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

Abstract

The application relates to application of Phellinus baumii exosomes in glioma treatment, an extraction method and a composition. Phellinus baumii is used as raw material, Phellinus baumii exosomes are provided through operations such as soaking, crushing and centrifugation, and it is verified that the Phellinus baumii exosomes have anti-glioma activity in vitro. Based on the research result, the application provides a new application of the plant exosomes from Phellinus baumii for glioma treatment, and provides a cost-effective potential active ingredient for glioma drug development.

Description

Technical Field

[0001] This invention relates to the field of plant exosome technology, and in particular to the use of a *Phellinus linteus* exosome and a composition thereof containing the *Phellinus linteus* exosome in the treatment of glioma. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Phellinus linteus, a traditional Chinese medicine with a long history of medicinal use, has seen its active ingredients extracted and applied, making it a hot research topic. Phellinus linteus exosomes, important bioactive substances within the herb, are small vesicles secreted by cells, typically ranging from 30 to 150 nm in diameter. Studies show that exosomes can carry various specific proteins, RNA, DNA, and other substances, playing a crucial role in regulating intracellular and extracellular environmental balance, signal transduction, and immune responses. They also facilitate intercellular communication and regulation through fluid circulation or intercellular transfer. Therefore, exosomes are a focus of contemporary research on many diseases.

[0004] Numerous studies have demonstrated that *Sanghuang* contains polysaccharides, flavonoids, and other bioactive components, which have shown inhibitory effects on various cancer cells under laboratory conditions. For example, polysaccharides in *Sanghuang* can stimulate the immune system and enhance the activity of the body's natural killer cells, thereby combating the proliferation of tumor cells. Flavonoids have antioxidant and anti-inflammatory effects, helping to reduce cell damage and prevent cancer. In addition to directly inhibiting cancer cells, *Sanghuang* can also enhance the body's anti-cancer ability by regulating the internal environment. *Sanghuang* can clear heat and dry dampness, promote blood circulation and remove blood stasis, thereby improving the internal environment and reducing the risk of cancer. However, no research has yet been found on the anti-cancer effects of *Sanghuang* exosomes. Whether *Sanghuang* exosomes have anti-tumor biological effects and their specific mechanisms of action remain unclear and require further investigation. Summary of the Invention

[0005] This invention uses *Phellinus linteus* exosomes as the research object, providing a method for extracting *Phellinus linteus* exosomes and demonstrating for the first time the in vitro anti-glioma activity of *Phellinus linteus* exosomes. Based on these research results, this invention provides a new application of plant exosomes derived from *Phellinus linteus* for the treatment of gliomas:

[0006] In a first aspect, the use of Phellinus linteus exosomes, or a composition containing Phellinus linteus exosomes, in the preparation of an anti-glioma product is provided.

[0007] The *Sanghuang* mentioned in the first aspect above is a fungus of the Polyporaceae family, *Phellinus tectorius*. Phellinus igniarius(L.ex Fr.) Quel.[ Fomes igniarius (L.) Fr.; Boletus igniarius L.; Polyporus igniarius The fruiting body of Fr.; the exosomes of Phellinus linteus are extracellular vesicles or exosome-like nanoparticles secreted by plant cells with a particle size of 30-150 nm, and the aforementioned exosomes can be extracted using fresh Phellinus linteus as raw material.

[0008] In one embodiment of the present invention, a method for extracting the above-mentioned *Sanghuang* exosomes is provided. The *Sanghuang* is chopped, soaked in a buffer salt solution, then ground at high speed and filtered to remove impurities, yielding a coarse filtrate. Extracellular vesicles or nanoparticles of suitable particle size in the coarse filtrate are screened based on the principle of size separation. In a preferred embodiment, a complex enzyme can be introduced into the coarse filtrate for enzymatic hydrolysis to enhance the release of cellular contents. The specific steps are as follows:

[0009] (1) Grind and extract the shrub fungus soaked in PBS, filter it through gauze to obtain coarse filtrate, centrifuge the coarse filtrate at high speed and retain the supernatant;

[0010] (2) Add the compound enzyme preparation to the supernatant obtained in step (1) to enzymatically hydrolyze to obtain the enzymatic hydrolysate; after freezing and centrifuging the enzymatic hydrolysate, filter it through 100 mesh to obtain the filtrate, filter the filtrate through tangential flow and concentrate it to obtain the final product.

[0011] In step (1):

[0012] Furthermore, the PBS soaking time is 5-7 hours; the coarse filtrate is centrifuged at 800-1200 rpm for 18-22 minutes.

[0013] In step (2):

[0014] Furthermore, the dosage ratio of the supernatant to the compound enzyme preparation is 0.1~0.3%.

[0015] Furthermore, the compound enzyme preparation is composed of enzymes in the following proportions: 20% wt. pectinase, 40% wt. cellulase, 10% wt. β-1,3-glucanase, and 30% wt. ligninase.

[0016] Furthermore, the tangential flow filter has a filter diameter of 30-600 nm.

[0017] The "composition containing Phellinus linteus" mentioned in the first aspect above refers to a composition containing Phellinus linteus exosomes prepared by the above extraction method, and should be an active dose. The purpose of application includes, but is not limited to, preventing, improving, treating, adjuvant treating or improving the prognosis of glioma. The present invention has confirmed the in vitro anti-glioma activity of Phellinus linteus exosomes. Under the premise of clear biological activity, knowing the specific value of the "active dose" is conventional technical content in the field.

[0018] In some embodiments, the above composition includes, in addition to an active dose of Phellinus linteus exosomes, other active ingredients. These "other active ingredients" are components with anti-glioma activity, and their specific types include, but are not limited to, small molecule entities, natural plant / animal extracts, polymeric compounds, or antibodies. Specific examples include temozolomide, carmustine, lomustine, gambogia, sesquiterpene lactones, bevacizumab, nimotuzumab, etc.

[0019] In some other embodiments, the "other active ingredients" are ingredients that assist in anti-glioma activity, and according to the activity classification, they include, but are not limited to, liver-protecting ingredients, immunomodulatory ingredients, side-effect-reducing ingredients, and analgesic ingredients.

[0020] In some other embodiments, the composition further includes a pharmaceutically acceptable carrier. According to the general understanding in the art, "pharmaceutically acceptable" means that the carrier is chemically and / or physically compatible with other components in the composition, or even has a beneficial effect, and is physiologically compatible with the subject of the composition or has the effect of preventing, improving or even treating diseases.

[0021] Furthermore, the aforementioned carriers include, but are not limited to, pharmaceutically acceptable liquid, gel, or solid carriers, aqueous media, non-aqueous media, antimicrobial agents, isotonic agents, buffers, antioxidants, suspending / dispersing agents, chelating agents, emulsifiers, diluents, adjuvants, excipients, auxiliary reagents, or combinations of one or more of the above components.

[0022] Furthermore, the aqueous medium is selected from sodium chloride injection, Ringer's injection, isotonic glucose injection, sterile water injection, or Ringer's glucose or lactate injection.

[0023] Furthermore, the non-aqueous medium is selected from non-volatile oils of plant origin, such as cottonseed oil, corn oil, sesame oil, or peanut oil.

[0024] Furthermore, the isotonic agent is selected from sodium chloride or dextrose.

[0025] Furthermore, the buffer solution is selected from phosphate or citrate buffer solutions.

[0026] Furthermore, the antioxidant is sodium bisulfate.

[0027] Furthermore, the suspending / dispersing agent is selected from sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, or polyvinylpyrrolidone.

[0028] Furthermore, the chelating agent is selected from EDTA and EGTA.

[0029] Furthermore, the emulsifier is polysorbate 80.

[0030] Furthermore, examples of the auxiliary reagents include fillers, binders, disintegrants, buffers, preservatives, lubricants, flavoring agents, thickeners, colorants, or emulsifiers.

[0031] The subjects of the above-mentioned composition include mammals or non-mammals. Mammals include rats, livestock, humans, dogs, rabbits, etc., and non-mammals include fish. The dosage of the composition varies depending on the subject's species, age, sex, weight, the specific disease or pathological state to be treated, the severity of the disease or pathological state, the route of administration, and the judgment of the prescribing person. This is also technical content that can be routinely determined by those skilled in the art.

[0032] In the first aspect mentioned above, the anti-glioma product includes, but is not limited to, drugs, modeling drugs, or kits that can detect, diagnose, prevent, improve, or treat gliomas.

[0033] The dosage forms of the drug include, but are not limited to, powders, granules, tablets, capsules, suspensions, emulsions, syrups, sprays, and other oral dosage forms, topical preparations, suppositories, or sterile injectable solutions, with injections being more preferred. Feasible routes of administration include oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, local, sublingual, or rectal administration. Oral or non-oral administration is preferred. Non-oral routes include subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intrasynovial, intrasternal, intrathecal, intralesional, and intracranial injection or infusion techniques.

[0034] Secondly, the use of *Sanghuang* exosomes, or compositions containing said *Sanghuang* exosomes, in the preparation of food, beverages, health products, or cosmetics.

[0035] Examples of the food products mentioned include biscuits, pastries, powders, and instant granules; examples of the beverages mentioned include compound fruit juices, concentrated liquids, carbonated drinks, or tea drinks; examples of the health products mentioned include tablets, capsules, oral liquids, powders, and granules; and the dosage forms of the cosmetics mentioned include aqueous solutions, emulsions, creams, films, powders, oils, or suspensions. The above examples are not intended to be specific limitations; any product form acceptable in the fields of food, beverages, health products, and cosmetics is within the scope of protection of this invention.

[0036] Compared with the prior art, the beneficial effects of the present invention are:

[0037] Compared with current research, this invention provides a plant-derived, all-natural exosome derived from the traditional Chinese medicine *Sanghuang* (Phellinus linteus), which can effectively inhibit the progression of gliomas and exhibit low cytotoxicity to microglia, along with its applications. *Sanghuang* exosome extraction is inexpensive, yields a large amount, effectively inhibits glioma progression, and exhibits low cytotoxicity to normal cells. *Sanghuang* exosomes can effectively inhibit glioma cell autophagy, thereby inducing glioma death, while showing no significant cytotoxicity to normal microglia at corresponding concentrations.

[0038] The findings of this invention provide new clinical applications for Phellinus linteus and offer novel potential active ingredients for the treatment of gliomas, which is of great significance for the development of anti-glioma drugs. Attached Figure Description

[0039] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0040] Figure 1 This is a schematic diagram of the preparation process of Sanghuang exosomes in this invention;

[0041] Figure 2 The particle size characterization results of the Sanghuang exosomes in Example 1;

[0042] in, Figure 2 Image A shows the NTA results of Sanghuang exosomes in Example 1. Figure 2 Image B is a TEM image of the exosomes of *Sanghuang* from Example 1;

[0043] Figure 3 This is a diagram showing the cell proliferation results after treating glioma (U87) and microglia (HMC3) with Sanghuang exosomes in Example 2.

[0044] in, Figure 3 Image A shows cell morphology images of the control group (NC) and the PELNs group for glioma (U87). Figure 3 In Figure B, the relative cell activity results are shown in the control group (NC-U87) and the exosome group (PELNs-U87) of glioma, and the control group (NC-HMC3) and the exosome group (PELNs-HMC3) of microglia.

[0045] Figure 4 The images show the scratch test results of the control group and the Sanghuang exosome treatment group in Example 2.

[0046] Figure 5This is a Western blotting result of PELNs regulating the AKT-MTOR-related pathway as described in Example 2. Detailed Implementation

[0047] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0048] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0049] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0050] Example 1

[0051] In this embodiment, an exosome of *Phellinus linteus* is provided, and its extraction method is as follows:

[0052] (1) Take fresh Phellinus linteus, wash it clean, and sterilize it by irradiating it with γ-rays at 3-5 kGy for 12-14 hours. Cut it into pieces less than 1 cm in size. 3 Soak the Sanghuang blocks in PBS solution for 6 hours.

[0053] (2) Transfer the soaked Sanghuang blocks and PBS solution from step (1) to a volumetric food processor for shearing, grinding and juicing. The speed is 3000 rpm / min and the grinding time is 5-15 min. The resulting grinding liquid is coarsely filtered through three layers of sterile gauze to obtain coarse filtrate. Transfer it to a 50 ml sterile centrifuge tube for centrifugation to remove impurities. The centrifugation speed is 1000 rpm and the centrifugation time is 20 min. The supernatant is retained.

[0054] (3) Add the compound enzyme preparation to the supernatant of step (2) for enzymatic hydrolysis to obtain the enzymatic hydrolysate; the enzymatic hydrolysis temperature is 40-50℃, and the enzymatic hydrolysis time is 4-8h; the dosage ratio of the supernatant to the compound enzyme preparation is 0.1-0.3%. The above compound enzyme preparation is composed of enzymes in the following proportions: 20%wt. pectinase, 40%wt. cellulase, 10%wt. β-1,3-glucanase, and 30%wt. ligninase.

[0055] (4) Transfer the enzymatic hydrolysate from step (3) to a cryogenic purification device, and perform cryogenic centrifugation and filtration to remove impurities. The filter screen is 100 mesh. Filter the obtained filtrate tangentially (filter diameter 30-600 nm) and concentrate it to obtain plant exosome-like vesicles. Irradiate them for sterilization to obtain the Sanghuang exosomes.

[0056] The particle size and electron microscopy analysis results of the above-mentioned Sanghuang exosomes are as follows:

[0057] (1) Particle size analysis

[0058] The sample cell was rinsed three times with pure water. After rinsing, the instrument was calibrated using a standard (100nm PS beads). Subsequent testing was then performed. The sample cell was rinsed with 1×PBS. The sample was diluted with 1×PBS and added to the sample cell. Real-time dynamic images of the exosome particles were observed on the computer screen. Information was collected, and a corresponding test report was generated. After the test, the sample cell was rinsed with 1×PBS before testing the next sample. A particle size distribution was plotted, as shown in the image. Figure 2 The particle size distribution diagram shown in Figure A indicates that the average diameter of the exosomes from *Sanghuang* is 137 nm.

[0059] Table 1. Particle size distribution of exosomes from *Phellinus linteus*

[0060]

[0061] (2) Transmission electron microscopy (TEM) analysis

[0062] For TEM imaging, 10 μL of *Sanghuang* exosome suspension was added to a pre-prepared Parafilm sealing film (the back of the sealing film adhered to the table). The copper mesh of the film was placed face down, allowing it to naturally absorb the suspension droplets for 10-15 minutes. Excess droplets were then removed with filter paper, and the film was allowed to dry slightly. 10 μL of 2% phosphotungstic acid solution was pipetted onto the sealing film. The copper mesh, now with the stained surface facing up, was inverted and allowed to stand for 3-5 minutes. Excess droplets were carefully removed with filter paper, and the film was allowed to dry under an incandescent lamp. After drying for 30 minutes, the sample was sent for analysis, and TEM images were taken. The resulting transmission electron microscope micrographs are shown below. Figure 2 As shown in B.

[0063] Example 2

[0064] This embodiment verifies the anti-glioma activity of the *Sanghuang* exosomes described in Example 1.

[0065] 1. Detection of anti-glioma cell proliferation by exosomes of Phellinus linteus

[0066] U87 glioma cell line and HMC3 microglia in logarithmic growth phase were seeded at 6000 cells / well in 96-well cell culture plates, and allowed to adhere overnight for 12 hours. A blank control group (NC) and a PELNs test group (2.2E+11 microparticles / mL) were set up. Nine replicates were performed, with three wells as parallel controls. Cells were cultured at 37°C, and cell viability was assessed at 0, 24, and 48 hours. The assay method was CCK8 (10 μL / well), incubated at 37°C for 4 hours, then incubated in the dark for 30 minutes. Cell viability was calculated by measuring the OD450 absorbance using a microplate reader. Figure 3 As shown.

[0067] 2. Detection of the anti-glioma cell repair effect of Phellinus linteus exosomes

[0068] U87 breast cancer cell line in logarithmic growth phase was used, and two groups were established: a blank control group and a group inoculated with *Sanghuang* exosomes. Cell suspension was seeded in 6-well plates. The blank control group was treated with complete culture medium, while the group inoculated with *Sanghuang* exosome solution at a concentration of 2.2E+11 particles / mL. Two replicates were performed, with two wells forming a parallel control group. Cells were incubated at 37°C for 24 hours. The 6-well plates were then placed in a live-cell imaging system. The blank control group was replaced with basal culture medium and scratch-marked. The group inoculated with *Sanghuang* exosome solution (2.2E+11 particles / mL) was again added. Live-cell imaging data were collected after 48 hours to analyze the glioma cell repair capacity.

[0069] The results are as follows Figure 4 As shown, in the control group, after 48 hours, the scratch boundaries of tumor cells became less clear, and tumor cells began to migrate towards the scratched area. In contrast, glioma cells in the exosome group showed significant aggregation and shrinkage, and the number of cells migrating to the scratched area was significantly less than in the control group. These results indicate that *Sanghuang* exosomes effectively inhibited the repair function of glioma cells.

[0070] 3. Detection of protein expression related to the inhibition of AKT / mTOR signaling pathway and apoptosis in gliomas by Sanghuang exosomes

[0071] U87 glioma cell line in logarithmic growth phase was seeded at 200,000 cells / well in 2 ml of cell suspension in a 6-well cell culture plate and allowed to adhere overnight for 12 h. A blank control group and a PELNs test group (2.2E+11 cells / mL) were set up, and cells were cultured for another 24 h. Cells were lysed with protein lysis buffer at 12,000 rpm for 15 min, and the supernatant was collected. Protein concentration was determined using a BCA assay kit: 30 μg of total protein was loaded per well, and an appropriate amount of 6× protein loading buffer was added. The mixture was then incubated in a boiling water bath for 10 min. A PAGE gel rapid gel preparation kit was used to prepare the separating and stacking gels: an appropriate volume of protein sample was added, and 6 μL of protein marker was added per well. The voltage was initially adjusted to a constant 90 V. When the bromophenol blue indicator of the electrophoresis sample crossed the separating gel, the voltage was changed to a constant 110 V until the bromophenol blue indicator reached the bottom of the separating gel. PVDF membranes of appropriate size were cut according to the number of samples and the size of the target protein and immersed in methanol for approximately 5 seconds. Immerse all materials in the transfer apparatus in the transfer solution: sponge pad-filter paper-PAGE gel-PVDF membrane-filter paper-sponge pad. After removing air bubbles, transfer the membrane at a constant current of 400mA for 45 minutes. After the transfer is complete, remove the PVDF membrane and incubate it in a shaker with 53% skim milk powder at room temperature for 1 hour.

[0072] Prepare a primary antibody solution with 5% skim milk powder and add it to the antibody incubation cassette. With the membrane facing up, incubate overnight at 4°C. After primary antibody incubation, wash the membrane three times (5 min each) with TBST. Incubate the membrane with secondary antibody at 37°C for 30 min, then wash three times (5 min each) with TBST. Prepare ECL chemiluminescence buffer and add it to the front side of the PVDF membrane. Develop and fix the membrane. Figure 5 As shown.

[0073] according to Figure 5 The results showed that in glioma cells cultured with Phellinus linteus exosomes, the expression levels of mTOR and phosphorylated p-mTOR and p-AKT were significantly reduced, effectively inhibiting the PI3K / AKT / mTOR signaling pathway in gliomas, indicating that Phellinus linteus exosomes have a therapeutic effect on gliomas.

[0074] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of *Phellinus linteus* exosomes or compositions containing *Phellinus linteus* exosomes in the preparation of anti-glioma products, characterized in that, The anti-glioma product is a drug for treating gliomas; the extraction method of the *Phellinus linteus* exosomes includes the following steps: (1) Grind and extract the mulberry fungus soaked in PBS, filter it through gauze to obtain coarse filtrate, centrifuge the coarse filtrate at high speed and retain the supernatant; the soaking time in PBS is 5-7 h; the centrifugation speed of the coarse filtrate is 800-1200 rpm and the centrifugation time is 18-22 min; (2) Add the compound enzyme preparation to the supernatant obtained in step (1) to enzymatically hydrolyze and obtain the enzymatic hydrolysate; freeze and centrifuge the enzymatic hydrolysate and filter it through 100 mesh to obtain the filtrate; filter the filtrate through tangential flow and concentrate it to obtain the supernatant; the dosage ratio of the supernatant to the compound enzyme preparation is 0.1~0.3%; the compound enzyme preparation is composed of the following enzymes in the following proportions: 20wt.% pectinase, 40wt.% cellulase, 10wt.% β-1,3-glucanase and 30wt.% ligninase.

2. The application as described in claim 1, characterized in that, In the composition, the exosomes of Phellinus linteus are in the active dose; In addition to an active dose of Phellinus linteus exosomes, the composition also includes other active ingredients, which are components with anti-glioma activity and are selected from temozolomide, carmustine, lomustine, gambogia, sesquiterpene lactones, bevacizumab or nimotuzumab.

3. The application as described in claim 1, characterized in that, The composition further includes pharmaceutically acceptable aqueous and non-aqueous mediators; the aqueous mediator is selected from sodium chloride injection, Ringer's injection, isotonic glucose injection, sterile water injection, Ringer's glucose or lactate injection; the non-aqueous mediator is selected from cottonseed oil, corn oil, sesame oil or peanut oil.

4. The application as described in claim 1, characterized in that, The composition also includes pharmaceutically acceptable isotonic agents, buffers, antioxidants, suspending / dispersing agents, chelating agents, or emulsifiers; The isotonic agent is selected from sodium chloride or dextrose; The buffer solution is selected from phosphate or citrate buffer; The antioxidant is sodium bisulfate; The suspending / dispersing agent is selected from sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose, or polyvinylpyrrolidone; The chelating agent is selected from EDTA and EGTA; The emulsifier is polysorbate 80.

5. The application as described in claim 1, characterized in that, The dosage form of the drug is selected from powder, granules, tablets, capsules, suspension, emulsion, syrup, spray, suppository or sterile injection solution; The administration site can be selected from the oral cavity, intravenous, intramuscular, intra-arterial, intramedullary, intrasheath, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, or intestinal tract.

6. The application as described in claim 5, characterized in that, The drug is in the form of a sterile injectable solution.

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