KR20250038281A - Composition for preventing, ameliorating or treating degenerative brain disease comprising caffeic acid as effective component - Google Patents

Abstract

The present invention relates to a composition for preventing, improving or treating degenerative brain diseases, comprising caffeic acid as an active ingredient. The caffeic acid has a neuroprotective effect as shown in behavioral analysis of memory function using an animal model; and changes in the expression levels of Aβ and BACE-1. The caffeic acid reduces oxidative stress, and reduces the expression levels of GFAP and Iba-1, which are neuroinflammatory factors in mouse hippocampal tissues. In addition, the caffeic acid has the effect of reducing the expression levels of p-NFκb, TNF-α, IL-1β and TLR4, and increases the expression levels of p-PI3K, p-AKT and BDNF. The caffeic acid also has the effect of increasing the expression levels of synaptic proteins (SNAP-25, SYN and SNAP-23 and PSD-95). Therefore, the caffeic acid can be usefully used as a medicine, health functional food or feed additive for preventing, improving or treating degenerative brain diseases.

Description

{Composition for preventing, ameliorating or treating degenerative brain disease comprising caffeic acid as effective component}

The present invention relates to a composition for preventing, improving or treating degenerative brain diseases, comprising caffeic acid as an active ingredient.

Alzheimer's disease (AD) is the most common cause of dementia, which impairs memory and cognitive function. AD is characterized by two major pathological features: the accumulation of amyloid beta (Aβ) peptides in the brain and the formation of neurofibrillary tangles (NFTs). The main enzyme responsible for Aβ production is β-secretase, also known as β-site amyloid precursor protein cleavage enzyme-1 (BACE-1), which produces toxic Aβ peptides that induce AD-related pathological changes and neurodegeneration. NFTs are formed by hyperphosphorylation of tau, a microtubule-associated protein that plays a key role in stabilizing neuronal microtubules and providing intracellular transport pathways. In a hyperphosphorylated state, tau loses its ability to bind to microtubules and cannot maintain cytoskeletal organization, resulting in misfolded proteins and NFTs. Accumulation of these two pathological proteins in the brain leads to oxidative stress, neuroinflammation, downregulation of neurotrophic factors in the brain, and synaptic dysfunction. Accumulation of Aβ in the brain induces the formation of reactive oxygen species (ROS) and lipid peroxidation, which destroys the cellular defense mechanism. The endogenous cellular antioxidant mechanism is regulated only by certain factors such as nuclear factor erythroid lineage 2-related factor 2 (Nrf2) and related genes. Nrf2 is a major signaling pathway that regulates reactive oxygen and redox signals by activating phase II detoxification enzymes. Nrf2 acts as a regulator of cellular antioxidant and detoxification defense mechanisms, and its activation can reduce cellular damage in various organs and tissues.

Increased oxidative stress associated with AD can induce transcription of specific inflammatory factors, such as NF-κB, a member of the mitogen-activated protein kinase family, and is known to play an important role in the accumulation of amyloid beta and subsequent neurodegeneration. In addition to oxidative stress, neuroinflammation can be caused by various factors, such as activated astrocytes and microglia. In particular, oxidative stress has been reported to be involved in the activation of astrocytes and microglia, and the activation of microglia and astrocytes is one of the key factors of neuroinflammation and is involved in the development of various neurodegenerative diseases, such as AD and Parkinson's disease (PD). The hippocampus, which is affected in the early stage of AD, plays a role in storing long-term and short-term memory. The role of growth factors and the PI3K/AKT signaling pathway in hippocampal plasticity and memory function has been extensively emphasized. The PI3K/AKT signaling pathway promotes cell survival, proliferation, and differentiation, which are important for normal cell activity. BDNF protein is known to be a key factor in the homeostasis of brain physiology and function, supporting neuronal survival, synaptic function, and hippocampal neurogenesis, and playing a role in improving cognitive ability.

Currently, there is no known treatment for AD, and only certain drugs can reduce the signs and symptoms associated with AD. Meanwhile, several natural compounds such as flavonoids, vitamins, phenolic acids, and polyphenols have received special attention in the management of this disease. Natural compounds have antioxidant and anti-inflammatory activities and also increase synaptic integrity, memory, and cognitive function. Phenolic compounds, which have antioxidant, anticancer, antibacterial, and anti-inflammatory properties, are a group of compounds mainly found in fruits and vegetables.

Meanwhile, as for prior art related to caffeic acid, Korean Patent Publication No. 2016-0144020 discloses a pharmaceutical composition for preventing or treating an itch-related disease containing caffeic acid, and Korean Patent Registration No. 1409111 discloses a composition for treating or preventing acne containing caffeic acid or a salt thereof. However, to date, no composition for preventing, improving, or treating a degenerative brain disease containing caffeic acid as an active ingredient has been disclosed.

The present invention was derived from the above needs, and the present invention provides a composition for preventing, improving or treating degenerative brain diseases, containing caffeic acid as an active ingredient, and has a brain neuroprotective effect through behavioral analysis of memory function using an animal model; and changes in the expression levels of Aβ and BACE-1, and has the effect of reducing oxidative stress, reducing the expression levels of GFAP and Iba-1, which are neuroinflammatory factors in mouse hippocampal tissue, as well as reducing the expression levels of p-NFκb, TNF-α, IL-1β and TLR4, and increasing the expression levels of p-PI3K, p-AKT and BDNF, and increasing the expression levels of synaptic proteins (SNAP-25, SYN and SNAP-23 and PSD-95), thereby completing the present invention.

To achieve the above purpose, the present invention provides a pharmaceutical composition for preventing or treating degenerative brain diseases, comprising caffeic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a health functional food composition for preventing or improving degenerative brain diseases, comprising caffeic acid or a food-wise acceptable salt thereof as an effective ingredient.

In addition, the present invention provides a feed additive for preventing or improving degenerative brain diseases, which contains caffeic acid or a food-wise acceptable salt thereof as an effective ingredient.

In addition, the present invention provides a veterinary composition for preventing or treating degenerative brain diseases, comprising caffeic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

In addition, the present invention provides a method for preventing or treating a degenerative brain disease, comprising a step of administering to an animal other than a human a composition containing caffeic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

The present invention relates to a composition for preventing, improving or treating a degenerative brain disease, comprising caffeic acid as an active ingredient, wherein caffeic acid has a neuroprotective effect as shown in behavioral analysis of memory function using an animal model; and changes in the expression levels of Aβ and BACE-1, and reduces oxidative stress and reduces the expression levels of GFAP and Iba-1, which are neuroinflammatory factors in mouse hippocampal tissue, as well as the expression levels of p-NFκb, TNF-α, IL-1β and TLR4, and increases the expression levels of p-PI3K, p-AKT and BDNF, and the expression levels of synaptic proteins (SNAP-25, SYN and SNAP-23 and PSD-95).

Figure 1 shows the chemical structure of caffeic acid.
Figure 2 shows the results of behavioral analysis on memory function using an animal model and the results of confirming the neuroprotective effect of caffeic acid through changes in the expression levels of Aβ and BACE-1 involved in memory function. (a) The percentage of spontaneous change behavior of experimental mice in the Y-maze; (b) The average escape latency to reach the hidden platform until the 5th day; The results of the probe test, (c) The time spent by the mouse in the quadrant where the platform was during training, (d) The number of times crossing the center, (e) The results of Western blot analysis of Aβ and BACE-1 in the hippocampus of the mouse, and (f) The results of DAPI staining (blue staining of Aβ) in the hippocampus (Cornu Ammonis (CA1) and Dentate Gyrus (DG)) of the mouse are confirmed by confocal microscopy images (magnification 10Х, scale bar = 50 μm) and their quantification graphs. Cont is the control group administered saline, CA is the group administered caffeic acid to the control group, Aβ is the group administered Aβ to induce AD, and Aβ+CA is the group administered caffeic acid and induces AD. ω indicates that there is a statistically significant difference between the measurement values of the control group and the measurement values of the Aβ group, and p<0.05, and σ indicates that there is a statistically significant difference between the measurement values of Aβ and the measurement values of the Aβ+CA group, and p<0.05.
Figure 3 shows the results of confirming the effect of caffeic acid on oxidative stress. (a, b) The results of confirming changes in ROS and LPO values in the mouse hippocampus, (c) The results showing the protein expression levels of Nrf2 and HO-1 confirmed by immunoblotting in the mouse hippocampus, and (d) The immunofluorescence image (magnification 10Х, scale bar = 50μm) confirming the expression level of Nrf2 in the mouse hippocampus [Cornu Ammonis (CA1) and Dentate Gyrus (DG)] and the graph quantifying it. ω indicates that there is a statistically significant difference between the measurement values of the control group and the measurement values of the Aβ group, with p<0.05, and σ indicates that there is a statistically significant difference between the measurement values of Aβ and the measurement values of the Aβ+CA group, with p<0.05.
Figure 4 shows the results of confirming the effect of caffeic acid of the present invention on reducing the activation of glial cells. (a) Western blot results of GFAP and Iba-1 in mouse hippocampus tissue, and (b) confocal microscopy images of GFAP confirmed in mouse hippocampus tissue and a graph quantifying the same. ω indicates that there is a statistically significant difference between the measurement values of the control group and the measurement values of the Aβ group, with p<0.05, and σ indicates that there is a statistically significant difference between the measurement values of Aβ and the measurement values of the Aβ+CA group, with p<0.05.
Figure 5 shows the results of confirming the effect of caffeic acid on inflammatory factors. (a) Western blot analysis of p-NFκb, TNF-α, and IL-1β in the mouse brain, and (b) TLR4 protein expression level confirmed by immunoblotting in the mouse hippocampus. ω indicates that there is a statistically significant difference between the measurement values of the control group and the measurement values of the Aβ group, with p<0.05, and σ indicates that there is a statistically significant difference between the measurement values of Aβ and the measurement values of the Aβ+CA group, with p<0.05.
Figure 6 is the result of Western blot confirming the effect of caffeic acid on the expression levels of p-PI3K, p-AKT, and BDNF in the hippocampus of AD mice induced by Aβ administration. ω indicates that there is a statistically significant difference between the measurement values of the control group and the measurement values of the Aβ group, with p<0.05, and σ indicates that there is a statistically significant difference between the measurement values of Aβ and the measurement values of the Aβ+CA group, with p<0.05.
Figure 7 shows the results of confirming the change in the expression level of synaptic proteins according to the administration of caffeic acid, namely (a) the Western blot results of SNAP-25, SYN, and SNAP-23 proteins and (b) the immunofluorescence analysis results of PSD-95. ω indicates that there is a statistically significant difference between the measurement values of the control group and the measurement values of the Aβ group, with p<0.05, and σ indicates that there is a statistically significant difference between the measurement values of Aβ and the measurement values of the Aβ+CA group, with p<0.05.

The present invention relates to a pharmaceutical composition for preventing or treating degenerative brain diseases, comprising caffeic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

The above caffeic acid is a compound having a structural formula disclosed in FIG. 1, and the degenerative brain disease is preferably one selected from dementia, Alzheimer's disease, and memory impairment, but is not limited thereto.

The above degenerative brain diseases are characterized by neuroinflammation or apoptotic cell death.

The above pharmaceutical composition may further contain a pharmaceutically acceptable carrier, excipient or diluent in addition to the active ingredient.

In addition to the effective ingredient of the pharmaceutical composition above, the pharmaceutical composition may further contain one or more carriers selected from pharmaceutically acceptable saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil, and in addition to the effective ingredient, the pharmaceutical composition may further contain one or more auxiliary agents selected from pharmaceutically acceptable antioxidants, buffers, bacteriostatic agents, diluents, surfactants, binders, lubricants, wetting agents, sweetening agents, flavoring agents, emulsifiers, suspending agents, and preservatives.

The above pharmaceutical composition can be administered orally or parenterally in a conventional manner, and when formulated, it is prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrating agents, and surfactants that are commonly used. Solid preparations for oral administration include tablets, pills, powders, granules, and capsules, and these solid preparations are prepared by mixing the composition with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, and gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. In addition, non-aqueous solutions and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol, macrogol, Tween 61, cacao butter, laurin butter, and glycerogelatin.

In addition, the present invention relates to a health functional food composition for preventing or improving degenerative brain diseases, comprising caffeic acid or a food-wise acceptable salt thereof as an effective ingredient.

The above health functional food composition is characterized by reducing the expression level of one or more proteins selected from among Aβ (Amyloid beta), BACE1 (beta-site amyloid precursor protein (APP) cleaving enzyme-1), GFAP (glial fibrillary acidic protein), Iba-1 (ionized calcium-binding adaptor molecule-1), p-NFκb (p-nuclear factor kappa B), TNF-α (tumor necrosis factor-alpha), IL-1β (interleukin 1 beta), and TLR4 (toll-like receptor 4) increased by a degenerative brain disease.

The above health functional food composition is characterized by increasing the expression level of one or more proteins selected from among Nrf2 (nclear factor erythroid-2-related factor 2), HO-1 (Heme oxygenase-1), p-PI3K (p-phosphatidylinositol 3-kinases), p-AKT (p-protein kinase B), BDNF (brain-derived neurotrophic factor), SNAP-25 (synaptosome associated protein of 25 kDa), SYN (synaptophysin), SNAP-23 (synaptosome associated protein of 23 kDa), and PSD-95 (postsynaptic density protein-95) reduced by a degenerative brain disease.

The above health functional food composition can be manufactured in any one formulation selected from powder, granules, pills, tablets, capsules, candy, syrup, and beverage, but is not limited thereto.

When the health functional food composition of the present invention is used as a food additive, the health functional food composition can be added as it is or used together with other foods or food ingredients, and can be used appropriately according to a conventional method. The amount of the effective ingredient can be used appropriately depending on its intended use (prevention or improvement). Generally, when manufacturing food or beverage, the health functional food composition of the present invention is added in an amount of 15 parts by weight or less, preferably 10 parts by weight or less, based on the total raw materials. However, in the case of long-term intake for health purposes, the amount can be less than the above range, and since there is no problem in terms of safety, the effective ingredient can be used in an amount greater than the above range.

There is no special limitation on the type of the above health functional food. Examples of foods to which the above health functional food composition can be added include meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea drinks, alcoholic beverages, and vitamin complexes, and include all health foods in the conventional sense.

In addition, the health functional food composition of the present invention can be manufactured as a food, particularly a functional food. The functional food of the present invention includes ingredients that are usually added during food manufacturing, and includes, for example, proteins, carbohydrates, fats, nutrients, and seasonings. For example, when manufactured as a drink, it may include natural carbohydrates or flavoring agents as additional ingredients in addition to the effective ingredient. The natural carbohydrate is preferably a monosaccharide (e.g., glucose, fructose, etc.), a disaccharide (e.g., maltose, sucrose, etc.), an oligosaccharide, a polysaccharide (e.g., dextrin, cyclodextrin, etc.), or a sugar alcohol (e.g., xylitol, sorbitol, erythritol, etc.). The flavoring agent can use a natural flavoring agent (e.g., thaumatin, stevia extract, etc.) and a synthetic flavoring agent (e.g., saccharin, aspartame, etc.).

In addition to the above health functional food composition, the composition may further contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. The ratio of the above added components is not particularly important, but is generally selected in the range of 0.01 to 0.1 parts by weight with respect to 100 parts by weight of the health functional food composition of the present invention.

In addition, the present invention relates to a feed additive for preventing or improving degenerative brain diseases, comprising caffeic acid or a food-wise acceptable salt thereof as an effective ingredient.

The feed additive of the present invention corresponds to supplementary feed under the Feed Management Act. The term 'feed' in the present invention may mean any natural or artificial diet, meal, etc., or ingredients of the meal, which are suitable for animals to eat, ingest, and digest. The type of the feed is not particularly limited, and feed commonly used in the relevant technical field may be used. Non-limiting examples of the feed include plant feeds such as grains, roots, food processing by-products, algae, fibers, pharmaceutical by-products, fats, starches, meal, or grain by-products; animal feeds such as proteins, inorganic substances, fats, minerals, fats, single-cell proteins, zooplankton, or food. These may be used alone or in combination of two or more.

In addition, the present invention relates to a veterinary composition for preventing or treating degenerative brain diseases, comprising caffeic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

The veterinary composition of the present invention may further comprise suitable excipients and diluents according to conventional methods. Excipients and diluents that may be included in the veterinary composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxy benzoate, talc, magnesium stearate, cetanol, stearyl alcohol, liquid paraffin, sorbitan monostearate, polysorbate 60, methylparaben, propylparaben, and mineral oil. The veterinary composition according to the present invention may further include fillers, anticoagulants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, etc., and the veterinary composition according to the present invention may be formulated using a method well known in the art so as to provide rapid, sustained or delayed release of the active ingredient after administration to an animal, and the formulation may be in the form of a powder, granule, tablet, capsule, suspension, emulsion, solution, syrup, aerosol, soft or hard gelatin capsule, suppository, sterile injectable solution, sterile external preparation, etc. The effective amount of the veterinary composition according to the present invention may be appropriately selected depending on the individual animal. It may be determined according to factors including the severity of the disease or condition, the sensitivity to the active ingredient of the present invention depending on the age, weight, health condition or sex of the individual, the route of administration, the period of administration, other compositions combined with or used simultaneously with the composition, and other factors well known in the physiological or veterinary fields.

In addition, the present invention relates to a method for preventing or treating a degenerative brain disease, comprising a step of administering to an animal other than a human a composition containing caffeic acid or a pharmaceutically acceptable salt thereof as an active ingredient.

Hereinafter, the present invention will be described in more detail using examples. It will be apparent to those skilled in the art that these examples are only intended to explain the present invention more specifically and that the scope of the present invention is not limited by these examples.

[Materials and Methods]

1. Compound

Caffeic acid (CAS 331-39-5), amyloid beta-peptide (Aβ), and all primary antibodies were purchased from Santa Cruz Biotechnology (Dallas, TX, USA). Information on the primary antibodies used in the examples of the present invention is disclosed in Table 1.

Table 1. List of primary antibodies

2. Intracerebral injection of amyloid-beta (Aβ) and grouping of mice

A stock solution (1 mg/mL) of human Aβ _1-42 peptide was dissolved in sterile saline and incubated at 37°C for 4 days to allow peptide aggregation. Mice were anesthetized with ketamine/xylene and placed in a stereotaxic frame. The bregma point of the skull was exposed. Aβ _1-42 (5 μL/5 min/mouse) was injected intracerebroventricularly (icv) using a Hamilton syringe (Trajan Scientific and Medical, Victoria Australia). The coordinates were adjusted to 2.4 mm dorsoventral (DV), 0.2 mm anteroposterior (AP), and 1 mm mediolateral (ML). Body temperature was maintained using a heating pad.

The mice were randomly divided into four groups (n=8/group) (Group 1: control group (normal mice administered saline); Group 2: normal mice administered caffeic acid (50 mg/kg/day) for 2 weeks); Group 3: AD-induced mice (Aβ _1-42 Group 4: AD mice induced with caffeic acid at a dose of 50 mg/kg/day for 2 weeks. The four groups of mice used in the present invention are identified as follows: Group 1: Cont; Group 2: CA; Group 3: Aβ; and Group 4: Aβ+CA. All experiments involving animals were performed in accordance with the guidelines and principles approved by the Institutional Animal Care and Use Committee (IACUC) of the Department of Applied Life Sciences, Gyeongsang National University, Republic of Korea (Approval ID: 125).

3. Behavioral Research

After drug treatment, the mice were conditioned for 2 days and then subjected to Y-maze test and Morris water maze (MWM) behavioral studies.

(1) Y-maze test

The Y-maze apparatus was made of transparent plastic and had three arms oriented at 120° angles to each other. The arms were 20 cm high, 10 cm wide, and 50 cm long. Mice were placed in the center of the maze and allowed to explore the three arms for 8 min. The movement of the mice within the maze was observed. Voluntary alternation was defined as the rodent entering an alternate arm, and the total number of arm entries was also considered.

The percentage of alternating behaviors (%) was calculated using the following formula: number of consecutive three arm entries/total number of arm entries -2 × 100. A higher percentage of spontaneous alternation behaviors was considered to indicate improved spatial working memory.

(2) Morris water maze test

The MWM test was performed in a circular tank with a diameter of 100 cm and a height of 40 cm. The tank was filled with opaque water to a depth of 15.5 cm at room temperature (23±0.5°C). A hidden platform was placed in one quadrant 1 cm below the water surface. The platform remained in the same position during the training session and was removed during the probe test. The MWM was performed as previously described. All mice were trained for 5 days, and the latency to reach the hidden platform in each trial was considered.

After training, the hidden platform was removed and a probe test was conducted. Mice were allowed to swim freely in the water tank for 1 min. The number of crossings and the time spent in the target quadrant were analyzed. Data were recorded using video tracking software (SMART V3.0 Panlab Harvard Apparatus Bioscience Company, Holliston, MA, USA).

4. Protein extraction for Western blot and biochemical analysis

After the behavioral study was completed, the mice were randomly divided into two groups: one group for Western blot and biochemical analysis and the other group for immunofluorescence study. For Western blot and biochemical analysis, the mice were anesthetized with ketamine/xylene, the brains were removed, and the hippocampus was carefully dissected. The hippocampal tissue was homogenized in PRO-PREP ^TM extraction solution (iNtRON Biotechnology, Inc., Seongnam, Korea). The supernatant was collected and centrifuged at 13,000 rpm for 25–30 min at 4°C, and then stored at -80°C for the experiment.

5. Brain collection for morphological analysis

Mice were anesthetized and perfused with 0.9% (w/v) saline and 4% (w/v) paraformaldehyde solution. The brains were carefully removed and fixed in cold 4% (w/v) neutral buffered paraformaldehyde (4°C for 72 h). After fixation, the brains were dehydrated in 20% (w/v) sucrose for 72 h. The brains were placed in optimal cutting temperature (O.C.T.) compound purchased from Finetek Japan C., Ltd., Tokyo, Japan, and then frozen. Sections of 14 μm were obtained on gelatin-coated slides using a microtome (Leica cryostat CM 3050S, Nussloch, Germany).

6. Western blot

Western blot was performed according to the conventional method. The protein concentration was measured using the Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA, USA). Equal amounts of protein were loaded onto SDS-PAGE (12-15%) gels using pre-stained protein markers (GangNam-STAIN, iNtRON Biotechnology, Dallas, TX, USA) and transferred to polyvinylidene-difluoride (PVDF) membranes. After blocking with 5% skim milk, the membranes were incubated with primary antibodies at 4°C. After incubation, the membranes were reacted with horseradish peroxidase (HRP)-conjugated secondary antibodies, and fluorescence was visualized using enhanced chemiluminescent (ECL) detection reagent (ATTO Corporation, Tokyo, Japan). The expression of bands was detected on X-ray films. ImageJ software (v. 1.50, NIH, Bethesda, MD, USA) was used for densitometry.

7. Immunofluorescence analysis

Immunofluorescence was performed according to the conventional method. The slides were washed with 1% phosphate-buffered saline (PBS) and reacted with Proteinase-K for 5 minutes. Next, a blocking solution containing 2% normal serum and 0.3% Triton X-100 in 1% PBS was incubated. The primary antibody was applied to the slides overnight at 4°C and then washed. The slides were then reacted with secondary antibodies labeled with tetramethylrhodamine isothiocyanate (TRITC) or fluorescein isothiocyanate (FITC) (Santa Cruz Biotechnology, Dallas, TX, USA). After secondary antibody treatment, the slides were reacted with DAPI (4,6-diamidino-2-phenylindole) to stain the nuclei. The slides were covered with a coverslip using a fluorescent mounting medium. Images were captured using a confocal microscope (FluoView FV 1000, Olympus, Tokyo, Japan).

8. Reactive oxygen species (ROS) analysis

Hippocampal brain homogenate was diluted to obtain a final concentration of 5 mg/mL tissue. A solution containing 1 mL of Lock buffer (pH ±7.4) and 0.2 mL of homogenate was mixed with 10 mL of 5 mM 7-dichlorodihydrofluorescein diacetate (DCFH-DA). The mixture was incubated at room temperature to form fluorescent DCF. The conversion of DCF to DCF was monitored using a microplate reader at 484 nm (excitation) and 530 nm (emission). A parallel blank was used to control the absorbance in the absence of homogenate (background fluorescence). ROS was expressed as the amount of DCF formed (pmol)/mg of protein.

9. Lipid Peroxidation (LPO) Analysis

Lipid peroxidation (LPO) assay was performed according to the instructions of the assay kit (catalog number K739-100 Biovision Incorporated, Milpitas, CA, USA). LPO was tested in the hippocampus of experimental animals. Malondialdehyde (MDA), a biomarker of LPO, was measured using thiobarbituric acid reactive substances (TBARS) to confirm the presence of LPO. TBARS content was measured spectrophotometrically (absorbance at 535 and 520 nm) using the standard substance TEP (1,1,3,3-tetraethoxypropane). Data were evaluated as relative MDA nmol/mg protein.

[Evaluation and statistical analysis]

In Western blot and immunofluorescence analyses, Image J software was used for densitometry. GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA) was used for graphing and statistical analysis.

Behavioral results were obtained from eight mice per group. For immunofluorescence analysis using Western blot and confocal microscopy, data were obtained from four mice per group and represent three experiments. Results are expressed as mean ± standard deviation and analyzed by one-way ANOVA followed by Tukey's multiple comparison test. A value of p < 0.05 was considered significant.

Example 1. According to caffeic acid administration Analysis of behavioral changes in AD mice, Aβ and Confirmation of the effect of reducing BACE-1 protein expression level

To determine the effect of caffeic acid on memory impairment associated with Alzheimer's disease (AD), Y-maze and Morris water maze (MWM) tests were performed. In the Y-maze, mice were placed in the center of the arm and allowed to explore the maze.

(1) Y-maze test

In the Y-maze test, the control group (Cont) entered the novel arm (an arm that had not been visited before in the maze) more frequently and spent more time, but the AD mice group (Aβ) injected with Aβ showed no preference for the novel arm, entered the arms randomly and spent the same amount of time in each arm. It was confirmed that the spontaneous alternation behavior rate of the caffeic acid-administered group (Aβ+CA) was enhanced compared to the AD mice group (Aβ) (Fig. 2a).

In addition, we conducted the MWM test to find a hidden platform to escape from the water. To do this, the mice were given a “spatial orientation map” in their brains. During the training period, the mice were able to find the hidden platform, and it was confirmed that the AD mice (Aβ) took longer to reach the hidden platform than the control group, and that the time was shortened when treated with caffeic acid (Fig. 2b). In the probe test, the latency to reach the platform location and the number of crossings on the hidden platform were reduced in the Aβ-injected mice compared to the control group. The AD mice administered caffeic acid increased the time in the target quadrant and the time crossing the platform (Fig. 2c and 2d).

The expression levels of Aβ and BACE-1 (beta-site amyloid precursor protein-cleaving enzyme-1) in the brains of the above mice were analyzed to confirm the effect of caffeic acid on amyloidogenic factors. Western blot results showed that the expression levels of Aβ and BACE-1 increased in the brain tissues of the AD mouse group (Aβ) compared to the control group treated with wild-type saline, and in contrast, the expression levels of Aβ and BACE-1 significantly decreased in the caffeic acid-administered group compared to the Aβ-injected mice (Fig. 2e).

In the results of immunofluorescence analysis, it was confirmed that the expression level of Aβ increased in the brain tissue (CA1 and DG) of the AD group compared to the control group, and the expression level of Aβ decreased in the group administered caffeic acid compared to the AD group (Fig. 2f).

Example 2. Confirmation of the effect of caffeic acid administration on reducing oxidative stress in the mouse hippocampus induced by Aβ

We evaluated the antioxidant activity of caffeic acid in oxidative stress induced by Aβ. First, we evaluated the level of reactive oxygen species (ROS) in the brain of mice. We confirmed that intracellular ROS increased after Aβ injection, and the increased ROS was reduced by caffeic acid administration (Fig. 3a). In addition, the results of lipid peroxidation (LPO) analysis showed that LPO increased in the brain of mice by Aβ injection, and the increased LPO was reduced by caffeic acid administration (Fig. 3b).

Nrf2, an endogenous oxidative stress regulator, is suppressed when ROS levels increase. In this Example 2, the expression levels of Nrf2 and its downstream target HO-1 were confirmed by Western blotting. The results showed that the expression levels of Nrf2 and HO-1 decreased by Aβ injection, and in contrast, when caffeic acid was administered, the expression levels of Nrf2 and HO-1 increased (Fig. 3c).

In addition, immunofluorescence analysis showed that the expression level of Nrf2 was reduced in the hippocampus of Aβ-injected mice compared to saline-treated control mice, whereas the expression level of Nrf2 was increased by caffeic acid administration (Fig. 3d).

Example 3. Confirmation of the effect of caffeic acid administration on the expression levels of GFAP and Iba-1 in the hippocampal tissue of AD mice induced by Aβ

Glial cells in the brain perform important activities in maintaining the homeostasis of neurons. When Aβ and oxidative stress accumulate in the brain, glial cells become overly activated, resulting in neuroinflammation and neurodegeneration. To analyze the effect of caffeic acid on activated glial cells induced by Aβ, we examined the expression levels of GFAP (glial fibrillary acidic protein), a marker of activated astrocytes, and Iba-1 (ionized calcium-binding adaptor molecule-1), a marker of activated microglia.

Western blot results showed that the expression levels of GFAP and Iba-1 were increased in the brains of AD mice induced by Aβ, and the expression levels of GFAP and Iba-1 were decreased in the hippocampus of mice administered caffeic acid (Fig. 4a).

In addition, immunofluorescence analysis using a confocal microscope showed that the expression level of GFAP increased in the hippocampus of AD mice, but decreased in the group administered caffeic acid (Fig. 4b).

Example 4. In the hippocampus of AD mice According to caffeic acid administration Inflammatory cytokines Check expression level change

To confirm the anti-inflammatory effect of caffeic acid, the expression levels of inflammatory cytokines, p-NFkB, TNF-α, and IL-1β, were confirmed by Western blot.

As a result, as disclosed in Fig. 5a, the expression levels of p-NFkB, TNF-α, and IL-1β increased in the brains of AD mice induced by injection of Aβ, and the expression levels of p-NFkB, TNF-α, and IL-1β decreased in the caffeic acid-administered group compared to the AD mice.

Meanwhile, the change in the expression level of TLR4 was confirmed through immunofluorescence analysis, and as a result, the expression level of TLR4 increased in the brain (CA1 and DG) of AD mice, and the expression level of TLR4β decreased in the caffeic acid-administered group compared to the AD mice (Fig. 5b).

Example 5. Confirmation of changes in the expression levels of PI3k/AKT and BDNF in the hippocampus of AD mice following caffeic acid administration

The PI3K (phosphatidylinositol 3-kinase)/AKT (portin kinase b) signaling pathway plays an important role in cell survival and proliferation. It has been reported that substances that activate the PI3K/AKT signaling pathway protect neurons from neurodegenerative diseases. BDNF (Brain-derived neurotrophic factor) is a neurotrophic factor of the CNS (central nervous system), and its activation protects neurons from Aβ-induced neurotoxicity. Considering the growth regulatory effect of caffeic acid, the expression levels of the PI3K/AKT signaling pathway and BDNF in the brains of experimental mice were analyzed by Western blot.

As a result, as disclosed in Fig. 6, the expression levels of pPI3K, p-AKT, and BDNF were significantly reduced in the hippocampus of AD mice compared to saline-treated mice (control group), and the expression levels of pPI3K, p-AKT, and BDNF increased in the caffeic acid-administered group compared to the AD mouse group (Fig. 6).

Example 6. According to caffeic acid administration Improved synaptic plasticity in the hippocampus of AD mice

Aβ is known to interfere with synaptic function, causing cognitive decline and memory impairment. To confirm the effect of caffeic acid on synaptic dysfunction, the expression levels of presynaptic factors, SNAP-25 (synaptosomal-associated protein 25) and SYN (synaptophysin), and the expression levels of postsynaptic factors, SNAP-23 and PSD-95 (postsynaptic density protein-95) proteins were confirmed.

The expression levels of SNAP-25, SYN, and SNAP-23 were analyzed by Western blot, and the protein expression level of PSD-95 was analyzed by immunofluorescence analysis.

As a result, as disclosed in Fig. 7a, the expression levels of SNAP-25, SYN and SNAP-23 in the AD-induced group were statistically significantly decreased compared to the control group, and the expression levels of SNAP-25, SYN and SNAP-23 in the caffeic acid-administered group were statistically significantly increased compared to the AD-induced group. As disclosed in Fig. 7b, the expression level of PSD-95 in the AD-induced group was statistically significantly decreased compared to the control group in the hippocampal tissue (CA1 and DG regions), and the expression level of PSD-95 in the caffeic acid-administered group was statistically significantly increased compared to the AD-induced group.

Claims (12)

A pharmaceutical composition for preventing or treating degenerative brain diseases, comprising caffeic acid or a pharmaceutically acceptable salt thereof as an active ingredient. A pharmaceutical composition for preventing or treating a degenerative brain disease, characterized in that in claim 1, the degenerative brain disease is any one selected from dementia, Alzheimer's disease, and memory impairment. A pharmaceutical composition for preventing or treating a degenerative brain disease, characterized in that in claim 1, the degenerative brain disease is caused by neuroinflammation or apoptotic cell death. A pharmaceutical composition for preventing or treating degenerative brain disease, characterized in that in claim 1, in addition to the effective ingredient, it further comprises a pharmaceutically acceptable carrier, excipient or diluent. A pharmaceutical composition for preventing or treating degenerative brain diseases, characterized in that in claim 1, the composition is manufactured in any one formulation selected from capsules, powders, granules, tablets, suspensions, emulsions, syrups, and aerosols. A health functional food composition for preventing or improving degenerative brain diseases, comprising caffeic acid or a food-wise acceptable salt thereof as an active ingredient. A health functional food composition for preventing or improving degenerative brain disease, characterized in that in claim 6, the composition is manufactured in any one formulation selected from powder, granules, pills, tablets, capsules, candy, syrup, and beverage. In claim 6, the composition is a health functional food composition for preventing or improving a degenerative brain disease, characterized in that it reduces the expression level of one or more proteins selected from among Aβ (Amyloid beta), BACE1 (beta-site amyloid precursor protein (APP) cleaving enzyme-1), GFAP (glial fibrillary acidic protein), Iba-1 (ionized calcium-binding adaptor molecule-1), p-NFκb (p-nuclear factor kappa B), TNF-α (tumor necrosis factor-alpha), IL-1β (interleukin 1 beta), and TLR4 (toll-like receptor 4) increased by the degenerative brain disease. In claim 6, the composition is a health functional food composition for preventing or improving a degenerative brain disease, characterized in that it increases the expression level of one or more proteins selected from among Nrf2 (nclear factor erythroid-2-related factor 2), HO-1 (Heme oxygenase-1), p-PI3K (p-phosphatidylinositol 3-kinases), p-AKT (p-protein kinase B), BDNF (brain-derived neurotrophic factor), SNAP-25 (synaptosome associated protein of 25 kDa), SYN (synaptophysin), SNAP-23 (synaptosome associated protein of 23 kDa), and PSD-95 (postsynaptic density protein-95) reduced by a degenerative brain disease. A feed additive for preventing or improving degenerative brain diseases, containing caffeic acid or a food-based salt thereof as an active ingredient. A veterinary composition for the prevention or treatment of degenerative brain diseases, comprising caffeic acid or a pharmaceutically acceptable salt thereof as an active ingredient. A method for preventing or treating a degenerative brain disease, comprising the step of administering to an animal other than a human a composition containing caffeic acid or a pharmaceutically acceptable salt thereof as an active ingredient.