Anti-aging composition using Basidiomycete exosomes and the functional compounds for improving cognition

Abstract

The present invention relates to an anti-aging functional composition derived from exosomes of basidiomycetes and a functional composition for inhibiting neuroinflammation or improving cognitive function derived from active compounds of exosomes. It was confirmed that exosomes released from basidiomycete mycelium can be utilized in skin-protecting health functional foods and cosmetics due to their major anti-aging functional improvement effects, and that active compounds obtained from the exosomes exhibit effective therapeutic effects for degenerative neurological diseases compared to existing drugs, thus being utilized in compositions for preventing neurological diseases or improving cognitive function.

Description

Technology Field

[0001] The present invention relates to an anti-aging functional composition by exosomes of basidiomycetes and a functional composition for inhibiting neuroinflammation or improving cognitive function by exosome-derived low molecular weight compounds. Background Technology

[0002] Exosomes are extracellular vesicles (hereinafter referred to as 'EVs') released by cells that play a role in intercellular information exchange and signal transmission. Because they are nanoparticles with a size of 50-300 nm, they are easily absorbed into cells, tissues, and skin. Utilizing these properties, they can be applied in a wide range of fields, such as pharmaceuticals and bio-cosmetics. In other words, if therapeutic ingredients are contained within the exosomes, they can be used in the pharmaceutical and bio fields, and if skin whitening and regenerating ingredients are contained within, they can provide anti-aging and skin improvement effects.

[0003] Since their first discovery, various human-derived exosomes have been researched and developed, but commercialization is not easy due to low production yields and difficulties in quality control, such as contamination or reduced purity. In particular, because there are many restrictions on using exosomes as skincare materials due to cosmetic safety standards, recent research has mainly focused on anti-aging, immune enhancement, and skin improvement using exosomes derived from natural products.

[0004] Prior art has reported various biological functionalities (antibacterial, anti-aging, immune enhancement, skin improvement) using fungal exosomes; however, research and commercialization regarding anti-aging related to basidiomycete-derived exosomes are still lacking. Furthermore, since exosomes consist of a mixture of various substances, material standardization (establishment of QC evaluation criteria and test methods) has not been achieved, so limitations in commercialization are expected even if they are developed.

[0005] To overcome this, it is necessary to enable the standardization of substances from exosomes derived from natural products, but profiling active compounds within exosomes and screening pharmacological candidate substances is the biggest hurdle. Among these, there have been no reported studies on anti-aging related to specific exosome-derived compounds, particularly regarding drugs for brain diseases and the improvement of dementia / memory.

[0006] As average human life expectancy increases worldwide, representative diseases affecting the central nervous system due to aging include epilepsy, neurodegenerative diseases, and stroke. Among these, neurodegenerative diseases are conditions characterized by degenerative changes in nerve cells of the central nervous system, leading to various symptoms due to the loss of intrinsic functions in the affected areas. As the incidence of these diseases increases in proportion to the growing elderly population, the social burden is accelerating.

[0007] Degenerative brain diseases can be classified based on major symptoms and affected brain regions, including Parkinson's disease (PD), Alzheimer's disease (AD), Huntington's disease (HD), frontotemporal dementia, corticobasal degeneration, progressive supranuclear palsy (PSP), multiple sclerosis, and amyotrophic lateral sclerosis (ALS).

[0008] These degenerative brain diseases are known to be caused by the aggregation of proteins and the death of nerve cells due to neurodegeneration associated with aging and genetic and environmental factors; however, the exact cause has not yet been identified, and basic research to determine the cause is currently being actively conducted. Among degenerative neurological diseases, multiple sclerosis has the largest market size, while Alzheimer's disease and Parkinson's disease are among those with significant social repercussions.

[0009] According to data from the Alzheimer's Association, a new case of Alzheimer's disease occurs every 68 seconds in the United States, and it is projected to occur every 33 seconds by 2050. Currently, the number of patients suffering from dementia worldwide amounts to 35.6 million, and this figure is estimated to reach 65.7 million by 2030. South Korea already entered an aging society in 2000, when the population aged 65 and older exceeded 7% of the total population. It is predicted to surpass 20% by 2026, reaching a super-aged society. If this happens, the issue of degenerative brain diseases is expected to emerge as a serious concern not only domestically but globally.

[0010] A more significant problem is that most central nervous system diseases represent an area of ​​unmet need, lacking effective treatments. Given Korea's ongoing super-aging population, the development of treatments for degenerative brain diseases and functional products that aid in their treatment is undoubtedly the most urgent task.

[0011] Currently, most dementia treatments are acetylcholinesterase inhibitors that merely delay the progression of the disease and have severe side effects; therefore, continuous research is being conducted on natural materials as an alternative for dementia treatment. In the absence of a fundamental cure capable of slowing down or halting the progression of Alzheimer's disease, recent rapid advancements in cellular and molecular technologies have led to the development of new treatments aimed at modifying the disease course, although these therapies are not yet fully perfected.

[0012] As the genetic, cellular, and molecular understanding of Alzheimer's disease increases, potential therapeutic targets have also expanded. Drugs are being developed in new directions, addressing a diverse range of targets including amyloid pathways, tau phosphorylation, neurons and synapses, inflammation, mitochondria, oxidative processes, reactive oxygen species, neurotransmitters and receptors, and statins.

[0013] Amyloid beta and tau, both identified as the primary causes of Alzheimer's disease, are peptides found in brain neurons; however, while tau appears inside the cell, amyloid beta appears only on the cell surface and aggregates to form plaques. Consequently, specific antibodies capable of targeting these molecules through surface recognition have been widely studied.

[0014] Biogen and Novartis maintain the leading group in related research. Although the amyloid beta-targeting antibody drugs Aducanumab and Lecanemab were recently approved for sale by the FDA, controversies regarding their therapeutic efficacy and side effects persist, and there are currently no other companies researching this field. In other words, there are currently no drugs capable of fundamentally curing Alzheimer's disease or halting its progression; they merely alleviate patient symptoms. Despite numerous efforts, the causes and pathogenesis of Alzheimer's disease have not yet been fully elucidated, and hypothetical treatments have formed the basis of drug therapy for the disease to this day.

[0015] As an alternative to this situation, a trend is also being observed to reposition already developed functional ingredients or pharmaceuticals, particularly compounds extracted from natural products, for the treatment of neurological disorders (e.g., Hericium erinaceus, Ganoderma lucidum, Reishi, Shiitake, Ceriporia, etc.).

[0016] For example, compounds extracted from the genus *Pterocarya* or *Pterocarya persica* have antibacterial and antifungal effects and have been used to treat vaginitis, nephritis, and edema. Among the compounds extracted from *Pterocarya persica*, PFC (C10H14O2), 5-(4-pentyl)-2-furaldehyde, and methyl-3-p-anisoloxypropionide have been reported as nematicidal components. In particular, PFC has been reported to strongly inhibit melanin production without cytotoxicity (IC50=8.4 μg / ml). However, until recently, there have been no cases of its use for functional purposes related to the treatment of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Prior art literature

[0017] M. L. Rodrigues, L. Nimrichter, From fundamental biology to the search for innovation: The story of fungal extracellular vesicles, European Journal of Cell Biology, 2022, 101, 2, 151205.J. Rizzo, M, L. Rodrigues, G. Janbon, Extracellular Vesicles in Fungi: Past, Present, and Future Perspectives. Front Cell Infect Microbiol. 2020, 10, 346. The problem to be solved

[0018] Therefore, the objective of the present invention is to elucidate the anti-aging functionality of exosomes derived from the mycelium of basidiomycetes, and furthermore, the objective of the present invention is to provide a functional composition for preventing neurological diseases or improving cognitive function containing the said active ingredient by discovering that the exosome-derived active compound has an effect on degenerative neurological diseases through the inhibition of neuroinflammation without side effects. means of solving the problem

[0019] To solve the above problem, the present invention provides a method for producing EV derived from basidiomycete mycelium containing PFC or PFCA.

[0020] In addition, the above mycelium is a mechanical insect mushroom ( Milkweed ), Monkey's Foot ( Ganoderma applanatum ), Reishi mushroom ( Ganoderma lucidum ), territory ( Reishi ), Lacerated Ceriporia , deer buttocks ( Hericium erinaceus ), shiitake ( Shiitake ) or truffle ( Truffles It is characterized by being selected from among mushroom mycelium.

[0021] According to one embodiment of the present invention, cosmetics, functional cosmetics, skin treatments, additives, raw materials, and health functional foods for anti-aging and skin improvement purposes are provided using EVs derived from Basidiomycete mycelium containing PFC or PFCA.

[0022] In addition, a pharmaceutical composition capable of improving cognitive function, enhancing memory, and preventing or treating degenerative neurological diseases is provided, comprising EV derived from Basidiomycete mycelium containing PFC or PFCA as an active ingredient.

[0023] In addition, a pharmaceutical composition capable of preventing or treating chronic liver diseases such as liver dysfunction, fatty liver, liver steatosis, NAFLD, and NASH is provided, comprising EV derived from basidiomycete mycelium containing PFC or PFCA as an active ingredient.

[0024] In addition, a pharmaceutical composition capable of improving cognitive function, enhancing memory, and preventing or treating degenerative neurological diseases is provided, comprising PFC or PFCA as an active ingredient.

[0025] In addition, a pharmaceutical composition capable of improving liver function and preventing or treating chronic liver diseases such as fatty liver, liver steatosis, NAFLD, and NASH is provided, comprising PFC or PFCA as an active ingredient.

[0026] In addition, functional ingredients and health functional foods for improving cognitive function / memory containing PFC or PFCA as active ingredients are provided. Effects of the invention

[0027] According to the present invention, exosomes released from basidiomycete mycelium exhibited a major anti-aging functional improvement effect, and it was confirmed that they can be utilized in health functional foods and cosmetics for skin protection.

[0028] In addition, the pharmaceutical composition according to the present invention, which includes PF as a basic skeletal structure, contains PFC and PFCA derived from the mycelium or exosomes of Basidiomycete, a natural product, so there is no cytotoxicity and the likelihood of side effects is low. It also has excellent anti-inflammatory and protective effects on human neurons and can be very effectively used for the improvement of cognitive function via the inhibition and regulation of fatty liver, and for the prevention or treatment of degenerative neurological diseases.

[0029] In particular, the pharmaceutical composition of the present invention, comprising the PFC, PFCA compound or a pharmaceutically acceptable salt thereof as an active ingredient, showed excellent effects in the passive avoidance test and Morris water maze test when administered orally for 4 weeks to a mouse model of memory impairment / Alzheimer's disease induced by beta-amyloid peptide (1-42), thus confirming that the composition of the present invention is effective against neurodegenerative diseases.

[0030] Meanwhile, the scope of the present invention is not limited by the effects described above. Brief explanation of the drawing

[0031] Figure 1 is a schematic diagram of the bio-transformation of PFCs contained in exosomes derived from basidiomycete mycelium to PFCA. Figure 2 shows TEM images of extracellular vesicles isolated from each mycelium, namely (a) IEV1, (b) IEV2, (c) GEV, and (d) CEV. Vesicular particle morphology of approximately 200 nm in size can be observed (scale bar = 500 nm). Figure 3 shows the results of NTA nanoparticle size analysis of extracellular vesicles isolated from each mycelium, namely (a) IEV1, (b) IEV2, (c) GEV, and (d) CEV. Figure 4 is a figure showing the immune macrophage activation effect of extracellular vesicles (EVs) of basidiomycetes. Figure 5 is a figure showing the immune-enhancing and anti-inflammatory effects through the measurement of (a) NO and (b) iNOS incidence rates of extracellular vesicles (EVs) of basidiomycetes. Figure 6 is a graph of PCR results regarding the inhibition of inflammatory cytokine expression by basidiomycete EVs samples, showing the anti-inflammatory effect by inhibiting the expression of (a) TNF-α, (b) IL-1β, (c) IL-6, and (d) MCP-1. Figure 7(a) is a graph of skin absorption of IEV2 derived from basidiomycetes over time. Figure 7(b) is a comparison chart of skin absorption of IEV1 and IEV2 derived from basidiomycetes. Figure 7(c) is a figure of skin absorption after 2 hours for each EV applied sample. Figure 8 is a figure showing the skin barrier moisturizing indicator (filagrin, fibrinogen, fibrillin) improvement effect of Basidiomycete EVs. Figure 9 is a graph showing the wrinkle-improving efficacy of Basidiomycete EVs through (a) collagen synthesis-promoting efficacy and (b) collagenase-inhibiting efficacy. Figure 10 is a graph showing the skin whitening effect of Basidiomycete EVs. Fig. 11 shows a mechanical worm mushroom ( I.lacteusHPLC analysis graphs of PFC and PFCA compounds extracted from mycelial cultures and exosome samples, respectively: (a) measurement after culture (PFC peak), (b) sample refrigerated for 30 days after culture (PFCA peak), (c) sample after IEV1 lysis (PFC peak), (d) sample after IEV2 lysis (PFCA peak), and (e) analysis results of the aqueous solution after EA extraction of the IEV2 lysis sample. Fig. 12 is I.lacteus As results of the chemical structure analysis of PFCA compounds extracted from, respectively (a) LC / MS, (b) 1 H-NMR in MeOD, (c)HSQC-dept in MeOD. Figure 13 shows the results of measuring the cell activity of human microglia with PFC and PFCA. Figure 14 is a graph showing the effects of PFC and PFCA on the expression of inflammation-inducing cytokines in human neurons. Figure 15 is a graph showing the efficacy evaluation of markers associated with cognitive / memory function in hippocampal cells of PFC and PFCA. Figure 16 is a graph showing the cognitive improvement effect of PFCA and the results of the passive avoidance test. Figure 17 is a graph showing the results of the Morris Water Maze test regarding the cognitive improvement effect of PFCA. Figure 18 shows the brain tissue of the PFC and PFCA. from life This is a graph showing the antioxidant superoxide dismutase activity effect. Figure 19 shows the brain tissue of the PFC and PFCA. from life This is a graph showing the anti-inflammatory effect. Figure 20 shows the liver tissue of PFC and PFCA. from life This is a graph showing the inhibitory effect on steatosis. Specific details for implementing the invention

[0032] The present invention will be described in detail below, focusing on the embodiments.

[0034] <Example 1>

[0035] 1-1. Culture of Basidiomycete Mycelium and Exosome Production

[0036] Basidiomycete mycelium, the basidiomycete is I. milky The medium for liquid culture of may include monopotassium phosphate (KH2PO4), dipotassium phosphate (K2HPO4), starch, sorghum flour, soybean flour, barley flour, glucose, sugar, magnesium sulfate (MgSO4), and water, and may have a hydrogen ion concentration (pH) of 4.0 to 6.0.

[0037] First, the mycelial culture step establishes an environment and provides optimal nutrients for mycelial proliferation. Liquid culture in the above culture step can be performed under a blue LED light source and maintained at a carbon dioxide concentration of 1,000 to 2,000 ppm. Meanwhile, as the mother strain in the culture step, one superior strain stored at 4°C in PDA (Potato dextrose agar) medium can be used, which has undergone a culture process for 7 to 10 days in a shaking incubator using PDB (Potato dextrose broth) medium while maintaining a constant temperature of 25°C. At this time, it is preferable that the amount of mycelium to be added as an inoculum be approximately 0.3-0.6% (w / v) based on the amount of solution to be cultured.

[0038] As the culture stage progresses and the mycelium proliferates, secondary metabolites are biosynthesized within the mycelium to increase cell viability and then loaded into endosomes. Additionally, for intercellular signaling, the mycelium releases the endosomes loaded with the metabolites through the cell wall to the outside of the mycelium, thereby generating exosomes in which the metabolites are extracellular endoplasmic reticulum (EV).

[0039] Exosomes derived from the mycelium of Basidiomycetes contain fat-soluble low-molecular-weight fatty acid compounds or intermediate compounds as secondary metabolites. Uniquely, in the present invention, it was confirmed that PFC compounds are detected when the culture medium and exosomes are extracted with ethylene acetate after cultivation.

[0040] In addition, it was discovered that PFC compounds undergo bio-transformation into PFCA through low-temperature storage at 4°C for about one month after the culture stage (see Fig. 1). Low molecular weight lipid compounds such as PFCs are mainly present in the double membrane layer of mycelia or exosomes, and it is presumed that the aldehyde functional group of PFCs bound to the double membrane layer of exosomes is converted into a carboxyl group by an enzyme present in the culture medium (e.g., aldehyde dehydrogenase) to form PFCA.

[0041] In the present invention, as a comparative group of mycelial cultures Ganoderma applanatum EV (GEV), Lacerated Ceriporia EVs (CEVs) were also manufactured using a process similar to the manufacturing method described in the present invention and used in experiments.

[0043] 1-2. Exosome Isolation and Purification

[0044] The exosome isolation and purification step consists of (a) a culture medium dispersion step, (b) a culture medium centrifugation step, and (c) an exosome filtering step. First, the culture medium containing exosomes undergoes (a) the culture medium dispersion step. At this time, the culture medium contains a mixture of mycelium, exosomes, and other substances.

[0045] Next, (b) the centrifugation step of the culture medium is a step for separating mycelia and exosomes, and centrifugation is performed at 10,000 to 15,000 rpm, at which time the heavy mycelia are distributed in the lower layer, and the relatively light exosomes are distributed in the supernatant in the form of colloidal dispersion.

[0046] Next, (c) the exosome filtering step is a step of separating and purifying the supernatant containing exosomes after the centrifugation step described above by using a 0.45 μm filter and a UF filter to filter the exosomes so that only nanoparticle exosomes pass through. It is desirable to vacuum freeze-dry the filtered exosomes so that they can be used for various purposes in the future. When stored under refrigeration at 4°C, they remain stable for more than 3 months without aggregation or denaturation, and they dissolve well in water.

[0047] The isolated and purified exosomes are analyzed by TEM and have a uniform particle size of approximately 100–300 nm and a spherical shape (see Fig. 2).

[0048] Nanoparticle track analysis (NTA) was performed to characterize the EVs isolated from each mycelium. As shown in Figure 3, particle concentration and size measurements were plotted and real-time imaging, and scattering distributions were presented for the nanoparticles in three consecutive 30 s runs (n=3). The four types of EVs were analyzed to have particle sizes of approximately 200 nm, and it can be seen that EVs originating from basidiomycetes exhibit similar particle characteristics.

[0050] <Example 2>

[0051] To confirm the immunogenicity and cellular activity of Basidiomycete EVs against immune cells, RAW 264.7 cells (ATCC) were cultured and seeded in 96-well plates at a density of 1 x 10⁴ cells / well, followed by an MTT assay (see Fig. 4). Treatment with Basidiomycete EVs at concentrations of 1, 10, 50, and 100 μg / mL resulted in no cytotoxicity and an overall increase in activity. Thus, the composition containing mycelium-derived exosomes from this study can possess the efficacy to protect the skin from disease or cell damage by enhancing the immunogenicity of immune cells (see Fig. 4).

[0052] As shown in Figure 5, to investigate the immune and anti-inflammatory effects by measuring the incidence of NO and iNOS, RAW 264.7 cells were pretreated with 10 µg / mL of EVs for 1 hour and then stimulated with 1 µg / mL of lipopolysaccharide (LPS). 10 µg / mL of α-lipoic acid (LA) (Sigma) and 10 µg / mL of dexamethasone (Sigma) were used as positive controls. After 24 hours of incubation, absorbance measurements were performed at 550 nm using a microplate reader (Multiskan GO instrument). A culture medium without LPS (normal group) was used as the negative control (NC). The measured standard values ​​were expressed in the form of mean + standard deviation, and statistical significance was determined using Student's t-test; a p-value of < 0.05 (*) was considered significant.

[0053] When each EV was treated at 10 μg / mL, the levels of immune and inflammation-related factors NO (a) and iNOS (b) were significantly reduced compared to dexamethasone (steroidal anti-inflammatory drug, DEX), and in particular, the reduction level caused by IEV was found to be almost similar to that of α-LA, a potent anti-inflammatory control (see Fig. 5). Through this, it was confirmed that EVs exert immune-enhancing / anti-inflammatory effects by regulating inflammatory signaling pathways through the inhibition of NO production and the expression of iNOS-related genes.

[0054] It was confirmed that when mycelial-derived EVs were treated to RAW 264.7 cells with LPS-induced inflammation, they reduced the expression levels of inflammation-related factors, specifically inflammatory cytokines (a) TNF-α, (b) IL-1β, (c) IL-6, and (d) MCP-1; notably, their anti-inflammatory effects were found to be superior compared to existing anti-inflammatory agents such as DEX and α-LA (see Fig. 6).

[0056] <Example 3>

[0057] To evaluate the skin absorption rate of EVs derived from basidiomycetes, skin permeability was measured using porcine skin tissue (see Fig. 7). By staining EV particles with Coomassie Brilliant Blue and applying them to the surface of the skin tissue, the permeated particles could be identified by observing a blue color. While the control group stained with BSA remained in the epidermal layer, EVs clearly penetrated into the dermis, subcutaneous fat layer, and muscle layer, as measured by the length of the stained cross-section, confirming that the skin permeability of EVs is relatively superior. Through this, EVs can be utilized as cosmetics and topical treatments that enhance skin absorption rates and anti-aging functionality.

[0059] <Example 4>

[0060] As indicators of anti-aging functionality, the constituent components filaggrin, fibronectin, and fibrillin gradually flatten the shape of skin cells, increase the strength of the skin barrier, and enhance intercellular cohesion on the skin surface. In this invention, to confirm the effect of EVs on skin barrier function, the mRNA expression levels of filaggrin, fibronectin, and fibrillin, which are skin barrier components, were measured by RT-PCR.

[0061] HDFa (ATCC) cells were cultured at 2 x 10⁴ cells / well in 24-well plates using DMEM medium containing 10% FBS, 1% penicillin, and 1% streptomycin, and then incubated for 24 hours in a 37°C, 5% CO₂ incubator. After removing the supernatant, each sample was placed in DMEM medium excluding FBS and incubated at 37°C, 5% CO₂ The culture was incubated in an incubator for 24 hours. After incubation, the supernatant was removed, and RNA was obtained using Easy Blue lysis reagent (iNtRON Biotechnology, Sungnam, Korea) according to the instructions. For cDNA synthesis, RT PreMix (BIONEER, Daejeon, Korea) was used at 42 ℃ for 60 minutes and at 95 ℃ for 5 minutes.

[0062] As shown in Figure 8, when HDFa cells were treated with EVs at concentrations of 1, 10, and 50 μg / mL, all skin indicators improved in a concentration-dependent manner, and in particular, at IEV 50 μg / mL, filaggrin expression levels increased significantly by more than 60% compared to the untreated group. This suggests that all EVs maintained skin barrier function and contributed to hydration or moisturizing functionality by upregulating the RNA expression rate of skin indicator proteins.

[0063] In addition, the efficacy of EVs on skin collagen formation and collagenase inhibition, which are key indicators of skin wrinkle improvement, was confirmed (see Fig. 9). HDFa cells were cultured and pretreated with EVs, and then the procollagen type I C-peptide EIA kit (Takara, Kusatsu, Japan) and the human pro-MMP-1 Quantikine ELISA kit were used (R&D System, Minneapolis, Minnesota, USA). 10 ng / mL of TGF-β (Sigma) was dissolved in the culture medium and used as a positive control, and the culture medium was used as a negative control. As a method to measure collagenase activity, an antibody against collagenase (MMP-1), an enzyme that degrades collagen, was used, and Phorbol 12-myristate 13-acetate (PMA) (Sigma) was used to activate the expression of MMP-1 as an endogenous inducer of superoxide production, which is a major ROS-generating substance and can increase MMP activity in dermal fibroblasts, thereby causing the degradation of extracellular matrix (ECM) proteins and premature skin aging.

[0064] As shown in Fig. 9(a), collagen synthesis increased in a concentration-dependent manner in the groups treated with EVs at concentrations of 1, 10, and 50 μg / mL. In particular, collagen synthesis was found to increase significantly by up to 50% at a concentration of 50 μg / mL of IEV1 and 2.

[0065] As shown in Fig. 9(b), it was confirmed that MMP-1 protein substantially increased after treatment with 50 nM PMA, whereas decreased in a concentration-dependent manner through treatment with EVs. Through this, it can be seen that EVs can be effective in improving wrinkles by inhibiting PMA-induced oxidative stress, which induces skin cell death, thereby reducing collagenase activity and reducing collagenase activity, through collagen synthesis and collagenase reduction in the skin ECM.

[0066] To observe the melanin inhibitory activity of Basidiomycete EV samples, B16 melanoma cells (ATCC) were 1 x 10⁴ Cells were cultured in 6-well culture plates containing DMEM medium with 10% FBS at a cell / well ratio. 1% penicillin-streptomycin was added under conditions of 37°C and 5% CO2. α-melanocyte-stimulating hormone (α-MSH), a melanin synthesis inducer, was prepared by dissolving it in 10% DMSO at a concentration of 50 μM. After incubation for 24 hours, EV samples were added and immediately treated with α-MSH (50 nM), followed by an additional 72 hours of incubation. Next, the cell plates were washed twice with PBS and trypsinized; the recovered cells were centrifuged at 5000 rpm for 10 minutes to remove the supernatant and obtain a cell pellet. Melanin was harvested by dissolving it in 2 N NaOH containing 10% DMSO at 60°C for 4 hours and transferred to a 96-well plate. DMSO (0.1% v / v) was used as the solvent for the control and test samples containing α-MSH, and the absorbance was measured at 475 nm using an ELISA reader.

[0067] After adding EVs (1, 10, 50 μg / mL) to α-MSH-induced melanocytes, it was confirmed that melanin synthesis was inhibited in a concentration-dependent manner (see Fig. 10). In particular, it was found that 50 μg / mL of IEVs downregulated more than 70% of melanin synthesis, confirming that it could inhibit melanin synthesis more efficiently than 50 μg / mL of arbutin (58%).

[0068] Although arbutin is recognized as a single whitening compound and is widely used as a skin whitening agent in the cosmetics market, it is glycosylated hydroquinone, which can cause cancer. Therefore, it is very encouraging that EVs not only exhibit a skin whitening effect by inhibiting melanin synthesis but also showed a whitening function superior to that of arbutin. As PFCs have been reported to strongly inhibit melanin production (IC50=8.4 μg / mL), it can be presumed that EVs contributed to the whitening functionality because they contain such compounds.

[0069] Meanwhile, the composition containing exosomes derived from basidiomycete mycelium of the present study can exhibit an anti-aging effect on the skin. According to one embodiment, the composition of the present study can be used for the prevention or treatment of skin aging by exhibiting an antioxidant effect through the scavenging of free radicals.

[0070] In particular, as shown in FIGS. 4 to 6, the exosome-containing composition of this study exhibited enhanced immune activity and anti-inflammatory effects, suggesting that the exosome component enhances / induces the cellular activity, anti-inflammatory power, and immunity of skin cells and immune cells against major external immune disruptors and inflammation-inducing factors on the skin, such as free radicals.

[0071] In addition, a composition containing exosomes, which is an embodiment of the present invention, can exhibit a skin moisturizing effect. Specifically, the composition of the present study uses a biological agent, resulting in minimal skin irritation, and exhibits excellent skin penetration and moisture retention capabilities in the stratum corneum, thereby providing excellent longevity of the moisturizing effect. According to one embodiment, the composition of the present study can exhibit an excellent skin moisturizing effect by promoting the expression of filaggrin, fibronectin, fibrillin, etc., which are major moisturizing indicators of the skin barrier.

[0072] In addition, a composition containing exosomes, which is an embodiment of the present invention, can exhibit a wrinkle improvement effect. Wrinkle improvement includes the prevention of wrinkles and the removal of wrinkles. According to one embodiment, the composition of the present study can exhibit an excellent wrinkle improvement effect through molecular mechanisms such as inhibition of collagenase activity and promotion of collagen synthesis.

[0073] In addition, a composition containing exosomes, which is an embodiment of the present invention, can exhibit a skin whitening effect. The skin whitening effect refers to the action of preventing or suppressing symptoms resulting from an increase in melanin as the production of melanin is inhibited. According to one embodiment, the composition of the present invention exhibits a skin whitening effect by inhibiting the production of melanin within cells, has high stability, and causes almost no side effects such as skin irritation.

[0074] A composition containing exosomes, which is an embodiment of the present invention, can be used safely for a long period of time as it has almost no toxicity or side effects in addition to the aforementioned effects. Meanwhile, a composition containing exosomes, which is an embodiment of the present invention, may be provided in the form of a cosmetic, and the cosmetic may include conventional auxiliary agents and carriers such as stabilizers, solubilizers, surfactants, vitamins, pigments, and fragrances, which are commonly used in cosmetics. The cosmetic may be manufactured in any formulation conventionally produced in the art, and may be formulated, for example, as a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing, oil, powder foundation, emulsion foundation, wax foundation, and spray. More specifically, it may be manufactured in the form of a softening lotion, a nourishing lotion, a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a mask pack, a spray, or a powder, but is not limited thereto. In addition, the composition containing exosomes may be provided in the form of a health functional food, and the food may be in the form of a powder, granule, tablet, capsule, film, or beverage, and may be candy, chocolate, beverage, gum, tea, vitamin complex, health supplement, etc.

[0075] When considering all the results of the present invention, the composition containing exosomes derived from basidiomycete mycelium is expected to fundamentally improve skin conditions, such as anti-aging, anti-inflammation, anti-wrinkle, whitening, and immune-boosting effects in the dermal layer, as it can be absorbed and reach the endothelium, unlike conventional cosmetic ingredients that cannot reach the dermis. Through this, it can be utilized as an additive for high-end functional cosmetics and topical therapeutic agents as a next-generation eco-friendly biomaterial based on exosomes.

[0076] Additionally, the present invention provides a pharmaceutical composition capable of improving cognitive function and preventing or treating degenerative neurological diseases through mechanisms of human neuronal protection, hepatoprotection, and hippocampal antioxidant effects, comprising PFC and PFCA extracted from basidiomycete mycelial exosomes as active ingredients.

[0077] The above PFC (C 10 H 14 O2) is a compound represented by the following chemical formula 1, with a molecular weight of 166.2 g / mol.

[0078] [Chemical Formula 1]

[0079]

[0080] The above PFCA (C 10 H 14 O3) is a compound represented by the following chemical formula 2, with a molecular weight of 182.2 g / mol.

[0081] [Chemical Formula 2]

[0082]

[0083] The composition according to the present invention may additionally contain one or more known substances having an effect of improving neurological diseases (cognitive improvement / memory improvement) in addition to PFC and PFCA. The known substances may be acetylcholinesterase, butyrylcholinesterase, beta-secretase, amyloid beta, tau protein, synuclein, inhibitors of neuroinflammation-related proteins / cytokines, inhibitors of adipocytokines / adiponectins, or promoters of antioxidant-related enzymes, but are not limited thereto.

[0084] In the present invention, the degenerative neurological disease may include Parkinson's disease (PD), Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), frontotemporal dementia, corticobasal degeneration, and progressive supranuclear palsy (PSP), and preferably, the degenerative neurological disease according to the present invention is dementia or Alzheimer's disease.

[0085] The term “Alzheimer’s disease” used in this invention refers to a condition in which brain nerve cells undergo degeneration and loss due to some cause, resulting in overall brain atrophy and loss of brain cells. It is reported that proinflammatory cytokines such as interleukin (IL)-1βIL-6 and tumor necrosis factor-α secreted by microglia and astrocytes in the brain, a decrease in acetylcholine and an increase in acetylcholinesterase (AChE), and free radicals are involved in the onset of the disease.

[0086] In the present invention, the degenerative neurological disease may be caused by beta-amyloid. The term “beta-amyloid” used in the present invention is also referred to as β-amyloid, amyloid beta, or Aβ. It is a peptide generally composed of 36 to 43 amino acids, and is known as a dementia-inducing substance that causes oxidative stress and inflammation in the brain. The aggregation of beta-amyloid is known to cause various degenerative neurological diseases such as Alzheimer's disease, Parkinson's disease, stroke, and Huntington's disease.

[0087] The composition according to the present invention may further contain one or more pharmaceutically acceptable additives selected from the group consisting of excipients, lubricants, emulsifiers, wetting agents, sweeteners, fragrances, and preservatives.

[0088] The composition according to the present invention can be formulated and used according to conventional methods. In particular, it can be formulated by adopting methods known in the art to provide rapid, sustained, or delayed release of the active ingredient after administration to mammals.

[0089] The method of administration of the composition according to the present invention can be easily selected according to the formulation and may be administered orally or parenterally. For example, it may be used via the dermal, intramuscular, peritoneal, intravenous, subcutaneous, nasal, epidural, and oral routes, but is not limited thereto. Solid formulations for oral administration may be tablets, pills, soft or hard capsules, pills, powders, or granules, but are not limited thereto. Meanwhile, forms for parenteral administration may be in the form of creams, lotions, ointments, ointments, liquids, sprays, patches, or injections, but are not limited thereto.

[0090] The dosage of the composition according to the present invention may vary depending on the patient's age, gender, body weight, severity of the condition, and route of administration, but generally, an amount of 0.5 to 100 mg / kg, 0.5 to 50 mg / kg, 0.5 to 10 mg / kg, or 0.5 to 5 mg / kg may be administered in divided doses from once to three times a day. However, the above dosage does not limit the scope of the present invention in any way.

[0091] In addition, the present invention provides a functional ingredient or health functional food for improving cognitive function / memory comprising PFC / PFCA as an active ingredient.

[0092] The above-mentioned PFCs and PFCAs can inhibit the activity of neuroinflammation-related enzymes and inflammatory cytokines induced by oxidative stress within nerve cells. Although drugs that inhibit the activity of these inflammation-related enzymes have been developed for the treatment of dementia and the improvement of cognitive function and memory in patients with dementia or cognitive decline (e.g., Donepezil, Memantine, Tacrine, Rivastigmine), there has been a demand for natural products with similar activity due to insufficient efficacy, various side effects, risk of recurrence, and difficulties in synthesis. Accordingly, the natural product-derived pharmaceutical composition of the present invention, which contains the above-mentioned PFCs / PFCAs as active ingredients, can be usefully employed for cognitive improvement and memory improvement without side effects by directly protecting nerve cells from oxidative stress.

[0093] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier, excipient, or diluent in addition to the active ingredient described above. Examples of the carrier, excipient, and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0094] The pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, and aerosols, external preparations, suppositories, or sterile injectable solutions, each according to conventional methods. Specifically, when formulating, it may be prepared using diluents or excipients such as fillers, weighting agents, binders, humectants, disintegrants, and surfactants that are commonly used. Solid formulations for oral administration include, but are not limited to, tablets, pills, powders, granules, and capsules.

[0095] These solid formulations may be prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients. In addition to liquids and liquid paraffin for oral administration, various excipients, such as humectants, sweeteners, flavorings, cosmetic agents, and preservatives, may be added.

[0096] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. As non-aqueous solvents and suspensions, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used. As materials for suppositories, Witepsol, Macrogol, Tween, cocoa paste, laurin paste, glycerogelatin, etc. may be used.

[0097] The above health functional food may contain the above PFC, PFCA and / or pharmaceutically acceptable salts thereof in an amount of 0.001 to 100 weight%, 0.001 to 50 weight%, 0.1 to 100 weight%, 0.1 to 50 weight%, or 0.1 to 40 weight% of the total weight of the health functional food.

[0098] The health functional food for improving cognitive function / memory according to the present invention may be in the form of powder, granules, tablets, capsules, or beverages, and may additionally include other natural or synthetic substances for health / function and additives for product formulation.

[0099] In the case where the above health functional food is a beverage, it may contain the above PFC, PFCA and / or pharmaceutically acceptable salts thereof in amounts of 0.001 to 2 g, 0.001 to 1 g, 0.01 to 2 g, 0.01 to 1 g, or 0.01 to 0.1 g per 100 mL. In addition, there are no special restrictions on the liquid components, and it may contain various flavoring agents, natural carbohydrates, etc., as additional ingredients in normal amounts, as in ordinary beverages.

[0100] Examples of the aforementioned natural carbohydrates include monosaccharides; disaccharides such as glucose and fructose; polysaccharides such as maltose and sucrose; oligosaccharides such as dextrin and cyclodextrin; and sugar alcohols such as xylitol, sorbitol, and erythritol. As for the aforementioned flavoring agents, natural flavoring agents (thaumatin, cinnamon, stevia extracts (e.g., rebaudioside A, glycirazine, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) may be used.

[0101] In addition, the health functional food for improving cognitive function / memory according to the present invention may contain various nutritional supplements, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickening agents (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. Furthermore, it may contain fruit pulp for the production of natural fruit juices, fruit juice beverages, and vegetable beverages. These ingredients may be used independently or in combination.

[0102] Health functional foods for improving cognitive function / memory that may contain the above-mentioned PFC, PFCA and / or pharmaceutically acceptable salts thereof include, for example, various food products, beverages, chewing gums, vitamin complexes, health supplements, etc.

[0103] The present invention will be explained in detail below by the following examples. However, the following examples are merely illustrative of the present invention and do not limit the present invention.

[0105] <Example 5>

[0106] 5-1: I. milky HPLC Analysis of PFC and PFCA Compounds via Extraction

[0107] I. milkyPFC and PFCA compounds were obtained by ethyl acetate (EA) extraction of the mycelial culture. As shown in Figure 11, during HPLC analysis of the extracted sample after the culture step, a peak (a) at 16.06 min disappeared after low-temperature storage at 4 ℃ for about a month, and a new peak (b) appeared at 12.94 min. This suggests that the PFC compound was bio-transformed into PFCA. Additionally, exosome samples (IEV1, IEV2) extracted from the two mycelial cultures described above were placed in a lysis solution (1 N NaOH + 0.1% SDS), dissolved by ultrasonication, and then extracted with ethyl acetate; it was confirmed that peaks corresponding to PFC (c) and PFCA (d) appeared at 16.08 min and 12.96 min, respectively.

[0108] On the other hand, when the water layer of the sample was recovered and analyzed after EA extraction, no effective peaks related to metabolites appeared (e). This indicates that not only the mycelial culture medium but also exosomes derived from mycelium contain PFC and PFCA compounds.

[0110] 5-2: I. milky Analysis of PFCA compounds via LC / MS and H-NMR through extraction

[0111] Specifically, LC / MS was performed to analyze the molecular weight of the 12.94 fraction sample of the extracted sample in Fig. 11(b). Bioactive compounds present in the I. lacteus mycelial culture were fractionated using a UHPLC analysis system. Analysis was performed using a Thermo Scientific Ultimate 3000 UHPLC system coupled with a tandem mass spectrometer (LCQ Fleet, Thermo Fisher Scientific, MA, USA) equipped with a SCINChrom C18G 100A column (100 mm × 2.1 mm, 1.8 µm, Scinco, Australia). Mass spectra were collected in full-scan mode over a mass range of 100–1000 Da. MS / MS spectra were obtained with an impact energy of 25 eV. As a result of comparing the UV-vis mass spectroscopy built into the Thermo Scientific Ultimate 3000 UHPLC system, it was confirmed that the sample in Fig. 11(b) is identical to the synthetic compound (5-pentyl furan carboxylic acid, molecular weight 182.2 g / mol, EN300-8132959, Enamine Ltd.) (see Fig. 12(a)).

[0112] Additionally, as shown in Figs. 12(b) and (c), NMR spectroscopy analysis was performed to analyze the molecular structure of the 12.94 fraction sample. NMR data were acquired using a 700 MHz Bruker Avance III HD NMR spectrometer and a room temperature TBO probe. Fraction sample 1 As a result of performing H-NMR in MeOD(b) and HSQC-dept in MeOD(c), it was confirmed that the molecular structure of the sample is identical to that of the synthetic compound (5-pentyl furan carboxylic acid, EN300-8132959, Enamine Ltd.).

[0114] <Example 6>

[0115] 6-1. Measurement of Neurocytotoxicity / Activity

[0116] To determine the cytotoxicity / activity of a composition containing PFC and PFCA as active ingredients, experiments were conducted using the following method.

[0117] Specifically, samples were prepared by diluting PFC and PFCA stock solutions dissolved in ethanol. As a control sample, PF (2-Pentylfuran, Sigma) reagent, a similar compound with cognitive-enhancing functionality, was used. Human microglial cells (HMC-3) (ATCC) were cultured at 0.8 x 10⁴ cells / well in Dulbeco's Modified Eagle's Media, Gibco (DMEM) high glucose medium (10% FBS, 1% Penicillin / Streptomycin). After replacing the medium with fresh FBS-free media, PF, PFC, and PFCA samples were added at various concentrations (1-50 μg / mL) and cultured for 24 hours. Subsequently, OD values ​​were quantified at 450 nm using EZ-cytox (Daeil Lab, Korea) and plotted as shown in Figure 13. A culture medium without the sample (Control) or a sample containing 1% ethanol in the culture medium was used as a negative control. As shown in Figure 13, it was confirmed that PF, PFC, and PFCA samples did not affect cell activity or toxicity when treated to various cell types.

[0119] 6-2. Evaluation of the Inhibitory Efficacy of Human Microglia on Inflammation

[0120] To determine the effect on neuronal inflammation, a major factor causing human neurological diseases, the expression rate of inflammatory cytokines was measured using human microglia (HMC-3) (see Fig. 14).

[0121] HMC-3 cells were cultured at 3 x 10⁵ cells / well, replaced with fresh FBS-free media, and then 0.5 mM H₂O₂ and various concentrations (1-10 μg / mL) of PF, PFC, and PFCA samples were added and incubated for 24 hours. Real-time PCR was performed after RNA prep, and the results confirmed that the expression levels of inflammation-related cytokines—TNF-α, IL-1βIL-6, and MCP-1—which were increased in the H₂O₂-treated group, decreased with the treatment of PF, PFC, and PFCA. In particular, the PFC and PFCA-treated groups showed a greater decrease in expression levels compared to the decrease in PF expression, and the reduction was statistically significant in a concentration-dependent manner. This suggests that neuronal damage was inhibited by suppressing the expression of inflammatory cytokines through the reduction of oxidative stress via the activation of antioxidant functions within neurons.

[0123] 6-3. Evaluation of Efficacy for Memory-Related Markers

[0124] Murine hippocampal neuronal cell line HT22 (Salk Institute, La Jolla, CA, USA) was cultured in a CO2 incubator (Sanyo, Osaka, Japan) using Dulbecco's Modified Eagle Medium (DMEM; Welgene, Gyeongsan, South Korea) supplemented with 10% fetal bovine serum (FBS; Gibco™ Fisher Scientific, Waltham, MA, USA) and 1% penicillin-streptomycin. Cultured cells were trypsinized with 0.05% trypsin-ethylenediamine tetraacetic acid solution (Welgene) at approximately 70% confluence and subcultured. HT22 cells transfected with an antioxidant response factor (ARE) conjugated luciferase reporter plasmid (HT22-ARE) were used for the ARE-luciferase reporter assay.

[0125] Referring to Figure 15, when 100 μg / mL of sulforaphane (SFN) was applied to HT22 mouse hippocampal neurons responsible for memory, ARE, which initiates the transcription of a group of genes involved in cellular antioxidant / defense responses, was activated approximately 11-fold. On the other hand, the PF, PFC, and PFCA treatment groups showed a concentration-dependent increase in activity, with the PFCA treatment group showing the strongest ARE-luciferase activity compared to the other groups. Considering the oxidative stress-inducing properties of amyloid beta in the mammalian brain, it is presumed that the intake of PFC and PFCA contributed to the alleviation of cognitive impairment or improvement of memory through the restoration of redox balance by antioxidant enzymes, including hippocampal region-related proteins (e.g., Nrf2, HO-1).

[0127] <Example 7>

[0128] 7-1. Preparation of Test Substance, Control Substance, Excipient, and Inducing Substance

[0129] The test substances PFC, PFCA (Enamine Ltd), the control substance Donepezil (Sigma), the excipient Corn oil (Sigma), and the inducing substance beta-amyloid peptide (1-42) (Sigma) were used in the experiment.

[0131] 7-2. Test Systems and Rearing Environment

[0132] In this invention, 40 male mice (C57BL / 6N, Orient Bio) aged 7 weeks were used in the experiment, and the temperature (23±3 ℃), humidity (60±10%), and light-dark cycle (12 hours) in the animal room were maintained constant. The experimental animals used were handled in accordance with “for the Care and Use of Laboratory Animals [Department of Health, Education, and Welfare Publication (National Institute of Health) 85-23, 1996]”. No abnormalities were observed during the experimental period that would affect the test results. Solid feed for rodents was provided to all animals for free consumption during the experiment, and groundwater disinfected using a UV sterilizer and a microfiltration device was placed in polycarbonate drinking bottles for free intake. Mice were housed in polycarbonate rearing boxes (W 170 x L 235 x H 125 mm) at a rate of one mouse per box during the acclimatization and administration periods, and the rearing boxes, bedding, and The water bottle was changed at least once a week.

[0134] 7-3. Test Group Composition, Dose Setting, Group Separation, and Administration Setting

[0135] Animal groups were separated by first excluding abnormal animals by performing a cued test on animals determined to be healthy during the acclimatization period, and secondly excluding animals without an eye blink test on the day after administering beta-amyloid peptide (1-42). After that, the animals' body weights were measured and ranked, and then randomly distributed so that the average body weight of each group was evenly distributed.

[0136] Beta-amyloid peptide (1-42) was prepared at a concentration of 1 μg / μL by dissolving it in sterile 0.1 M phosphate-buffered physiological saline (pH 7.4), and then stored at 37°C for about 1 week before administration to induce agglutination, and administered into the third chamber of the mouse brain (intracerebroventricular injection). Specifically, mice were anesthetized by administering a mixture of zoletil and rompun (4:1, v / v) at a dose of 1 mL / kg using a stereotaxic apparatus, and the intracerebral administration coordinates were fixed at anterior / posterior (AP) -1.0 mm, mediolateral / lateral +1.0 mm, and dorsal / ventral -2.5 mm relative to the bregma, after which 5 μL of aggregated beta-amyloid peptide (1-42) was administered at a rate of 2 μL / min. After allowing all administration groups to recover for 3 days, the test substance was administered. Each substance was administered orally using a 1 mL syringe after fixing the dorsal and cervical regions of the mice.

[0137] The composition of the test groups is as follows. Normal: Normal group (corn oil, n=8), MI: Beta-amyloid administration group (n=8), Donepezil: Control substance administration group (1 mg / kg / day, n=8), PFC: Test substance administration group (1 mg / kg / day, n=8), PFCA: Test substance administration group (1 mg / kg / day, n=8).

[0138] As shown in the test schedule below, the administration was performed once a day for 4 weeks (a total of 25 times), and the dosage was calculated as 5 mL / kg based on the measured body weight.

[0139]

[0141] 7-4. Mouse Symptom Observation

[0142] During the administration and observation period, the mortality of mice, the type of general symptoms, the date of onset, and the severity of symptoms were observed once a day and recorded for each individual. The administration start date was set as Day 3, and the mice were observed for 4 weeks after the administration of the test substance. The body weight of the mice was measured at the time of entry, at the time of group separation, and once a week during the experiment.

[0143] To examine the harmfulness of the composition of the present invention, a test substance was administered according to the method described in Examples 7-2 and 7-3 above, and general symptoms of mice were observed. As a result, no mice died or showed abnormal symptoms due to the administration of the test substance.

[0145] 7-5. Passive Avoidance Test

[0146] To evaluate the learning ability improvement effects of the PFC and PFCA of the present invention (influence on conditioned memory of the hippocampus and memory related to the limbic system, and working memory ability) through passive avoidance experiments using mice prepared as in 7-2, 7-3, and 7-4 of Example 7, an avoidance learning box (Jeongdo BNP Co., Ltd., Seoul, Korea) was used.

[0147] Specifically, the passive avoidance test consists of two boxes (20×20×20 cm) made of white and black polyvinyl plastic, respectively, with the floors designed to allow electricity to flow through a grid with 2 mm spacing. A 50 W light bulb was installed in the white box to create a bright environment, and a guillotine door (5×5 cm) was installed to allow movement to the black box.

[0148] The mouse was placed in the light section, and after a 10-second search period, the guillotine door was opened to allow entry into the dark section. The time from when the guillotine door opened until the mouse entered the dark section was measured. If the mouse did not enter the dark section within 60 seconds after the guillotine door opened, the mouse was induced to enter the dark section, and the time taken to enter was recorded as 60 seconds.

[0149] When the mouse entered the dark compartment, the guillotine door was closed, and an electric stimulus of 0.5 mA was immediately applied through the grid for 3 seconds, after which the mouse was moved to the cage. 24 hours after the electric stimulus was applied, the mouse was placed back in the bright box and given a 10-second exploration time, after which the guillotine door was opened and the time it took for the mouse to enter the dark compartment (Step-through latency time) was measured up to 300 seconds.

[0151] 7-6. Statistical Analysis

[0152] Statistical analysis was performed using SPSS Statistics 12 for Medical Science, and significance with the MI control group was tested. The significance of all treatment groups was tested using Student's t-test. Statistical significance regarding the mean difference between groups was indicated as significant when the p-value < 0.05.

[0153] To confirm the working memory improvement effect of PFC and PFCA in relation to long-term memory, a passive avoidance test was conducted, yielding the results shown in Figure 16. As shown in Figure 16, there was no difference in the time spent in the dark room among all experimental groups in the Acquisition trial; however, in the Test trial, the time spent in the dark room by the Normal group (normal rats) was 290.5±6.7 seconds, while the MI group was 120.75±29.4 seconds, which was significantly reduced (p<0.001).

[0154] In the experimental groups administered beta-amyloid along with 1 mg / kg of PFC and PFCA, the times were 221±16.3 seconds and 243.5±15 seconds, respectively, while the positive control group administered donepezil showed 205±21.3 seconds, confirming that PFC and PFCA statistically significantly increased the time spent in the dark room that had been reduced by beta-amyloid. Each disease model was restored to 76% and 84% of the normal rat level, respectively, and compared to the MI disease-induced group, the test substance treatment groups (PFC (182.6%), PFCA (201.2%)) showed an improved learning ability of 13.2% and 18.6%, respectively, compared to the positive control group (169.4%).

[0156] <Example 7>

[0157] The effects of the PFC and PFCA of the present invention on improving degenerative brain diseases were evaluated through the Morris water maze test using mice prepared as in Examples 7-2 and 7-3. Specifically, this experiment was conducted to improve hippocampus-dependent spatial learning and long-term memory. A escape platform with a diameter of 9 cm and a height of 25 cm was installed in one quadrant of a circular tank with a diameter of 90 cm and a height of 45 cm. Clean water at 20 ± 2 ℃ was filled about 1 cm above the escape platform, and the time taken for the experimental animals to find the escape platform was measured.

[0158] Four signs were installed in a circular tank to divide it into four quadrants, and the entry point was varied. The experiment was repeated four times a day for 60 seconds; if the experimental animals found the escape platform within 60 seconds, the experiment was completed, and if they did not find it, their location was guided by hand to stay for 10 seconds. On the last day of the experiment, the escape platform was removed, and the time spent in the zone where the platform had been was measured for 60 seconds to assess learning ability. All experiments were recorded and measured using the Ethovision program (Noldus, Netherlands).

[0159] To confirm the working memory improvement effect of PFC and PFCA in relation to long-term spatial memory, the above Morris water maze test results were obtained as shown in Fig. 17. It was confirmed that Alzheimer's disease was induced, based on the fact that the time taken to find the escape platform was fast at 12.1±2 seconds for the Normal group (normal mice) as learning progressed, whereas the MI group injected with amyloid beta was delayed to 58.2±4.7 seconds.

[0160] In contrast, in the PFC and PFCA administration groups, the time required to find the escape band shortened as learning progressed, and the donepezil group, acting as a positive control, also showed a shortened time, confirming that PFC and PFCA reduced the time required to find the escape band caused by amyloid beta. The two compounds significantly restored the disease model to levels of 55.32% and 46.27%, respectively, and demonstrated improved learning ability effects of 4.98% and 14.03%, respectively, compared to the positive control group (60.3%).

[0161] Through this, it was confirmed that the PFC and PFCA of the present invention improve short-term, long-term, and spatial learning ability and memory in an amyloid beta-induced MI / amnesia model.

[0163] <Example 8>

[0164] 8-1. Cerebrum From life Antioxidant test

[0165] After the completion of the Morris water maze experiment (day 28), mice were euthanized by inhalation anesthesia, and their brains were extracted. The cerebral antioxidant defense system was observed by removing the cortex and measuring SOD enzyme activity. After weighing the isolated cerebral cortex, it was homogenized with a buffer solution containing 0.1 M MEDTA (pH 7.4), 10 mM sucrose, and 10 mM Tris-HCl, and centrifuged at 12,000×g for 15 minutes. The homogenate was centrifuged at 14,000 rpm for 30 minutes at 4 ℃, the supernatant was transferred to a new tube, and an antioxidant Superoxide Dismutase (SOD) activity test was performed using an antioxidant Superoxide Dismutase (SOD) assay kit.

[0166] As shown in Figure 18, SOD activity involved in the antioxidant / defense response of cerebral cells was significantly reduced in the MI-administered group, whereas the activity of the PF, PFC, and PFCA-treated groups was significantly increased, with the PFCA-treated group exhibiting the strongest cellular antioxidant activity compared to the other groups. This supports the ARE activity results in Figure 15, and considering the oxidative stress-inducing characteristics of amyloid beta in the mammalian brain, it is presumed that the intake of PFC and PFCA contributes to the alleviation of cognitive impairment or improvement of memory through the restoration of redox balance by SOD antioxidant enzymes, including proteins related to the cerebral cortex or hippocampus (e.g., Nrf2, HO-1).

[0167] Recently, as it has been verified that reactive oxygen species are associated with various types of neurodegenerative diseases, research on the in vivo generation and inhibition of reactive oxygen species is being actively conducted. Reactive oxygen species are molecules derived from oxygen that have a greater oxidizing power than oxygen, including superoxide anion, hydrogen peroxide, hydroxyl radical, singlet oxygen, and peroxy radical, and are generated from various enzymes such as the mitochondrial electron transport chain, cytochrome P450, NADPH oxidase, xanthine oxidase, and cyclooxygenase.

[0168] In particular, studies have shown that beta-amyloid, a causative agent of dementia, aggregates to generate various reactive oxygen species, while conversely, cytotoxicity caused by beta-amyloid is reduced when antioxidant components and antioxidant enzymes are artificially increased. Therefore, the results of the present invention demonstrate the potential to utilize naturally derived compounds such as PFC and PFCA for cognitive improvement through the inhibition or reduction of reactive oxygen species.

[0170] 8-2. Mouse cytokine array kit analysis

[0171] To analyze the effects of excised cerebral tissue on inflammation-related markers, an assay was performed using a mouse cytokine array kit (R&D systems, Cat #ARY006). It was used to simultaneously detect 40 mouse cytokines in excised brain samples according to the manufacturer's instructions. Using an Amersham Imager 600 (GE Healthcare Life Sciences, Pittsburgh, Pennsylvania), the intensity (pixel density) of each spot was quantified using ImageJ software, corrected for background intensity, and normalized to positive for analysis.

[0172] As shown in Figure 19, regarding the relative expression levels of each cytokine, it was confirmed that the expression levels of inflammation-related cytokines IL-1α, IL-1β, CXCL1, and MCP-1 increased in the MI-induced group, whereas they significantly decreased in the PFC and PFCA-treated groups. In particular, the PFC and PFCA-treated groups showed a higher level of reduction in expression compared to the Donepezil group. This is in accordance with Figures 14 and 15 in vitro In a context similar to the test results, this supports the inhibition of neuronal damage by suppressing inflammatory cytokine expression through the reduction of oxidative stress via the activation of antioxidant function within neurons.

[0173] In summary, considering that the results are similar to those of the study by Zhang et al., which showed improved results in passive avoidance and water maze tests when turmeric extract was administered to mice with brain damage induced by amyloid beta (1-42), and the study showing the cognitive function improvement effects of curcumin and curcuminoid, which are the main active ingredients of turmeric (Eun et al., 2017), it is judged that these results are strong evidence that, at least under the conditions of this experiment, PFC and PFCA at a dosage of 1 mg / kg show an equivalent or superior memory improvement effect compared to donepezil 1 mg / kg in a mouse model of amyloid beta-induced memory impairment.

[0175] <Example 9>

[0176] Liver tissues from mice excised in animal experiments were prepared into frozen sections and slides. To investigate fatty liver disease, the sectioned slides were fixed with 4% paraformaldehyde, stained with Oil red O solution for 15 minutes, and then washed with 60% isopropanol. The nuclei were stained with Haematoxylin, washed with distilled water, mounted, and examined under a microscope.

[0177] As shown in Figure 20, severe steatosis was observed in the liver tissue of mice injected with amyloid beta compared to normal mice, and it was confirmed that the liver tissue of PFC and PFCA showed a significant fatty liver inhibitory effect of more than 50% compared to donepezil.

[0178] The liver plays a major role in removing amyloid beta from the periphery, but it is known that if this removal function is impaired, it causes hepatic steatosis and intrahepatic inflammatory responses, progresses to NAFLD or NASH, and accumulates in the brain, leading to an increased risk of dementia.

[0179] Therefore, it can be inferred that in the present invention, PFC and PFCA inhibit NAFLD / NASH and neurodegenerative diseases by protecting hepatocytes and neurons through anti-neuroinflammation or the regulation of metabolism-related circuits. Additionally, it can be suggested that PFC and PFCA can be utilized as therapeutic agents / preventive agents for NAFLD / NASH or AD, health functional foods, etc.

[0180] A notable point in the comprehensive analysis of the embodiments of the present invention is that PFC and PFCA restored the disease model to 76% and 84% of normal mice, respectively, in the passive avoidance test, thereby restoring memory and behavioral abilities to almost the same level as normal mice, and verified that in the water maze test, the disease group with reduced cognitive ability, learning ability, and behavioral ability due to Alzheimer's dementia was restored to a high level of 55.32% and 46.27%, respectively.

[0181] Considering that research on cause-modifying therapies (DMTs) accounts for the majority of recent clinical trials for Alzheimer's dementia treatments, and given that the world is seeking methods for the fundamental treatment of Alzheimer's disease, the value of this invention, which is based on the premise of curing rather than temporarily alleviating symptoms, is considered very high. PFC and PFCA are natural exosome-derived substances that possess not only anti-aging functionality but also advanced capabilities that activate brain nerve cell functions through a mechanism of action differentiated from existing dementia treatments to suppress or cure the fundamental causes of cognitive impairment. Therefore, they are expected to be effective not only for Alzheimer's disease but also for other degenerative brain diseases, and through continuous development in the future, they are expected to develop into global innovative new drugs capable of providing patients with a wider range of treatment options.

Claims

Claim 1 The method comprises the steps of: culturing the mycelium of the *Merticulum vulgare* in a culture medium; releasing extracellular vesicles from the mycelium into the culture medium; and centrifuging the culture medium and then filtering to separate and purify the extracellular vesicles; wherein the extracellular vesicles are Pentylfuran-2-carbaldehyde (PFC, C 10 H 14 O2) or Pentylfuran-2-carboxylic acid (PFCA, C 10 H 14 A method for preparing an extracellular vesicle derived from the mycelium of *Irpex lacteus*, characterized by containing O3. Claim 2 A cosmetic composition for skin improvement containing an extracellular vesicle produced by the manufacturing method of claim 1 as an active ingredient. Claim 3 A cosmetic composition according to paragraph 2, characterized in that the composition exhibits an effect of inhibiting or improving skin aging through inhibition of reactive oxygen species (ROS) in the skin, promotion of collagen synthesis, inhibition of melanin, and improvement of wrinkles. Claim 4 A health functional food containing extracellular vesicles produced by the manufacturing method of claim 1 as an active ingredient. Claim 5 A pharmaceutical composition for improving cognitive function, enhancing memory, or preventing or treating degenerative neurological diseases, containing an extracellular vesicle prepared by the manufacturing method of claim 1 as an active ingredient. Claim 6 A pharmaceutical composition for the prevention or treatment of fatty liver, non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH), containing an extracellular vesicle prepared by the manufacturing method of claim 1 as an active ingredient. Claim 7 A pharmaceutical composition for improving cognitive function, enhancing memory, or preventing or treating degenerative neurological diseases, containing as an active ingredient PFC or PFCA isolated from extracellular vesicles prepared by the manufacturing method of claim 1. Claim 8 A pharmaceutical composition for the prevention or treatment of fatty liver, non-alcoholic fatty liver disease (NAFLD), and non-alcoholic steatohepatitis (NASH), containing as an active ingredient PFC or PFCA isolated from extracellular vesicles prepared by the manufacturing method of claim 1.

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