Preparation method and application of ganoderma lucidum outer vesicles without sporocarp waste source

Abstract

The invention discloses a preparation method and application of ganoderma lucidum outer vesicles without sporocarp waste sources. The ganoderma lucidum external vesicles are used for preparing medicines for treating ischemic cerebral apoplexy sequelae, and can improve core symptoms such as dyskinesia (including limb paralysis, imbalance and gait abnormity), cognitive decline and the like after apoplexy. The spore-free sporocarp waste sourced ganoderma lucidum outer vesicles are prepared from spore-free sporocarp waste which is completely released by spores after a ganoderma lucidum powder spraying period through raw material pretreatment, differential centrifugation, tangential flow concentration and filter membrane filtration. According to the preparation method, the waste is used as a raw material to prepare the high-value medical outer vesicle, green circulation of agricultural waste-high-added-value medical raw materials is achieved, the ganoderma lucidum outer vesicle can effectively penetrate through the blood-brain barrier, brain-targeted delivery is achieved, and the problems that traditional ganoderma lucidum active ingredients are low in blood-brain barrier penetration rate and poor in blood-brain barrier penetration rate are solved. The effective treatment concentration in the brain tissue is difficult to achieve, and the traditional drug delivery bottleneck is broken through.

Description

Technical Field

[0001] This invention belongs to the field of traditional Chinese medicine and relates to a method for preparing Ganoderma lucidum exovesicles derived from non-spore fruiting body waste and their application. Background Technology

[0002] Ganoderma extracellular vesicles (GLEVs) are a class of extracellular vesicles (EVs) with a particle size between 30-150 nm, extracted and isolated from the fruiting body of Ganoderma lucidum. GLEVs carry Ganoderma-specific active ingredients such as polysaccharides and triterpenes, and have the characteristics of good biocompatibility and strong targeted delivery ability of active substances, making them an important carrier for the development of natural active preparations. Currently, the relevant technologies for Ganoderma lucidum vesicles mainly focus on the optimization of preparation methods and applications in specific fields. For example, patent CN120361065A discloses the application of Ganoderma lucidum vesicles in anti-breast cancer products; patent CN202510420349.9 discloses "a method for preparing Ganoderma lucidum vesicles containing medicinal and edible active ingredients", which improves the yield of vesicles through extraction process improvement. In terms of applications, it has been expanded to corneal damage repair (CN202511353631.6), skin care (CN202511223696.9), liver protection (CN202510798017.4) and other fields, but these applications do not involve the treatment of stroke sequelae such as behavioral disorders.

[0003] There are two core problems in the current preparation of Ganoderma lucidum exovesicles: First, the raw materials do not utilize industrial waste. Current technologies mostly use fresh Ganoderma lucidum mycelium or fruiting bodies that have not released spores as raw materials, while the "non-spore fruiting body waste" generated after Ganoderma lucidum powdering is usually discarded, failing to achieve high-value utilization. Second, the current extraction process of Ganoderma lucidum exovesicles mostly adopts differential centrifugation, which has low extraction efficiency, long cycle, and poor product purity, making it impossible to achieve large-scale preparation. Third, the application scenarios are limited. Current technologies have not explored the potential of Ganoderma lucidum exovesicles in the fields of neuroprotection and stroke sequelae intervention. Existing treatments for stroke sequelae (such as unilateral limb dysfunction) (such as Ginkgo biloba extract injection) have shortcomings such as weak blood-brain barrier penetration and significant side effects, and there is a lack of highly effective intervention agents from natural sources. Summary of the Invention

[0004] The purpose of this invention is to address the aforementioned shortcomings of the prior art by providing an application of Ganoderma lucidum exovesicles derived from non-spore-forming fruiting body waste.

[0005] Another objective of this invention is to provide a method for preparing Ganoderma lucidum exovesicles derived from sporeless fruiting body waste.

[0006] The objective of this invention can be achieved through the following technical solutions:

[0007] The application of Ganoderma lucidum exovesicles derived from non-spore fruiting body waste in the treatment of sequelae of ischemic stroke, wherein the non-spore fruiting body waste Ganoderma lucidum exovesicles are prepared from non-spore fruiting body waste after the complete release of spores during the Ganoderma lucidum powdering period through raw material pretreatment, differential centrifugation, tangential flow concentration, and membrane filtration.

[0008] Preferably, the sequelae of ischemic stroke include core symptoms such as post-stroke motor dysfunction and cognitive decline.

[0009] The Ganoderma lucidum vesicles described in this invention are used to prepare drugs for the treatment of sequelae of ischemic stroke, which can improve the core symptoms of post-stroke motor dysfunction (including limb paralysis, balance disorder, gait abnormality) and cognitive decline; at the same time, they can be extended to prepare adjuvant drugs for the treatment of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease, and alleviate the accompanying behavioral disorders and neurological deficits.

[0010] The Ganoderma lucidum vesicles described in this invention treat sequelae of stroke through a multi-pathway synergistic effect of anti-oxidative stress, anti-inflammation, and repair of damaged nerve cells.

[0011] The Ganoderma lucidum vesicles described in this invention are administered via tail vein injection at a dose of 1×10⁻⁶. 7 ~1×10 10 Particles / time, once daily, for 7-14 consecutive days.

[0012] Preferably, the Ganoderma lucidum exovesicles derived from the waste of aspore-free fruiting bodies are prepared by the following method: Aspore-free fruiting body waste of Ganoderma lucidum is taken, washed with sterile water, cut into pieces, mechanically crushed, and impurities removed. PBS is added at a material-to-liquid ratio of 1-4 L / kg, and the mixture is soaked at 4℃ and 60-120 rpm for 8-12 hours to fully dissolve the exovesicles. The mixture is then filtered through three layers of gauze to remove coarse residue. The filtrate is centrifuged at 2000-4000g at 4℃ for 15-25 min, the precipitate is discarded, and the supernatant is passed through a 60-mesh sieve. The supernatant is then centrifuged again at 10000-12000g at 4℃ for 30-60 min, and the supernatant is collected to further remove macromolecular impurities and cell debris. The supernatant is collected and sequentially passed through a hollow fiber column with a pore size of 0.22-0.45 μm and a molecular weight cutoff of 30-100 kDa. The tangential flow concentration and filtration was performed using a hollow fiber column: First, the supernatant was passed through a hollow fiber column with a filter diameter of 0.22~0.45μm, and the permeate was collected; then, the permeate was concentrated by passing it through a hollow fiber column with a molecular weight cutoff of 30~100kDa, and the concentrate was collected; finally, the concentrate obtained after tangential flow concentration and filtration was further concentrated by centrifugation at 3500g for 15min using a 0.45μm, 30kDa ultrafiltration tube, and the concentrate was collected. The concentrate was then filtered through a 0.22μm filter membrane for sterilization, thus obtaining the Ganoderma lucidum exovesicles.

[0013] A method for preparing Ganoderma lucidum exovesicles from non-spore-forming fruiting body waste includes the following steps: Ganoderma lucidum non-spore-forming fruiting body waste is taken, washed with sterile water, cut into pieces, mechanically crushed, and impurities removed. PBS is added at a material-to-liquid ratio of 1-4 L / kg, and the mixture is soaked at 4℃ and 60-120 rpm for 8-12 hours to fully dissolve the exovesicles. The mixture is then filtered through three layers of gauze to remove coarse residue. The filtrate is centrifuged at 2000-4000g at 4℃ for 15-25 min, the precipitate is discarded, and the supernatant is passed through a 60-mesh sieve. The supernatant is then centrifuged again at 10000-12000g at 4℃ for 30-60 min, and the supernatant is collected to further remove macromolecular impurities and cell debris. The supernatant is collected and sequentially passed through a hollow fiber column with a pore size of 0.22-0.45 μm and a molecular weight cutoff of 30-100 kDa. The hollow fiber column was used for tangential flow concentration and filtration. Finally, the concentrate obtained after tangential flow concentration and filtration was further concentrated by centrifugation at 3500g for 15min using a 0.45μm, 30KDa ultrafiltration tube. The concentrate was collected and filtered through a 0.22μm filter membrane for sterilization to obtain the Ganoderma lucidum exovesicles.

[0014] Preferably, the hollow fiber column tangential flow concentration filtration is performed by first passing the supernatant through a hollow fiber column with a filter diameter of 0.22~0.45μm and collecting the permeate; then, the permeate is concentrated by passing it through a hollow fiber column with a molecular weight cutoff of 30~100kDa and collecting the concentrate.

[0015] Further preferably, the preparation method includes the following steps: Take the sporeless Ganoderma lucidum fruiting body waste after the spore release period, wash it, cut it into pieces, mechanically crush it, add PBS at a ratio of 1:4 (w / v), soak it at 4℃ and 120rpm for 12 hours to fully dissolve the exovesicles, filter it through three layers of gauze to remove coarse residue, centrifuge the filtrate at 4000g for 20 min, discard the precipitate, filter the supernatant through a 60-mesh sieve to obtain a clear filtrate, and then centrifuge it at 10000g and 4℃... Centrifuge for 40 min to further remove macromolecular impurities and cell debris, and collect the supernatant. The collected supernatant is then subjected to tangential flow concentration filtration through a hollow fiber column with a pore size of 0.45 μm and a hollow fiber column with a molecular weight cutoff of 30 kDa. First, the supernatant is passed through a hollow fiber column with a pore size of 0.45 μm, and the permeate is collected. Then, the permeate is concentrated by passing it through a hollow fiber column with a molecular weight cutoff of 30 kDa, and the concentrate is collected. The concentrate obtained after tangential flow concentration filtration is centrifuged at 3500 g for 15 min using a 0.45 μm, 30 kDa ultrafiltration tube, and the concentrate is collected. The concentrate is then centrifuged at 110,000 g at 4 °C for 70 min, and the precipitate is collected. The precipitate is resuspended in PBS and filtered through a 0.22 μm filter membrane to obtain the Ganoderma lucidum exovesicles.

[0016] Ganoderma lucidum exovesicles derived from sporeless fruiting body waste were prepared according to the preparation method described above.

[0017] The aforementioned non-spore-forming fruiting body waste is derived from Ganoderma lucidum exovesicles, with a particle size distribution of 30~150nm and a concentration of not less than 1×10⁻⁶. 10 The membrane contains particles per mL and exhibits a typical cup-shaped vesicle structure with good membrane stability, meeting pharmaceutical requirements.

[0018] A pharmaceutical composition for treating sequelae of ischemic stroke, comprising Ganoderma lucidum exovesicles derived from the waste of the aspore-free fruiting body.

[0019] Beneficial effects:

[0020] This invention uses the waste of non-spore-forming fruiting bodies after the powdering period of Ganoderma lucidum as raw material, and prepares Ganoderma lucidum exovesicles (GLEVs) through an optimized tangential flow concentration and filtration process. These GLEVs are then applied to the treatment of sequelae of ischemic stroke. Compared with traditional Ganoderma lucidum preparations, animal-derived exovesicles, and existing clinical drugs, this invention possesses significant technical advantages and application value. On the one hand, it achieves resource recycling and significantly reduces production costs. Non-spore-forming fruiting bodies after the powdering period of Ganoderma lucidum are discarded in large quantities due to their low content of active ingredients, resulting in resource waste and environmental pollution. This invention uses this waste as raw material to prepare high-value medicinal exovesicles, achieving a green cycle of "agricultural waste - high-value-added pharmaceutical raw materials." A single batch processing of 1 kg of waste can produce exoves with a concentration of not less than 1×10¹. 0 GLEVs with particles / mL significantly reduce raw material costs compared to using first-harvest Ganoderma lucidum fruiting bodies, while also saving Ganoderma lucidum cultivation enterprises on waste disposal costs. Furthermore, this invention employs a combined process of "gradient centrifugation-tangential flow concentration-membrane filtration," which, compared to differential centrifugation and differential centrifugation-polymer precipitation methods, increases GLEVs yield by over 50%, achieving a concentration of up to 1×10⁻⁶. 10 The particle size is above 10 ... More importantly, the GLEVs of this invention have been experimentally proven to have antioxidant and neuroprotective effects, capable of repairing nerve cells damaged after stroke and promoting the recovery of patients' behavior and cognition. Furthermore, the GLEVs of this invention have a wide therapeutic window; administration 24 hours after reperfusion in MCAO / R model mice still significantly improves neurological deficits and balance, and the therapeutic effect can last up to 6 weeks after administration, significantly extending the therapeutic window and benefiting more patients with sequelae who missed acute thrombolytic therapy. In terms of safety, the GLEVs of this invention are isolated from the traditional Chinese medicine Ganoderma lucidum, possessing advantages such as high safety from natural sources and low immunogenicity. In summary, the GLEVs of this invention can not only be used for the treatment of sequelae of ischemic stroke, but also, based on their neuroprotective properties, can be extended to the adjunctive treatment of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Simultaneously, based on their antioxidant activity, they can be developed into high-end functional food or cosmetic raw materials, forming a diversified application pattern of "medicine + big health," with broad market prospects. Attached Figure Description

[0021] Figure 1Flowchart of the process exploration for separating Ganoderma lucidum exovesicles

[0022] Figure 2 Transmission electron micrograph of Ganoderma lucidum exovesicles

[0023] Figure 3 Average concentration of Ganoderma lucidum exovesicles

[0024] Figure 4 Purity of Ganoderma lucidum exovesicles

[0025] Figure 5 The yield of Ganoderma lucidum exovesicles

[0026] Figure 6 Antioxidant capacity of four types of Ganoderma lucidum exovesicles

[0027] A: Quantitative results of BCA protein in four types of Ganoderma lucidum exovesicles; B: DPPH free radical scavenging capacity of four types of Ganoderma lucidum exovesicles; C: Hydroxyl free radical scavenging capacity of four types of Ganoderma lucidum exovesicles.

[0028] Figure 7 The optimal concentration of Ganoderma lucidum exovesicles (TFF-GLEVs) for scavenging DPPH and hydroxyl radicals

[0029] A: Vitamin C (Vc) positive control standard curve; B: Scavenging rates of DPPH and hydroxyl radicals by different concentrations of TFF-GLEVs.

[0030] Figure 8 In vivo distribution diagram of Ganoderma lucidum exovesicles

[0031] A: In vivo fluorescence intensity at 4, 8, 12, and 24 hours post-injection; B: Quantitative fluorescence intensity graph.

[0032] Figure 9 Ganoderma lucidum exovesicles significantly improved pathological changes in a middle cerebral artery occlusion / reperfusion (MCAO / R) model.

[0033] Figure 10 Ganoderma lucidum exovesicles significantly improved oxidative stress levels in a middle cerebral artery occlusion / reperfusion (MCAO / R) model.

[0034] A: Malondialdehyde (MDA) content determination results; B: Superoxide dismutase (SOD) activity determination results; C: Glutathione peroxidase (GSH-Px) activity determination results.

[0035] Figure 11 Ganoderma lucidum exovesicles significantly improved neurological function in mice with a middle cerebral artery occlusion / reperfusion (MCAO / R) model.

[0036] Figure 12 Ganoderma lucidum exovesicles significantly improved balance ability in mice with a middle cerebral artery occlusion / reperfusion (MCAO / R) model.

[0037] Figure 13 Ganoderma lucidum vesicles significantly improved forelimb muscle strength in mice with a middle cerebral artery occlusion / reperfusion (MCAO / R) model.

[0038] Figure 14 Ganoderma lucidum exovesicles significantly improved motor speed in a middle cerebral artery occlusion / reperfusion (MCAO / R) mouse model.

[0039] Figure 15 Ganoderma lucidum exovesicles significantly improved the gait regularity index in mice with a middle cerebral artery occlusion / reduction (MCAO / R) model.

[0040] Figure 16 Ganoderma lucidum exovesicles significantly reduced bilateral limb symmetry in a middle cerebral artery occlusion / reperfusion (MCAO / R) mouse model.

[0041] Figure 17 Effects of the first treatment time point of Ganoderma lucidum exovesicles on neurological function in a middle cerebral artery occlusion / reperfusion (MCAO / R) mouse model

[0042] Figure 18 Effect of the first treatment time point of Ganoderma lucidum exovesicles on balance ability in mice with middle cerebral artery occlusion / reperfusion (MCAO / R) model

[0043] Figure 19 Effect of the time point of first treatment with Ganoderma lucidum exovesicles on body weight in mice with middle cerebral artery occlusion / reperfusion (MCAO / R) model Detailed Implementation

[0044] Example 1: Exploration of the extraction process of Ganoderma lucidum exovesicles (GLEVs)

[0045] Take 1 kg of sporeless Ganoderma lucidum fruiting body waste after the spore release period, wash, cut into pieces, and mechanically crush. Add phosphate-buffered saline (PBS) at a ratio of 1:4 (w / v) and soak at 4℃ and 120 rpm for 12 h to fully dissolve the exovesicles. Filter through three layers of gauze to remove coarse residue. Centrifuge the filtrate at 4000g for 20 min, discard the precipitate, and filter the supernatant through a 60-mesh sieve to obtain a clear filtrate. Centrifuge again at 10000g and 4℃ for 40 min to further remove macromolecular impurities and cell debris, and collect the supernatant.

[0046] Then, four methods were used for parallel separation. Figure 1 ):

[0047] A. Differential centrifugation method (UC-GLEVs): The collected supernatant was centrifuged again at 4°C and 10,000g for 40 min, and the supernatant was collected. The supernatant was then centrifuged at 3500g for 15 min using a 0.45μm, 30kDa ultrafiltration tube, and the concentrate was collected. The concentrate was then ultracentrifuged at 110,000g for 70 min, and the precipitate was collected. The precipitate was resuspended with PBS and filtered through a 0.22μm filter membrane to obtain UC-GLEVs.

[0048] B. Differential centrifugation-polymer precipitation method (UC-PEG GLEVs): The collected supernatant was centrifuged again at 10,000g for 40 min, and the supernatant was collected. The supernatant was then centrifuged at 3500g for 15 min using a 0.45μm, 30kDa ultrafiltration tube. The concentrate was collected, and an equal volume of polyethylene glycol (PEG) precipitant was added. The mixture was allowed to stand overnight at 4°C. The next day, the mixture was centrifuged at 10,000g at 4°C for 40 min, and the supernatant was discarded. The precipitate was replenished with PBS and then ultracentrifuged at 110,000g at 4°C for 70 min. The precipitate was resuspended in PBS and filtered through a 0.22μm filter membrane to obtain UC-PEG GLEVs.

[0049] C. Tangential Flow Concentration-Ultrafiltration Centrifugation (TFF-UC GLEVs): The collected supernatant was sequentially passed through a hollow fiber column with a pore size of 0.45 μm and a hollow fiber column with a molecular weight cutoff of 30 kDa for tangential flow concentration and filtration. First, the supernatant was passed through a hollow fiber column with a pore size of 0.45 μm, and the permeate was collected. Then, the permeate was passed through a hollow fiber column with a molecular weight cutoff of 30 kDa for concentration, and the concentrate was collected. The concentrate obtained after tangential flow concentration and filtration was centrifuged at 3500 g for 15 min using a 0.45 μm, 30 kDa ultrafiltration tube to further concentrate to 10 mL, and the concentrate was collected. The concentrate was centrifuged at 110,000 g at 4 °C for 70 min, the precipitate was collected, the precipitate was resuspended with PBS, and filtered through a 0.22 μm filter membrane to obtain TFF-UC GLEVs.

[0050] D. Tangential Flow Concentration Filtration Method (TFF-GLEVs): The collected supernatant was sequentially passed through a hollow fiber column with a pore size of 0.45 μm and a hollow fiber column with a molecular weight cutoff of 30 kDa for tangential flow concentration filtration. First, the supernatant was passed through a hollow fiber column with a pore size of 0.45 μm, and the permeate was collected. Then, the permeate was passed through a hollow fiber column with a molecular weight cutoff of 30 kDa for concentration, and the concentrate was collected. Finally, the concentrate obtained after tangential flow concentration filtration was centrifuged at 3500g for 15 min using a 0.45 μm, 30 kDa ultrafiltration tube to further concentrate to 10 mL, and the concentrate was collected. The concentrate was then filtered through a 0.22 μm filter membrane for sterilization to obtain high-purity Ganoderma lucidum exovesicles (TFF-GLEVs).

[0051] Transmission electron microscopy showed that the particles obtained by the four methods all exhibited typical cup-shaped vesicle structures with intact lipid bilayer membranes. Figure 2 Nanoflow cytometry analysis showed that the particle size of all GLEVs was mainly distributed in the range of 30-150 nm, consistent with the morphological characterization of exovesicles. The nanoflow cytometry results showed that the particle concentration of TFF-GLEVs was significantly higher than that of the other three methods. Figure 3 Using the particle number / total protein ratio as a purity indicator, there was no significant difference in the purity of the products obtained by the four methods. Figure 4 Calculations of the yield per unit weight of Ganoderma lucidum raw material show that the TFF-GLEVs method has the highest extraction efficiency. Figure 5 ).

[0052] The above results indicate that the tangential flow concentration filtration (TFF-GLEVs) method can significantly improve the extraction yield while maintaining the typical morphology and purity of GLEVs, and is therefore determined to be the optimal extraction process.

[0053] Example 2: Scavenging ability of Ganoderma lucidum exovesicles against DPPH and hydroxyl radicals

[0054] Prepare a 20 μg / mL DPPH methanol working solution. Quantify the protein content of the four types of Ganoderma lucidum exovesicles isolated. Figure 6 -A), diluted with PBS to a concentration of 1 mg / mL, mixed thoroughly, and dispersed by filtration through a 0.22 μm filter membrane. The reaction system was set up as follows: 50 μL sample + 150 μL DPPH working solution. The reaction was carried out at room temperature in the dark for 30 min, and the absorbance was measured at 515 nm. A standard curve was prepared using vitamin C (Vc) as a positive control.

[0055] A hydroxyl radical detection kit was used, and the procedure was strictly followed according to the instructions. The hydroxyl radical scavenging ability of GLEVs extracted by four methods was compared.

[0056] The results showed that all four types of Ganoderma lucidum exovesicles exhibited varying degrees of DPPH and hydroxyl radical scavenging abilities. Among them, at the same protein concentration, TFF-GLEVs demonstrated the strongest scavenging ability for both DPPH and hydroxyl radicals, with a hydroxyl radical scavenging capacity of 92.3% ± 2.7%, significantly superior to other methods (P<0.01). Figure 6 -B, Figure 6 -C).

[0057] Vitamin C (Vc) showed a linear correlation with DPPH free radical scavenging capacity in the concentration range of 10 μg / mL to 30 μg / mL. Figure 7-A); further investigation was conducted to determine the optimal concentration of TFF-GLEVs for scavenging DPPH and hydroxyl radicals. The results showed that the ability of TFF-GLEVs to scavenge DPPH and hydroxyl radicals increased in a concentration-dependent manner. Figure 7 -B). TFF-GLEVs achieved a clearance rate of 55% (equivalent to 20 μg / mL Vc) at a concentration of 3.18 × 10⁹ particles / mL; and a clearance rate of 97.1% (equivalent to 62.5 μg / mL Vc) at a concentration of 4.00 × 10¹⁰ particles / mL.

[0058] The above experiments show that GLEVs extracted by the TFF-GLEVs method have the most significant dose-dependent free radical scavenging ability, and their antioxidant activity is comparable to that of high concentrations of vitamin C.

[0059] Example 3: In vivo brain targeting evaluation of Ganoderma lucidum exovesicles

[0060] TFF-GLEVs (DiR-GLEVs) were labeled with DiR fluorescent dye. Free DiR dye (Free-DiR) and DiR-labeled synthetic liposomes (DiR-LPs) served as controls. A middle cerebral artery occlusion / reperfusion (MCAO / R) model was established using C57BL / 6J male mice, and mice were randomly assigned to groups 24 h after modeling.

[0061] Mice in each group were injected with an equal dose (in DiR) of the marker via the tail vein. Whole-body fluorescence imaging was performed using a small animal in vivo imaging system at 4, 8, 12, and 24 h post-injection, with a focus on observing fluorescence signals in the brain. The fluorescence intensity of brain regions was quantitatively analyzed using software.

[0062] The results showed that the DiR-GLEVs group exhibited a weak signal in the brain 4 hours after injection, which then continued to increase, reaching a peak at 24 hours, with significant fluorescence enrichment in the brain. At the same time point, the fluorescence intensity in the brain of the DiR-GLEVs group was significantly higher than that of the DiR-LPs group and the Free-DiR group (P < 0.01), while the brain signals of the latter two remained weak throughout. Figure 8 The results of this experiment show that GLEVs have good blood-brain barrier penetration ability and brain targeting specificity, and their targeting efficiency is better than that of conventional synthetic liposomes.

[0063] Example 4: Pathological protective effect of Ganoderma lucidum exovesicles against cerebral ischemia-reperfusion injury

[0064] MCAO / R model mice were randomly divided into: model group (MCAO, physiological saline), positive control group (YKT, Ginkgo biloba extract injection 10 mg / kg), and low-dose GLEVs group (GLEVs-L, 1×10⁻⁶). 7particles / animal), high-dose GLEVs group (GLEVs-H, 1×10 10 (particles / animal), sham-operated group. All groups received the drug via tail vein once daily for 7 consecutive days. Brains were harvested after the last administration for TTC and HE staining.

[0065] HE staining results showed that the cell structure in the Sham group was normal; the MCAO group showed disordered cell arrangement, increased interstitial spaces, and severe nuclear pyknosis; the pathological damage in the GLEVs treatment group was reduced, and the cell morphology and arrangement in the GLEVs-H group were close to normal. Figure 9 -A).

[0066] TTC staining results showed that the brain tissue in the Sham group was uniformly red with no infarct foci; the MCAO group showed obvious pale infarct areas and significantly increased infarct volume; compared with the MCAO group, the infarct area in the GLEVs treatment group (especially the high-dose group) was significantly smaller, and the percentage of cerebral infarct volume was significantly reduced (P < 0.01). Figure 9 -B).

[0067] Example 5: Regulatory effect of Ganoderma lucidum exovesicles on oxidative stress in brain tissue

[0068] Same grouping and administration as in Example 4. After the last administration, the brain was harvested, and the infarcted hemisphere was separated to prepare a homogenate supernatant. The following were measured strictly according to the instructions of the commercially available kit: superoxide dismutase (SOD) activity, glutathione peroxidase (GSH-Px) activity, and malondialdehyde (MDA) content. Protein concentration was determined using the BCA method for data standardization.

[0069] The results showed that, compared with the Sham group, the MCAO group had significantly lower SOD and GSH-Px activities and significantly higher MDA content (both P < 0.01). GLEVs treatment could reverse this trend, with the GLEVs-H group showing the most significant effect. Figure 10 Experimental results show that GLEVs effectively alleviate oxidative stress damage after cerebral ischemia-reperfusion by enhancing the activity of endogenous antioxidant enzymes and inhibiting lipid peroxidation.

[0070] Example 6: The promoting effect of Ganoderma lucidum exovesicles on the recovery of nerve function

[0071] The modified neurological deficit score (mNSS) was used to score mice in a blinded manner at 24 h after modeling (before drug administration) and on day 7 (after drug administration). The score covered motor, sensory, and balance reflexes (details are shown in Table 1).

[0072] Table 1

[0073]

[0074] Seven days after administration, the MCAO score in the MCAO group decreased slightly but remained high. Compared with the MCAO group, the mNSS scores in the GLEVs-L, GLEVs-H, and YKT groups were significantly lower (P<0.01). Furthermore, the improvement in the GLEVs-H group was superior to that in the GLEVs-L group, showing a dose-dependent effect (P<0.05). Figure 11 ).

[0075] Example 7: Effects of Ganoderma lucidum exovesicles on motor coordination and muscle strength

[0076] Training was conducted before testing. The fall latency of mice on the rotundus was recorded before administration (24 h after modeling) and on day 7 after administration. The grip strength of the mice's forelimbs was measured using a grip dynamometer and normalized by dividing by the body weight on that day.

[0077] Seven days after administration, the fall latency was shortened in the MCAO group. The latency was significantly prolonged in all treatment groups, with the GLEVs-H group showing the largest increase, superior to the YKT group (P<0.05). Figure 12 The normalized grip strength in the MCAO group decreased significantly on day 7, while GLEVs treatment significantly restored forelimb muscle strength. The recovery rate in the GLEVs-H group was close to that in the Sham group and significantly better than that in the YKT group (P<0.01). Figure 13 Experimental results showed that GLEVs could effectively improve motor coordination, balance, and muscle weakness in MCAO / R mice.

[0078] Example 8: The effect of Ganoderma lucidum exovesicles on gait function

[0079] Starting 7 days prior to surgery, mice were allowed to walk freely three times daily on a gait analysis track, with 15-minute intervals between each session, to familiarize them with the environment. Formal gait data was collected one day before surgery as a normal control. Gait tests were performed 24 hours after modeling (before drug administration) and 24 hours after the last drug administration (day 7 post-surgery). The track glass plate was kept clean for each test. At least three complete, uninterrupted gait sequences were recorded for each mouse, and the system automatically recorded and analyzed the parameters.

[0080] On postoperative day 7, the mean movement speed of mice in the MCAO model group was significantly lower than that in the Sham group (P < 0.01). Compared with the MCAO group, the movement speed of mice in all treatment groups was significantly improved (P < 0.05). Among them, the GLEVs-H group showed the most significant improvement, with its movement speed recovering to a level close to that of the Sham group, and significantly better than that of the YKT positive drug group (P < 0.05). Figure 14 ).

[0081] On postoperative day 7, the MCAO model caused a sharp decline in the gait regularity index in mice (P < 0.01). After treatment with GLEVs, the regularity index significantly recovered. The significant improvement in the GLEVs-H group (P < 0.01) indicates that high-dose GLEVs can effectively restore the coordination and rhythm of walking movements. Figure 15 In addition, the Sham group mice exhibited basically symmetrical hindlimb movement. After MCAO surgery, due to unilateral brain injury, the bilateral asymmetry index of the model group mice was significantly increased (P < 0.01). After drug treatment, the GLEVs-H group was able to correct this asymmetry to the greatest extent, and its asymmetry index was significantly lower than that of the MCAO group and the YKT group. Figure 16 This suggests that it can effectively promote the recovery of function in the affected limb and rebuild bilateral motor balance.

[0082] Example 9: Exploration of the therapeutic time window of Ganoderma lucidum exovesicles

[0083] After successful MCAO / R modeling, participants were randomly assigned to a model group (MCAO) and four treatment groups (n=3). All treatment groups received their first tail vein injection of GLEVs-H at different time points after reperfusion (4 h, 8 h, 12 h, 24 h) at a dose of 1×10⁻⁶. 10 Particles / each), then once daily for 6 weeks. mNSS scoring and rotarod experiments were performed weekly, and weight changes were recorded.

[0084] The results showed that, at all time points, the mNSS score and rotator latency were significantly better in the treatment groups than in the MCAO model group at the same time point (P<0.05). Even when administration was started 24 h after reperfusion, GLEVs still showed clear efficacy. Figures 17-18 In the experiment, the body weight of mice in the MCAO group continued to decrease within 2 weeks, while the body weight of mice in all treatment groups stopped decreasing and started to increase within one week of administration, showing a trend of first decreasing and then increasing. Figure 19 The results indicate that GLEVs treatment helps promote the overall recovery of the patient's condition. These findings demonstrate that GLEVs treatment initiated within 24 hours of cerebral ischemia-reperfusion injury effectively promotes the long-term recovery of neurological and motor function, exhibiting a relatively wide treatment time window.

Claims

1. The application of Ganoderma lucidum exovesicles derived from non-spore-forming fruiting body waste in a drug for treating sequelae of ischemic stroke, characterized in that... The aforementioned non-spore fruiting body waste is derived from Ganoderma lucidum exovesicles, which are prepared from non-spore fruiting body waste after the complete release of spores during the Ganoderma lucidum powdering period through raw material pretreatment, differential centrifugation, tangential flow concentration, and membrane filtration.

2. The application according to claim 1, characterized in that, The sequelae of ischemic stroke include core symptoms such as post-stroke motor dysfunction and cognitive decline.

3. The application according to claim 1, characterized in that, The Ganoderma lucidum exovesicles derived from the waste of non-spore-forming fruiting bodies are prepared by the following method: Non-spore-forming fruiting bodies of Ganoderma lucidum are washed with sterile water, cut into pieces, mechanically pulverized, and impurities removed. PBS is added at a material-to-liquid ratio of 1-4 L / kg, and the mixture is soaked at 4℃ and 60-120 rpm for 8-12 hours to fully dissolve the exovesicles. The mixture is then filtered through three layers of gauze to remove coarse residue. The filtrate is centrifuged at 2000-4000g at 4℃ for 15-25 min, the precipitate is discarded, and the supernatant is passed through a 60-mesh sieve. The supernatant is then centrifuged again at 10000-12000g at 4℃ for 30-60 min, and the supernatant is collected to further remove macromolecular impurities and cell debris. The supernatant is collected and sequentially passed through a hollow fiber column with a pore size of 0.22-0.45 μm and a molecular weight cutoff of 30-100 kDa. The tangential flow concentration and filtration was performed using a hollow fiber column: First, the supernatant was passed through a hollow fiber column with a filter diameter of 0.22~0.45μm, and the permeate was collected; then, the permeate was concentrated by passing it through a hollow fiber column with a molecular weight cutoff of 30~100kDa, and the concentrate was collected; finally, the concentrate obtained after tangential flow concentration and filtration was further concentrated by centrifugation at 3500g for 15min using a 0.45μm, 30kDa ultrafiltration tube, and the concentrate was collected. The concentrate was then filtered through a 0.22μm filter membrane for sterilization, thus obtaining the Ganoderma lucidum exovesicles.

4. A method for preparing Ganoderma lucidum exovesicles derived from sporeless fruiting body waste, characterized in that, The process includes the following steps: Take the sporeless fruiting body waste of Ganoderma lucidum, wash with sterile water, cut into pieces, mechanically crush to remove impurities, add PBS at a material-to-liquid ratio of 1-4 L / kg, and soak at 4℃ and 60-120 rpm for 8-12 hours to fully dissolve the exovesicles. Filter through three layers of gauze to remove coarse residue. Centrifuge the filtrate at 2000-4000g at 4℃ for 15-25 min, discard the precipitate, and pass the supernatant through a 60-mesh sieve. Centrifuge again at 10000-12000g at 4℃ for 30-60 min, collect the supernatant, and further remove macromolecular impurities and cell debris. Collect the supernatant and pass it sequentially through a hollow fiber column with a pore size of 0.22-0.45 μm and a molecular weight cutoff of 30-100 kDa. The hollow fiber column was used for tangential flow concentration and filtration. Finally, the concentrate obtained after tangential flow concentration and filtration was further concentrated by centrifugation at 3500g for 15min using a 0.45μm, 30KDa ultrafiltration tube. The concentrate was collected and filtered through a 0.22μm filter membrane for sterilization to obtain the Ganoderma lucidum exovesicles.

5. The preparation method according to claim 4, characterized in that, The hollow fiber column tangential flow concentration filtration process involves first passing the supernatant through a hollow fiber column with a filter diameter of 0.22~0.45μm and collecting the permeate; then, the permeate is concentrated by passing it through a hollow fiber column with a molecular weight cutoff of 30~100kDa and collecting the concentrate.

6. The preparation method according to claim 4, characterized in that, The process includes the following steps: Collect sporeless Ganoderma lucidum fruiting bodies after the spore release period, wash, cut into pieces, and mechanically crush. Add PBS at a ratio of 1:4 (w / v), and soak at 4°C and 120 rpm for 12 hours to fully dissolve the exovesicles. Filter through three layers of gauze to remove coarse residue. Centrifuge the filtrate at 4000g for 20 min, discard the precipitate, and filter the supernatant through a 60-mesh sieve to obtain a clear filtrate. Then, centrifuge at 10000g and 4°C... Centrifuge for 40 min to further remove macromolecular impurities and cell debris, and collect the supernatant. The collected supernatant is then subjected to tangential flow concentration filtration through a hollow fiber column with a pore size of 0.45 μm and a hollow fiber column with a molecular weight cutoff of 30 kDa. First, the supernatant is passed through a hollow fiber column with a pore size of 0.45 μm, and the permeate is collected. Then, the permeate is concentrated by passing it through a hollow fiber column with a molecular weight cutoff of 30 kDa, and the concentrate is collected. The concentrate obtained after tangential flow concentration filtration is centrifuged at 3500 g for 15 min using a 0.45 μm, 30 kDa ultrafiltration tube, and the concentrate is collected. The concentrate is then centrifuged at 110,000 g at 4 °C for 70 min, and the precipitate is collected. The precipitate is resuspended in PBS and filtered through a 0.22 μm filter membrane to obtain the Ganoderma lucidum exovesicles.

7. Ganoderma lucidum exovesicles derived from non-spore-forming fruiting body waste are prepared according to the preparation method of any one of claims 4-6.

8. The Ganoderma lucidum exovesicles derived from non-spore-forming fruiting body waste according to claim 7, characterized in that, Its particle size distribution is 30~150nm, and its concentration is not less than 1×10⁻⁶. 10 The membrane contains particles per mL and exhibits a typical cup-shaped vesicle structure, demonstrating good membrane stability.

9. A pharmaceutical composition for treating sequelae of ischemic stroke, characterized in that, It contains Ganoderma lucidum exovesicles derived from non-spore-forming fruiting body waste as described in claim 7 or 8.

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