materials 1. Experimental cells Human umbilical vein endothelial cells (HUVECs) were purchased from the Cell Bank of the Chinese Academy of Sciences.

[0211] 2. Experimental reagents TNF-α cytokine (PeproTech); Ganoderma lucidum exosomes (self-made in this invention, purity ≥95%, concentration 1 mg / mL); CD54 antibody, CD144 antibody (Abcam, dilution ratio 1:500); fluorescent secondary antibody (Cy3-labeled goat anti-rabbit IgG, Servicebio, dilution ratio 1:1000); 4% paraformaldehyde solution; PBS buffer (pH 7.4); DAPI staining solution; fetal bovine serum for cell culture, DMEM medium.

[0212] 3. Experimental Apparatus Cell culture incubator (ThermoFisher); laser confocal fluorescence microscope (OlympusFV3000); clean bench; high-speed refrigerated centrifuge; electronic balance.

[0213] (II) Experimental Methods 1. Cell Culture and Grouping Logarithmic growth phase HUVEC cells were harvested, digested with trypsin, and then a homogeneous cell suspension was prepared using complete culture medium. The cell density was adjusted to 1×10⁻⁶ cells / cells. 5 Cells were seeded per well in confocal culture dishes, with 2 mL of complete culture medium added to each dish; the cells were cultured at 37°C and 5% CO2 until they were completely adhered and confluent.

[0214] The cells were divided into 6 groups: normal group, TNF-α model group (model group), Ganoderma lucidum exosome intervention group (Example 1 group), H-EXO group (exosomes derived from human umbilical cord mesenchymal stem cells), Ganoderma lucidum exosome + TNF-α group (Example 1 + TNF-α group), and H-EXO + TNF-α group (exosomes derived from human umbilical cord mesenchymal stem cells + TNF-α group).

[0215] 2. Model Establishment and Intervention Treatment The normal group received no treatment, and the complete culture medium was replaced as usual. The TNF-α model group was replaced with serum-free culture medium containing 10 ng / mL TNF-α and cultured for another 24 hours to construct a cell inflammation model. The Ganoderma lucidum exosome intervention group (Example 1 group) had TNF-α added to the complete culture medium at a concentration of 10 ng / mL. 7 Ganoderma lucidum exosomes at 1000000 mL; Ganoderma lucidum exosomes + TNF-α group (Example 1 + TNF-α group) consisted of Ganoderma lucidum exosomes prepared in Example 1. 7 Particals / mL and 10 ng / mL TNF-α serum-free medium were co-incubated for 24 h; the H-EXO group was prepared by adding 10 ng / mL TNF-α to complete medium. 7 Human umbilical cord mesenchymal stem cell exosomes with a concentration of 10 / mL, H-EXO+TNF-α group: human umbilical cord mesenchymal stem cell exosomes 7Particals / mL and serum-free medium containing 10 ng / mL TNF-α were co-incubated for 24 h.

[0216] 3. Cell viability assay Cell viability was assessed using a CCK-8 assay kit. Cells were divided into three groups: a normal group, a TNF-α model group (model group), and a Ganoderma lucidum exosome intervention group (G-EXO group). The concentration of Ganoderma lucidum exosomes was 10⁻⁶ g / L. 7 Particles / each, 5×10 7 microparticles / each, 10 8 Particles / each, 5×10 8 Particle / unit.

[0217] 4. Immunofluorescence staining After incubation, the culture medium was discarded, and the cells were washed three times with pre-cooled PBS buffer for 5 min each time. The cells were fixed with 4% paraformaldehyde at room temperature for 15 min, and washed three times with PBS. The cells were permeabilized with 0.3% Triton X-100 for 10 min, washed with PBS, and blocked with 5% bovine serum albumin blocking solution at room temperature for 1 h. The blocking solution was discarded, and CD54 primary antibody and CD144 primary antibody were added respectively, and the cells were incubated overnight at 4°C. The next day, the unbound primary antibody was thoroughly washed away with PBS, the corresponding fluorescent secondary antibody was added, and the cells were incubated at room temperature in the dark for 1 h. After washing with PBS, the cells were stained with DAPI for 5 min, washed three times with PBS, and then observed and images were acquired under a laser confocal fluorescence microscope.

[0218] (III) Experimental Results In the normal group, HUVEC cells maintained intact morphology, with weak CD54 fluorescence signal and strong and uniformly distributed CD144 fluorescence signal, and tight intercellular connections. In the TNF-α model group, cells were shrunken, with significantly enhanced CD54 fluorescence signal and significantly weakened CD144 fluorescence signal, indicating broken and impaired cell connection structures. After intervention with Ganoderma lucidum exosomes prepared in this patent, HUVEC cell morphology returned to normal, CD54 fluorescence intensity decreased significantly, CD144 fluorescence signal rebounded significantly, and intercellular connection structures became more intact and tight. The results indicate that the Ganoderma lucidum exosomes prepared in this patent can effectively inhibit TNF-α-induced inflammatory responses in HUVEC cells, protect cell barrier structures, and have good anti-inflammatory effects.

[0219] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. The application of Ganoderma lucidum exosomes in the preparation of anti-allergic rhinitis products, characterized in that, The method for preparing Ganoderma lucidum exosomes includes the following steps: (1) Cultivating secondary seed culture; (2) Expanded culture and acquisition of Ganoderma lucidum mycelium fermentation broth; (3) Separate, extract and purify the fermentation broth of Ganoderma lucidum mycelium to obtain Ganoderma lucidum exosomes.

2. The application according to claim 1, characterized in that, The specific method for cultivation described in step (1) is as follows: (1.1) Inoculate high-yield triterpenoid Ganoderma lucidum mycelia into PDA medium for slant culture until the Ganoderma lucidum mycelia cover the slant test tubes; (1.2) Pour water into a slant test tube covered with Ganoderma lucidum mycelium, scrape the Ganoderma lucidum mycelium on the culture medium into the water to make a mycelium suspension; inoculate the suspension into a seed culture medium to obtain a primary seed solution; (1.3) Shake the primary seed liquid prepared in step (1.2) to break up the mycelial balls, and inoculate the broken mycelium into a deep liquid fermentation medium to obtain the secondary seed liquid.

3. The application according to claim 2, characterized in that, The high-yielding triterpenoid Ganoderma lucidum mycelium mentioned in step (1.1) is laboratory-screened strain 14021, whose ganoderic acid A content is ≥1.2mg / g.

4. The application according to claim 2, characterized in that, The PDA culture medium described in step (1.1) comprises: 180-220 g / L potato extract, 18-22 g / L glucose and 15-20 g / L agar powder; The pH of the PDA culture medium is 5.5-6.0; The temperature for slant culture is 26-37℃; The slant culture time is 7-10 days.

5. The application according to claim 2, characterized in that, The seed culture medium described in step (1.2) comprises: glucose 30-40 g / L, tryptone 4-6 g / L, yeast extract 2-3 g / L, KH2PO4 0.8-1.2 g / L and MgSO4·7H2O 0.4-0.6 g / L; The pH of the seed culture medium is 5.5-6.

0.

6. The application according to claim 2, characterized in that, The culture temperature described in step (1.2) is 26-28℃; The endpoint of the culture is determined by: OD 600 Value reaches 1.8-2.0, viable count ≥10 8 CFU / mL.

7. The application according to claim 2, characterized in that, The submerged liquid fermentation medium described in step (1.3) includes: glucose 30-40 g / L, soybean meal powder 8-15 g / L, KH2PO4 1.2-1.8 g / L, and MgSO4·7H2O 0.8-2.5 g / L; The pH of the deep liquid fermentation medium is 5.5-6.0; The culture temperature is 26-28℃; The cultivation time is 1-5 days.

8. The application according to claim 1, characterized in that, The specific method for expanding the culture and obtaining the fermentation broth in step (2) is as follows: (2.1) The secondary seed liquid prepared in step (1.3) is inoculated into a fermenter for culture and scale-up culture to obtain the primary Ganoderma lucidum strain; (2.2) The primary Ganoderma lucidum strain prepared in step (2.1) is then inoculated into a fermenter for culture. After the culture is completed, the mixture is filtered to obtain Ganoderma lucidum mycelium fermentation broth.

9. The application according to claim 1, characterized in that, The specific methods for separation, extraction and purification described in step (3) are as follows: After gradient centrifugation and filtration of the Ganoderma lucidum mycelium fermentation broth, the precipitate was washed, then suspended in buffer solution and filtered again to obtain purified Ganoderma lucidum exosomes. Preferably, the specific operation of the gradient centrifugation is as follows: centrifugation at 800-1200×g for 8-15 minutes, centrifugation at 1800-2200×g for 15-25 minutes, centrifugation at 3500-4500×g for 25-40 minutes, and finally ultracentrifugation at 90000-120000×g at 4℃ for 1.0-2.0 hours.

10. The application according to claim 1, characterized in that, The dosage of the Ganoderma lucidum exosomes is 1×10⁻⁶. 8 -1×10 9 Particle / unit.

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