Extraction method and application of fungus-derived exosome-like nano vesicles

Abstract

The invention discloses an extraction method and application of fungus source exosome-like nano vesicles. The extraction method comprises the following steps: preparing a fungus filtrate by a hot water method, carrying out centrifugation and filtration purification on the fungus filtrate, and then carrying out ultrafiltration interception to obtain the fungus-derived exosome-like nano-vesicles. The extracted fungus-derived exosome-like nano-vesicles have a lipid bilayer structure, are uniform in particle size distribution, are rich in lipid, nucleic acid and protein, are non-toxic, and have a remarkable anti-inflammatory effect; the extraction method provided by the invention is suitable for extracting exosome-like nano vesicles in various fungal samples, and has high yield and high activity.

Description

Technical Field

[0001] The present invention belongs to the technical field of natural nanomedicines, and specifically relates to a method for extracting fungal exosome-like nanovesicles and applications thereof. Background Art

[0002] Extracellular vesicles are defined by the International Extracellular Vesicle Society as "particles that are naturally released from cells, encapsulated by a lipid bilayer, and cannot replicate, i.e., do not contain a functional core". Extracellular vesicles can be released by various types of cells, including cells of plants, animals, and microorganisms. A large amount of evidence shows that extracellular vesicles play an important role in many biological processes, including toxicity processing, intercellular communication, and pathogen defense. However, the biological heterogeneity of extracellular vesicles has hindered their clinical application. This heterogeneity is affected by both intracellular biological processes and interference from human manipulation.

[0003] Among various types of vesicles, plant-derived cell vesicles are considered to be less risky because most of them are extracted from edible and medicinal plants. Because their shape is consistent with mammalian cell-derived exosomes, they are called exosome-like. Excitingly, exosome-like nanovesicles secreted by plant cells are becoming the next generation of nanotherapeutics due to their low toxicity, low immune risk, biocompatibility, eco-friendliness, low cost, and easy mass production. Plant-derived exosome-like nanovesicles were first recorded by transmission electron microscopy in the 1960s. Although early observations failed to prove their importance, in 2013, Zhang's team successfully extracted vesicles from grapefruit through a scalable method and conducted biological application research. Due to their high stability and broad application prospects, research on plant-derived exosome-like nanovesicles has attracted widespread attention. The biological composition of plant-derived exosome-like nanovesicles is mainly divided into proteins, lipids, nucleic acids, and secondary metabolites. There are differences in the composition of exosome-like nanovesicles obtained from plants of different origins, and they have different biological effects and organ targeting.

[0004] Current research mostly uses gymnosperms and angiosperms, but these plants take a long time to grow and are affected by seasons and origins, resulting in instability between batches of plant exosomes, changes in composition and content, and unstable biological activity. Therefore, it is costly to obtain quality-controlled plant exosomes, which greatly limits their clinical application and industrialization. Summary of the invention

[0005] In view of the deficiencies in the prior art, the object of the present invention is to provide a method for extracting exosome-like nanovesicles from fungi and their application.

[0006] Edible fungi in the Fungi phylum, such as Auricularia auricula, Lentinula edodes, and Flammulina velutipes, have a wide range of origins, short growth cycles, and mature artificial cultivation techniques, with high quality controllability; the bioactive components and content of edible fungal exosomes are stable, supporting large-scale extraction. The method for extracting exosome-like nanovesicles of the present invention is generally applicable to edible fungi, provides a method for extracting exosome-like nanovesicles from fungi, and expands its biomedical applications.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] A method for extracting fungal exosome-like nanovesicles comprises the following steps:

[0009] (1) tearing the fungus into pieces, adding the pieces to a solution and soaking them, and then heating and boiling the pieces to obtain a fungal liquid;

[0010] (2) Squeeze the fungal liquid at 60-100° C. in step (1), and filter through gauze to obtain a fungal solution;

[0011] (3) filtering the fungal solution at 30-80° C. in step (2) through a 70 μm±10 μm filter, centrifuging at 500-1000 g for 5-20 minutes, and collecting the supernatant;

[0012] (4) centrifuging the supernatant at 20-60° C. in step (3) at 10,000-25,000 g for 15-80 minutes and collecting the supernatant;

[0013] (5) filtering the supernatant obtained in step (4) first through a filter membrane with a pore size of 0.45 μm ± 0.1 μm, and then through a filter membrane with a pore size of 0.22 μm ± 0.05 μm, and collecting the filtrate;

[0014] (6) The filtrate obtained in step (5) is subjected to a first ultrafiltration at 0-40° C., and the filtrate is subjected to a second ultrafiltration to obtain a concentrated solution, which is the fungal exosome nanovesicles; the first ultrafiltration uses an ultrafiltration membrane with a cutoff pore size of 90-100 kDa, and the second ultrafiltration uses an ultrafiltration membrane with a cutoff pore size of 10-30 kDa.

[0015] The present invention selects fresh edible fungi, prepares fungal liquid by hot water method, and intercepts exosome-like nanovesicles by temperature control, filtration, centrifugation and ultrafiltration.

[0016] Preferably, in step (1), the soaking time is 6-24h; the soaking temperature is 0-40°C; the pH range of the solution is 5.8-7.8; the solution is selected from at least one of water (deionized water), phosphate buffer, Tris buffer, Tris-HCl buffer, TBS buffer, TBST buffer, TE buffer and Good's buffer.

[0017] Further preferably, in step (1), the soaking time is 8-12 hours; the soaking temperature is 4-10° C.; and the pH range of the solution is 7.0 to 7.6.

[0018] Preferably, in step (1), the mass volume ratio of the fungus to the solution is 100-200 g:500 mL.

[0019] Preferably, in step (1), the fungus is an edible fungus. The fungus is selected from the whole plant, including the cap and the stipe.

[0020] Preferably, in step (1), the fungus is selected from at least one of shiitake mushrooms, straw mushrooms, mushrooms, black fungus, white fungus, hericium erinaceus, bamboo fungus, pine mushroom (Matsutake), white mushroom, red mushroom, ganoderma lucidum, thunder ball, big red mushroom, truffle, white lingzhi mushroom, bolete, morel, saddle mushroom, truffle, dry mushroom, cordyceps sinensis, white ginseng mushroom, chicken mushroom, tiger milk mushroom, purple lilac mushroom, chanterelle, pine lactarius, Ode mushroom (water chicken), hydrangea, blue-headed mushroom, coral mushroom, yellow-handled chanterelle (trumpet mushroom), shuttle-handled milk mushroom (old man's head), Agaricus bisporus, long root mushroom, puffball, Poria, Polyporus, hairy headed coprinus, golden ear, versicolor, tree tongue, pseudo boletus, purple ganoderma, Flammulina velutipes, Pleurotus eryngii, Oyster mushroom, pine mushroom, hazel mushroom, bamboo yellow, honey fungus, and mulberry yellow.

[0021] Preferably, in step (1), the boiling time is 5-80 minutes.

[0022] Further preferably, in step (1), the boiling time is 15-45 minutes.

[0023] A fungus-derived exosome-like nanovesicle is prepared by the above extraction method.

[0024] A lipid nanovesicle is prepared by extracting lipids from the fungus-derived exosome-like nanovesicle and then adopting a thin film hydration method.

[0025] Use of the fungus-derived exosome-like nanovesicles or the lipid nanovesicles in the preparation of drugs.

[0026] Preferably, fungus-derived exosome-like nanovesicles or lipid nanovesicles are used as drug carriers or active pharmaceutical ingredients.

[0027] Preferably, fungal exosome nanovesicles and lipid nanovesicles are used as drug carriers, and the drug also includes a delivery drug, and the delivery drug includes at least one of proteins, antibodies, peptides, small molecule drugs, traditional Chinese medicines, nucleic acids, exosomes, inorganic nanoparticles, and organic nanoparticles;

[0028] Preferably, fungal exosome nanovesicles and lipid nanovesicles are used as drug carriers; the drug is at least one of an autoimmune disease drug, an acute injury drug, a wound repair drug, a tumor treatment drug, and a medical aesthetic anti-aging drug.

[0029] Preferably, fungus-derived exosome-like nanovesicles and lipid nanovesicles are used as active ingredients of the drug; the drug is one of a drug for preventing alcoholic liver damage or a drug for treating psoriasis.

[0030] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0031] The fungal exosome nanovesicles of the present invention can be combined with plant exosome nanovesicles, liposomes, extracellular vesicles or exosomes derived from mammalian cells for treatment.

[0032] The fungus-derived exosome nanovesicles of the present invention can be prepared into hybrid nanovesicles with plant-derived exosome nanovesicles, liposomes, extracellular vesicles or exosomes derived from mammalian cells by extrusion, freeze-thaw and ultrasound methods.

[0033] The edible fungal exosomes extracted by the present invention have a lipid bilayer structure, uniform particle size distribution, are rich in lipids, nucleic acids, proteins and secondary metabolites, can overcome the gastrointestinal barrier, maintain good physiological activity in the complex and changeable gastrointestinal environment, and are non-toxic. It can be used to extract edible fungal exosomes from large-volume, low-concentration samples, filling the gap in the research of edible fungal exosomes.

[0034] The beneficial effects of the present invention are:

[0035] The present invention provides a method for extracting edible fungal exosome nanovesicles with strong universality, simple extraction method and low cost. The extracted edible fungal exosome nanovesicles have the advantages of high biocompatibility, safety, low toxicity, low cost and easy availability. At the same time, edible fungal exosome nanovesicles can not only be taken up by cells and deliver internal active substances, induce a variety of cytokines for biological applications such as intercellular communication and disease treatment, but also be used as an excellent delivery carrier for a variety of drug delivery. In addition, it also supports biological and chemical modifications and combined treatments. Edible fungal exosome nanovesicles can cross-border regulate the expression of multiple genes and proteins in mammalian cells, thereby exerting therapeutic effects. This provides a new strategy for cross-border applications between animals and plants. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 The particle size and potential of shiitake mushroom exosome nanovesicles and shiitake mushroom exosome-derived lipid nanovesicles.

[0037] Figure 2It is the particle size of exosome nanovesicles from Flammulina velutipes, Pleurotus eryngii, White mushroom, Alternaria albuminosa, and Oyster mushroom.

[0038] Figure 3 This is the potential of exosome nanovesicles from Flammulina velutipes, Pleurotus eryngii, White button mushroom, Alternaria albuminosa, and Pleurotus ostreatus.

[0039] Figure 4 These are the experimental results of the antioxidant stress effect of edible fungal exosome nanovesicles on RAW264.7 cells.

[0040] Figure 5 The antioxidant stress effect of shiitake mushroom exosome-derived nanovesicles obtained by different preparation methods.

[0041] Figure 6a Lipid composition of shiitake mushroom exosome nanovesicles.

[0042] Figure 6b is the lipid content of shiitake mushroom exosome nanovesicles.

[0043] Figure 7 The oral stability and in vivo retention time of shiitake mushroom exosome nanovesicles and shiitake mushroom exosome-derived lipid nanovesicles.

[0044] Figure 8 To investigate the effects of shiitake mushroom exosome nanovesicles and shiitake mushroom exosome-derived lipid nanovesicles in preventing alcoholic liver injury in in vivo experiments.

[0045] Fig. 9 The effects of shiitake mushroom exosome nanovesicles and shiitake mushroom exosome-derived lipid nanovesicles on preventing alcohol-induced intestinal barrier damage in in vivo experiments.

[0046] Fig.10 To investigate the effects of shiitake mushroom exosome nanovesicles and shiitake mushroom exosome-derived lipid nanovesicles in preventing drug-induced liver injury in in vivo experiments. DETAILED DESCRIPTION

[0047] The following is a detailed and complete description of the concept of the present invention and the technical effects produced in combination with the embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only a part of the present invention, rather than all embodiments. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without paying creative work all belong to the protection scope of the present invention.

[0048] Example 1: Preparation of edible fungal exosome nanovesicles

[0049] Six edible fungi (Lentinus edodes, Flammulina velutipes, Pleurotus ostreatus, Agaricus bisporus, King oyster, and Termitomyces albuminosus) were selected to prepare exosome-like nanovesicles.

[0050] (1) Take 5-7 fresh edible fungi (shiitake mushrooms, enoki mushrooms, oyster mushrooms, white mushrooms, king oyster mushrooms, and Alstroemeria), weighing about 150 g, rinse the surface stains and rinse with ultrapure water. Tear the edible fungi into pieces and add them to 500 mL PBS buffer (0.01 M, pH 7.2-7.4), and soak them at 4°C overnight (8-12 h);

[0051] (2) heating the edible fungus solution soaked overnight in step (1) to boiling, and keeping boiling for 30 minutes to obtain an edible fungus liquid;

[0052] (3) Squeeze the edible fungus liquid at 90-70° C. in step (2) together with the edible fungus fragments, and filter with gauze to obtain a fungus solution;

[0053] (4) filtering the fungal solution at 70-50° C. in step (3) through a 70 μm filter, centrifuging at 1000 g for 20 minutes, and collecting the supernatant;

[0054] (5) taking the supernatant from step (4) (temperature about 30-50°C), centrifuging at 20,000 g for 30 minutes at room temperature, and collecting the supernatant;

[0055] (6) taking the supernatant in step (5) (temperature is room temperature), filtering it with filter membranes with pore sizes of 0.45 μm and 0.22 μm in turn, and collecting the filtrate;

[0056] (7) Take the filtrate in step (6), perform the first ultrafiltration at 4°C, collect the filtrate; then perform the second ultrafiltration, and collect the concentrate. The first ultrafiltration uses an ultrafiltration membrane with a cutoff pore size of 100 kDa (molecular weight in the filtrate is 0-100 kDa), and the second ultrafiltration uses an ultrafiltration membrane with a cutoff pore size of 10 kDa (molecular weight in the concentrate is 10-100 kDa). The final concentrate is the edible fungus-derived exosome nanovesicles.

[0057] Finally, the hydrated particle size of shiitake mushroom exosome nanovesicles (MELN) was 126.3±23.7nm, the potential was -7.78±0.12mV, the hydrated particle size of Flammulina velutipes exosome nanovesicles was 286.2±12.0nm, the potential was -13.9±0.61mV, and the hydrated particle size of Pleurotus ostreatus exosome nanovesicles was 353.6±10.27nm, the potential was -12.1±2. 21mV, the hydrated particle size of white mushroom exosome nanovesicles was 233.6±41.3nm, the potential was -9.3±1.125mV, the hydrated particle size of Pleurotus eryngii exosome nanovesicles was 216.8±18.74nm, the potential was -7.22±1.14mV, the hydrated particle size of Alternaria albuminosa exosome nanovesicles was 395.8±62.9nm, the potential was -10.1±0.56mV( Figure 1 The left and right pictures in Figure 2 , Figure 3 ).

[0058] Preparation of Lentinus edodes exosome-derived lipid vesicles (MELV) The total lipids of Lentinus edodes exosome nanovesicles (MELN) were extracted according to the Bligh-Dyer method. The specific steps are as follows: 1 mL of the above-prepared Lentinus edodes exosome nanovesicle PBS solution was added with 5 mL of 2:1 (v / v) MeOH:CHCl 3 Mix the solution and vortex. Then add 2.5 mL CHCl 3 and 2.5 mL ddHO 2 O and vortex. The mixture was centrifuged in a glass bottle at 2,000 rpm for 10 minutes at 22°C. After centrifugation, the mixture was separated into an aqueous phase and an organic phase. The organic phase was aspirated into a new glass bottle, and the residual chloroform was removed using a vacuum pump for 10-15 minutes. The dried lipids were immediately suspended in PBS and, after 10 minutes of ultrasonic treatment, filtered using a 0.22 μm microporous membrane to obtain MELV.

[0059] The particle size of MELV is 281.6±19.6nm and the potential is -2.18±0.13mV( Figure 1 (center and right images in Figure 2).

[0060] Example 2: Yields of Lentinus edodes exosome nanovesicles obtained by different preparation methods

[0061] In this example, different experimental groups were designed based on the controlled variable method to compare the preparation methods and yields of Lentinus edodes exosome nanovesicles.

[0062] Experimental group 1: prepared according to the operation method of Example 1;

[0063] Experimental group 2: Based on Example 1, the juice was directly squeezed and filtered without boiling;

[0064] Experimental group 3: Based on Example 1, the juice was directly boiled and filtered without soaking overnight at 4°C;

[0065] Experimental Group 4: Based on Example 1, after the bacterial liquid was boiled, all subsequent experimental operations were performed at 4°C for juicing, filtration and purification.

[0066] The obtained shiitake mushroom exosome nanovesicles were quantitatively analyzed by the BCA method, and the yields were compared.

[0067] The final yields were: 0.32±0.082 g / kg for experimental group 1, 0.018±0.0093 g / kg for experimental group 2, 0.25±0.031 g / kg for experimental group 3, and 0.17±0.014 g / kg for experimental group 4 (Table 1). The experimental results show that different preparation methods have a significant effect on the yield of shiitake exosome nanovesicles.

[0068] Table 1

[0069]

[0070] Example 3: Study on the in vitro antioxidant stress effect of edible fungal exosome nanovesicles

[0071] The DCFH-DA was used to detect hydrogen peroxide (H 2 O 2 )-induced oxidative stress response of RAW264.7 cells. DCFH-DA itself has no fluorescence and can freely penetrate the cell membrane. After entering the cell, DCFH-DA is hydrolyzed by the esterase in the cell to generate DCFH, which cannot pass through the cell membrane, making it easier to load the probe into the cell. Reactive oxygen in the cell can oxidize the non-fluorescent DCFH to generate fluorescent DCF, thereby realizing the detection of oxidative stress.

[0072] (1) Preparation of DCFH mother solution concentration: Dissolve 50 mg of DCFH-DA probe powder in 1.026 mL of DMSO to prepare a 100 mM DCFH-DA probe solution. Dilute it 100 times to obtain a 1 mM DCFH-DA probe solution (using DMSO for dilution).

[0073] (2) Establish H 2 O 2 Induced RAW264.7 cell oxidative stress model. First, RAW264.7 cells were plated at a density of 50% and cultured overnight. When the cell density reached 70%, H 2 O 2, incubated for 6 h, and then directly added the edible fungal exosome nanovesicles prepared in Example 1, and continued to incubate for 12 h.

[0074] (3) Add DCFH-DA probe, incubate at 37°C for 30 min, and observe green fluorescence using a fluorescence microscope.

[0075] The fluorescence intensity of each group was observed by fluorescence microscopy. The results showed that the fluorescence of the exosome-like nanovesicles (MELN) group derived from Lentinus edodes was the weakest, indicating that it has a strong anti-ROS effect ( Figure 4 ).

[0076] Example 4: Antioxidative stress effects of shiitake mushroom exosome nanovesicles prepared by different preparation methods

[0077] In this example, different experimental groups were designed by the control variable method to prepare nanovesicles derived from shiitake mushroom exosomes. The specific experimental groups are as follows: Experimental Group 1: prepared according to the operation method of Example 1; Experimental Group 2: based on Example 1, the boiling operation was not performed, and the juice was directly squeezed and filtered; Experimental Group 3: based on Example 1, the juice was directly boiled and filtered after not soaking overnight at 4°C; Experimental Group 4: based on Example 1, after the bacterial liquid was boiled, all subsequent experimental operations were performed at 4°C for juicing, filtration and purification.

[0078] The nanovesicles derived from Lentinus edodes exosomes prepared in each experimental group were subjected to the experimental procedures of Example 3 to explore their anti-oxidative stress effects. Figure 5 The results showed that the fluorescence intensity of each group was observed under a fluorescence microscope. The fluorescence of experimental group one was the weakest, and the fluorescence of experimental group two was the strongest, indicating that the nanovesicles derived from shiitake mushroom exosomes prepared by the method of experimental group one had a good anti-ROS effect.

[0079] Combined with the experimental conclusions of Example 2, the method for preparing bacterial liquid by a special hot water method and preparing edible fungal exosome nanovesicles by temperature-controlled filtration provided by the present invention not only maximizes the yield, but also has the best biological function of resisting oxidative stress.

[0080] Example 5: Analysis of lipid composition and content of shiitake mushroom exosome nanovesicles

[0081] Liquid chromatography-mass spectrometry (LC-MS) was used to analyze the lipid composition and content of the shiitake mushroom exosome nanovesicles prepared in Example 1. The samples were separated by a UHPLC Nexera LC-30A ultra-high performance liquid chromatography system using a C18 chromatographic column, with the column temperature set at 45°C and a flow rate of 300 μL / min. During the entire analysis process, the samples were placed in an autosampler at 10°C.

[0082] Figure 6a and Figure 6b The results of lipid composition analysis are shown, and a total of 32 lipid subclasses were identified, covering 765 different lipid molecular types.

[0083] Example 6: Oral Stability and In Vivo Retention Time of Lentinus edodes Exosome Nanovesicles

[0084] First, the shiitake mushroom exosome nanovesicles (MELN) and derived lipid vesicles (MELV) prepared in Example 1 were placed in buffer solutions of different pH (pH = 1.5; pH = 6.5; pH = 8). Incubate at 37 ° C for 2, 4, and 6 hours, and observe the changes in particle size to explore the stability of shiitake mushroom exosome nanovesicles. Then, DiD-labeled MELN and MELV were injected into the esophagus of mice by gavage (at a dose of 3 mg / kg). After gavage, mice were subjected to in vivo fluorescence imaging for five consecutive days to observe the fluorescence distribution and intensity.

[0085] Figure 7 The results showed that MELN and MELV showed good stability in different pH buffers. According to the fluorescence intensity analysis of in vivo imaging, it was found that MELN and MELV could still detect obvious fluorescence signals on the fourth day, indicating that the oral vesicles remained stable in the gastrointestinal tract and could accumulate in the intestine.

[0086] Example 7: Oral administration of shiitake mushroom exosome nanovesicles to prevent alcohol-induced liver damage

[0087] C57BL / 6J male mice were used to construct an acute alcohol-induced liver injury model. Two days before modeling, the mice were gavaged with shiitake mushroom exosome nanovesicles (MELN) and derived lipid vesicles (MELV), once every 12 hours, at a dose of 40 mg / kg (concentration 10 mg / ml), for a total of four times. After the last gavage, the mice fasted for 12 hours. After the fasting, the mice were perfused with 6g / kg of 50% ethanol aqueous solution at one time to construct an acute liver injury model. 12 hours after modeling, ALT, AST, MDA and inflammatory-related cytokines were detected by orbital blood sampling to determine the success of the modeling. The mice were killed 24 hours after modeling, and the mouse livers were taken for detection of relevant gene expression levels and flow cytometry to observe changes in the levels of resident immune cells and recruited circulating immune cells in the liver.

[0088] Figure 8It was shown that in the acute liver injury model, MELN and MELV can reduce the degree of acute liver dysfunction and oxidative stress caused by alcohol. HE staining results showed that mice perfused with MELN and MELV in advance had only mild injuries and a small amount of inflammatory cell infiltration. Both MELN and MELV can inhibit the expression of proinflammatory factors in the liver and upregulate the level of anti-inflammatory factors. In addition, these vesicles can also upregulate the expression of antioxidant and anti-inflammatory genes, thereby reducing liver damage in many aspects. Patients with alcoholic liver injury are often accompanied by immune disorders. Alcohol stimulates the recruitment of circulating immune cells in the liver, and infiltrating neutrophils produce proinflammatory mediators, leading to hepatocyte dysfunction. MELN and MELV can reduce alcohol-induced neutrophil infiltration and reduce lesion inflammation. Alcohol intake affects the function of NK cells, resulting in a decrease in their number. Oral administration of MELN and MELV can restore the NK cell population to a certain extent. In mice orally administered with MELN and MELV, the anti-inflammatory subtype of CD4 T cells increased, enhancing the anti-inflammatory effect. Alcohol can also cause imbalances in liver oxidation levels, further increasing the expression of ROS and promoting inflammation. The expression of related genes indicates that oral vesicles can effectively prevent the generation of ROS and improve the liver's antioxidant capacity.

[0089] Example 8: Oral administration of shiitake mushroom exosome nanovesicles to prevent alcohol-induced intestinal barrier damage

[0090] The intestines were collected from the mice sacrificed in Example 7, paraffin sections were made and tissue cytokine detection was performed, and the qPCR expression levels of related genes were analyzed.

[0091] Fig. 9 It was shown that alcohol damages the intestinal mucosal layer, causing it to become thinner and accompanied by inflammatory cell infiltration. Mice that took shiitake mushroom exosome nanovesicles (MELN) and derived lipid vesicles (MELV) had intact intestinal mucosa and less inflammatory cell infiltration. This shows that oral administration of MELN and MELV can protect the intestinal barrier, which is also proven by the downregulation of proinflammatory cytokines in colon tissue. By detecting the gene expression levels of tight junction proteins and intestinal mucus proteins in colon tissue, it was found that these genes were significantly upregulated after oral administration of MELN, especially Reg3g, which was the most significantly upregulated, and the MELV group was slightly inferior to the MELN group. This preliminarily shows that the vesicles play a liver protective role by repairing the intestinal barrier.

[0092] Example 9: Oral administration of shiitake mushroom exosome nanovesicles to prevent drug-induced liver injury

[0093] C57BL / 6J male mice were selected to establish an acute drug-induced liver injury model. Two days before modeling, mice were gavaged with shiitake mushroom exosome nanovesicles (MELN) and derived lipid vesicles (MELV), once every 12 hours, 40 mg / kg each time (concentration 10 mg / ml), for a total of four times. After the last gavage, the mice fasted for 12 hours. After the fasting, the mice received 300 mg / kg of acetaminophen at one time to construct an acute drug liver injury model. 12 hours after modeling, ALT, AST, MDA and inflammatory-related cytokines were detected by orbital blood sampling to determine the success of the modeling. The mice were killed 24 hours after modeling, and the mouse liver was taken for pathological section analysis.

[0094] Fig.10 It was shown that in the drug liver injury model constructed by acetylphenol, through preliminary in vivo biochemical index analysis, MELN and MELV can play a protective effect against drug-induced liver injury, and similar anti-inflammatory effects were also observed in the intestine.

[0095] In summary, the present invention creatively proposes an extraction method and application of exosome-like nanovesicles from edible fungi. Edible fungus-like exosomes and derived lipid vesicles from sources such as shiitake mushrooms were developed, and it was proved that they have good oral stability and long in vivo half-life. The application was explored in an acute liver injury model, and it was preliminarily confirmed that oral shiitake-derived plant vesicles and their derived lipid vesicles can repair the intestinal barrier and protect the liver.

[0096] The extraction method and application of the exosome-like nanovesicles derived from edible fungi of the present invention have the following characteristics: first, various types of edible fungus liquids are prepared by hot water method, and second, the exosome-like nanovesicles are extracted by differential centrifugation combined with ultrafiltration. The surface potential of the obtained edible fungi-derived exosome-like nanovesicles is negatively charged, and has a certain ability to resist active oxygen. The exosome-like nanovesicles derived from shiitake mushrooms have high oral stability and good anti-inflammatory effect. The present invention fills the gap in the preparation method of exosome-like nanovesicles derived from fungi, broadens its application scenarios, and opens up a new path for natural nanovesicles.

[0097] The above specific embodiments describe the present invention in detail, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. A method for extracting fungal exosome-like nanovesicles, characterized in that: The following steps are involved: (1) tearing the fungus into pieces, adding the pieces to a solution and soaking them, and then heating and boiling the pieces to obtain a fungal liquid; (2) Squeeze the fungal liquid at 60-100° C. in step (1), and filter through gauze to obtain a fungal solution; (3) filtering the fungal solution at 30-80° C. in step (2) through a 70 μm±10 μm filter, centrifuging at 500-1000 g for 5-20 minutes, and collecting the supernatant; (4) centrifuging the supernatant at 20-60° C. in step (3) at 10,000-25,000 g for 15-80 minutes and collecting the supernatant; (5) filtering the supernatant obtained in step (4) first through a filter membrane with a pore size of 0.45 μm ± 0.1 μm, and then through a filter membrane with a pore size of 0.22 μm ± 0.05 μm, and collecting the filtrate; (6) The filtrate obtained in step (5) is subjected to a first ultrafiltration at 0-40° C., and the filtrate is subjected to a second ultrafiltration to obtain a concentrated solution, which is the fungal exosome nanovesicles; the first ultrafiltration uses an ultrafiltration membrane with a cutoff pore size of 90-100 kDa, and the second ultrafiltration uses an ultrafiltration membrane with a cutoff pore size of 10-30 kDa.

2. The extraction method according to claim 1, characterized in that In step (1), the soaking time is 6-24 hours; the soaking temperature is 0-40° C.; the pH range of the solution is 5.8-7.8; and the solution is selected from at least one of water, phosphate buffer, Tris buffer, Tris-HCl buffer, TBS buffer, TBST buffer, TE buffer and Good's buffer.

3. The extraction method according to claim 1, characterized in that In step (1), the fungus is selected from at least one of shiitake mushrooms, straw mushrooms, mushrooms, black fungus, white fungus, hericium erinaceus, bamboo fungus, matsutake mushrooms, white mushrooms, red mushrooms, ganoderma lucidum, thunder balls, big red mushrooms, truffles, white lingzhi mushrooms, boletus, morels, saddle mushrooms, truffles, dry mushrooms, cordyceps sinensis, white ginseng mushrooms, Alpinia ternata, tiger milk mushrooms, purple lilac mushrooms, chanterelles, pine lactarius, Ode mushrooms, hydrangea, blue-headed mushrooms, coral mushrooms, yellow-handled chanterelles, shuttle-handled milk mushrooms, Agaricus bisporus, long root mushrooms, puffballs, Poria cocos, Polyporus umbellatus, hairy-headed coprinus, golden ear, versicolor, tree tongue, pseudo boletus, purple ganoderma, Flammulina velutipes, Pleurotus eryngii, Pleurotus ostreatus, pine mushrooms, hazel mushrooms, bamboo yellow, honey fungus, and mulberry yellow.

4. The extraction method according to claim 1, characterized in that In step (1), the boiling time is 5-80 minutes.

5. A fungal exosome-like nanovesicle, characterized in that: It is prepared by the extraction method according to any one of claims 1 to 4.

6. A lipid nanovesicle, characterized in that: The lipids are extracted from the fungal exosome-like nanovesicles as described in claim 5, and then prepared by a thin film hydration method.

7. Use of the fungal exosome-like nanovesicles according to claim 5 or the lipid nanovesicles according to claim 6 in the preparation of drugs.

8. The use according to claim 7, characterized in that: Fungal exosome-like nanovesicles or lipid nanovesicles as drug carriers or active pharmaceutical ingredients.

9. The use according to claim 8, characterized in that: Fungal exosome nanovesicles and lipid nanovesicles are used as drug carriers, and the drugs also include delivery drugs, which include at least one of proteins, antibodies, peptides, small molecule drugs, traditional Chinese medicine, nucleic acids, exosomes, inorganic nanoparticles, and organic nanoparticles; Fungal exosome nanovesicles and lipid nanovesicles are used as drug carriers, and the drugs are at least one of autoimmune disease drugs, acute injury drugs, wound repair drugs, tumor treatment drugs, and medical beauty and anti-aging drugs.

10. The use according to claim 8, characterized in that: Fungus-derived exosome-like nanovesicles and lipid nanovesicles are used as active ingredients of the drug; the drug is one of the drugs for preventing alcoholic liver damage or treating psoriasis.

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