WO2021242044A1 - Composition comprising hibiscus, rosemary, and grape seed extract as active ingredient for preventing or treating degenerative brain disease - Google Patents

Abstract

The present invention relates to a composition comprising hibiscus, rosemary, and grape seed extracts as active ingredients for preventing or treating a degenerative brain disease and, more specifically, to: a pharmaceutical composition comprising hibiscus, rosemary, and grape seed extracts as active ingredients for preventing or treating a degenerative brain disease; a method for treating degenerative brain disease by using the pharmaceutical composition; a health functional food composition comprising the extracts as active ingredients for preventing or improving a degenerative brain disease; a health functional food composition comprising the extracts as active ingredients for improving memory or cognitive functions; and a feed composition comprising the extracts as active ingredients for preventing or alleviating a degenerative brain disease. The composition comprising hibiscus, rosemary, and grape seed extracts as active ingredients of the present invention has the effect of preventing, treating, or alleviating a degenerative brain disease and improving memory or cognitive functions and as such, can be utilized for developing a composition for therapy of a degenerative brain disease.

Description

Composition for preventing or treating degenerative brain disease comprising hibiscus, rosemary, and grape seed extract as an active ingredient

The present invention relates to a composition for preventing or treating degenerative brain disease comprising hibiscus, rosemary, and grape seed extract as an active ingredient, and specifically, to prevent or treat degenerative brain disease comprising hibiscus, rosemary, and grape seed extract as an active ingredient. therapeutic pharmaceutical compositions; a method for preventing or treating degenerative brain disease using the pharmaceutical composition; Health functional food composition for preventing or improving degenerative brain disease comprising the extract as an active ingredient; A health functional food composition for improving memory or cognitive function comprising the extract as an active ingredient; And it relates to a feed composition for preventing or improving degenerative brain disease comprising the extract as an active ingredient.

As we enter a rapidly modernized and aging society, degenerative brain disease, a geriatric disease, is increasing, and among them, the incidence of senile dementia is increasing. In the case of Korea, the rate of aging among OECD countries for the past 40 years has been ranked first, and the number of dementia patients is also increasing. Due to the increase in dementia patients, the socio-economic cost of dementia treatment has already reached 10 trillion won in 2012, and it is predicted that the cost will be 13.46 trillion won by 2050, and the size of the dementia treatment market is also increasing. am.

Dementia is a type of degenerative brain disease, characterized by abnormal protein aggregation in a specific part of the brain, and symptoms such as cognitive impairment, memory loss, and loss of motor ability due to the death of nerve cells. Dementia is classified according to the cause, and among them, Alzheimer's dementia has the highest incidence. Alzheimer's dementia may be caused by aggregation of amyloid-β peptide aggregates and Tau protein. In the amyloid beta peptide aggregate, a lot of peptides consisting of 40 or 42 amino acids, which are cleaved by β-secretase and γ-secretase due to genetic and environmental factors, are formed in the amyloid precursor protein. Structurally, it has a β-sheet secondary structure, so aggregation between peptides occurs and a huge aggregate called plaque is formed. Tau protein is a protein involved in structural stabilization of microtubules present in the axon of nerve cells, and when excessive phosphorylation occurs, tangling occurs. Tau protein aggregates not only damage the structure of neurons, but also induce cytotoxicity, leading to neuronal cell death.

When Aβ peptide and tau protein aggregates are produced in brain tissue, a large amount of reactive oxygen species is generated and an inflammatory reaction occurs by microglia. IL-1, TNF-α, PGE2, NO, NOO-, H ₂ O ₂ and the like are secreted from microglia to promote the death of surrounding neurons. Reactive oxygen species are generated by oxidative stress and cause apoptosis by releasing cytochrome C and activation of caspase-3 in mitochondria. In addition, the aggregation of Aβ peptide is increased, and the aggregation of Tau protein, which stabilizes microtubules present in neurons, is also induced. When the microtubules collapse, the axons and dendrites of the nerve cells atrophy and the degeneration of the cranial nerve cells occurs, and the tau protein aggregates and accumulates in the cells, causing disturbances in nerve signal transmission such as material transport in the nerve cells and causing apoptosis. do.

As such, the representative symptom of degenerative brain disease appears as an increase in protein aggregates in brain tissue. Due to protein aggregation, an inflammatory immune response occurs in brain tissue and spreads to surrounding tissues. In addition, the function of organelles within cells is broken and the apoptosis reaction proceeds, leading to degeneration of the brain tissue.

Alzheimer's disease, which accounts for about half of geriatric dementia, currently does not have an effective treatment for the cause, nor is there a preventive method. There are five types of drugs approved by the U.S. Food and Drug Administration (FDA) and prescribed for Alzheimer's disease patients, including acetylcholine esterase inhibitors and glutamic acid receptor inhibitors, but all of them only relieve symptoms.

For degenerative brain disease, long-term use of the drug is required, so it is essential to develop a substance that is not toxic and has good efficacy. Therefore, side effects can be minimized by developing substances that can be treated and prevented from natural products used for food.

On the other hand, Korean Patent Publication No. 10-1424547 and Korean Patent Publication No. 10-2015-0047687 disclose a composition for the treatment of degenerative brain diseases including lactic acid bacteria or a product fermented by lactic acid bacteria, but these are degenerative diseases such as dementia. There was a limit to its commercial application because the therapeutic effect of brain diseases was not high.

Under this background, the present inventors have continued their earnest efforts to develop a preventive or therapeutic agent for degenerative brain disease, and as a result, the hibiscus, rosemary, and grape seed complex extracts are excellent in preventing and treating degenerative brain diseases. was completed.

One object of the present invention is to provide a pharmaceutical composition for preventing or treating degenerative brain disease comprising hibiscus, rosemary, and grape seed extract as an active ingredient.

Another object of the present invention is to provide a method for preventing or treating degenerative brain disease comprising administering the pharmaceutical composition to a subject other than a human.

Another object of the present invention is to provide a health functional food composition for preventing or improving degenerative brain disease comprising hibiscus, rosemary, and grape seed extract as an active ingredient.

Another object of the present invention is to provide a health functional food composition for improving memory or cognitive function comprising hibiscus, rosemary, and grape seed extract as an active ingredient.

Another object of the present invention is to provide a feed composition for preventing or improving degenerative brain disease comprising hibiscus, rosemary, and grape seed extract as an active ingredient.

The composition comprising the hibiscus, rosemary, and grape seed extract of the present invention as an active ingredient has the effect of preventing, treating or improving degenerative brain disease, and has the effect of improving memory or cognitive function, and is used to develop a composition for the treatment of degenerative brain disease can be

1 is a schematic diagram showing a method for producing an extract.

Figure 2 is a graph showing the cell viability by the extract.

3 is a figure and graph showing the presence or absence of genotoxicity of cells by the extract.

4 is a graph showing the antioxidant efficacy according to the concentration of the extract.

5 is a graph showing the effect of inhibiting intracellular nitrogen production according to the concentration of the extract.

6A is a schematic diagram showing a Y-shaped maze test procedure.

6B is a graph evaluating the short-term memory of mice by group through the Y-shaped maze test.

7 is a graph measuring spatial memory in Alzheimer's-induced mice through the Morris water maze test.

8A and 8B show the measurement of the time spent in the place where the platform for each group was through the Morris underwater maze test.

8C is a graph measuring the total movement distance of mice for each group through the Morris underwater maze test.

9 is a graph showing the synergistic effect on memory improvement of the composite extract by measuring the short-term memory of each group of mice through the Y-shaped maze-type test.

10 is a graph showing the effect on memory improvement by concentration of the complex extract.

11A is a photograph and graph showing the degree of deposition of beta-amyloid aggregates in the hippocampus of a mouse model.

11B is a photograph and graph showing the degree of deposition of beta-amyloid aggregates in the cerebral cortex of a mouse model.

12A is a photograph and graph showing the degree of gliosis in the hippocampal region of a mouse model.

12B is a photograph and graph showing the degree of gliosis in the cerebral cortex of a mouse model.

13A is a graph showing the amount of soluble beta-amyloid protein in mouse brain tissue.

13B is a graph showing the amount of insoluble amyloid protein in mouse brain tissue.

This will be described in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be considered that the scope of the present invention is limited by the specific descriptions described below.

One embodiment of the present invention for achieving the above object provides a pharmaceutical composition for preventing or treating degenerative brain disease comprising hibiscus, rosemary, and grape seed extract as an active ingredient.

Another aspect of the present invention provides a pharmaceutical composition for improving memory or cognitive function comprising hibiscus, rosemary, and grape seed extract as an active ingredient.

In addition, the present invention provides the use of extracts of hibiscus, rosemary, and grape seed for treating degenerative brain diseases.

Specifically, the extract may have a ratio of hibiscus, rosemary, and grape seed extract of 2:1:1, but is not limited thereto.

As used herein, the term "Hibiscus (Hibiscus sabdariffa L.)" is a plant belonging to the mallow family and mainly grows in tropical and subtropical regions, and its thick and red cup-shaped calyx is consumed as tea as well as cold drinks. High blood pressure, fever, liver disease, It is effective in inflammation, gallstones and obesity. Hibiscus extract has been reported to reduce total lipids, cholesterol, and triglycerides, and is known to have excellent anti-hyperlipidemia, antioxidant and anti-cancer effects.

The term of the present invention, "Rosmarinus officinalis" is native to the Mediterranean, but is now grown in California, Russia, the Middle East, England, France, Spain, Portugal, Yugoslavia, Morocco, etc. Rosemary is a leafy shrub that grows up to 2 m tall. The leaves are gray-green, needle-shaped, and the flowers are pale blue. It is a plant with a strong aroma.

As used herein, the term “grape seed” refers to grape seed, and proanthocyanidin in grape seed has antiviral, antibacterial, anti-inflammatory, and anti-allergic properties. Grape seed extract can be effective for about 3 days in the human body, and it is known that the effect is 20 times that of vitamin C and 50 times that of vitamin E. In addition, literature on excellent preventive and therapeutic effects for rheumatoid arthritis and osteoarthritis has been reported (Korean Patent No. 10-1044433, Korean Patent No. 10-1099021).

As used herein, the term “extract” refers to a substance obtained by extracting a certain substance, specifically an extract obtained by extraction treatment, a diluted or concentrated solution of the extract, a dried product obtained by drying the extract, and the extract It includes extracts of all formulations that can be formed using the extract itself and the extract, such as a crude product, a purified product, or a mixture thereof.

The extraction method is not particularly limited, and extraction may be performed according to a method commonly used in the art. Non-limiting examples of the extraction method include a solvent extraction method, an ultrasonic extraction method, a filtration method, a reflux extraction method, and the like, and these may be performed alone or in combination of two or more methods.

The type of the extraction solvent used for extraction in the present invention is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the extraction solvent include water, an alcohol having 1 to 4 carbon atoms, or a mixed solvent thereof, and these may be used alone or in combination of one or more. Specifically, the extraction solvent may be hot water.

As used herein, the term “complex extract” refers to an extract in which two or more extracts are combined into one.

In the present invention, the complex extract may be a combination of two or more extracts selected from the group consisting of hibiscus, rosemary, and grape seeds, or may be extracted by mixing two or more substances selected from the group, However, the present invention is not limited thereto.

The prevention, improvement, or therapeutic efficacy of the hibiscus, rosemary, and grape seed complex extracts for the prevention, improvement, or treatment of degenerative brain diseases was not known, and was first identified by the present inventors.

As used herein, the term "neurodegenerative disorders" is a disease that causes various symptoms while degenerative changes appear in nerve cells of the central nervous system. Representative degenerative brain diseases include Alzheimer's disease, Parkinson's disease (Parkinson's disease), Progressive supranuclear palsy, Multiple system strophy, Olivopontocerebellar atrophy (OPCA), Shy-Drager syndrome, striatum -Striatonigral degeneration, Huntington's disease, amyotrophic lateral sclerosis (ALS), essential tremor, cortico-basal ganlionic degeneration, diffuse Diffuse Lewy body disease, Parkinson-ALS-dementia complex of Guam, Pick's disease, and the like.

Specifically, the degenerative brain disease of the present invention may be due to aggregation of soluble or insoluble betamyloid (Aβ) protein, and specifically, dementia, Alzheimer's disease, Huntington's disease, Parkinson's disease, multiple systemic dystrophy (Multiple system) atrophy), multiple sclerosis, tauopathies, brain tumor, Pick's disease, or Creutzfeldt-Jakob disease may be selected from the group consisting of.

As used herein, the term “Alzheimer's disease” is a degenerative brain disease, which is a representative disease that mainly causes dementia in the elderly. The disease is characterized by the accumulation of beta-amyloid (Aβ) and the formation of paired helical filaments (PHF) composed mostly of hyperphosphorylated tau protein. It is known that nerve cells are killed by extracellular beta-amyloid deposition throughout the brain and intracellular hyperphosphorylated tau protein, which is a disease that causes progressive memory impairment, cognitive deficits, and personality changes. Alzheimer's disease of the present invention is meant to include Alzheimer's dementia.

In one embodiment of the present invention, the cytotoxicity of hibiscus, rosemary, and grape seed extracts was confirmed ( FIGS. 2 and 3 ), and their antioxidant efficacy ( FIG. 4 ) and NO inhibitory activity were confirmed ( FIG. 5 ), Alzheimer's-induced mice Using the model, the memory improvement effect of the extract was confirmed through the y-shaped maze test and the Morris water test ( FIGS. 6 to 9 ), and when the combined extract of the present invention was administered compared to the single extract, It was confirmed that the synergistic effect of improving memory loss due to the extract combination appeared in a concentration-dependent manner (Fig. 10), and by analyzing the level of beta-amyloid aggregation and reactive gliosis of astrocytes through immunohistochemical analysis and ELISA analysis (Fig. 11 to 13), although the extract of the present invention is harmless due to no cytotoxicity, the effect of improving memory by reducing beta-amyloid aggregation was confirmed.

Through the examples of the present application, it was confirmed that the memory ability was restored to a level almost similar to that of the normal control group when the complex extract was administered. confirmed that there is.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent, which may include a non-naturally occurring carrier.

More specifically, carriers, excipients and diluents that may be included in the pharmaceutical composition 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, propylhydroxybenzoate, talc, magnesium stearate, polycaprolactone, polylactic acid (Poly Lactic Acid), poly-L-lactic acid (poly-L-lactic acid), mineral oil, and the like.

The pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., external preparations, suppositories and sterile injection solutions according to conventional methods, respectively. The carrier may include various amorphous carriers, microspheres, nanofibers, and the like.

In the case of formulation, it can be prepared using a diluent or excipient such as a filler, extender, binder, wetting agent, disintegrant, surfactant, etc. commonly used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations include at least one excipient in the extract and its fractions, for example, starch, calcium carbonate, It may be prepared by mixing sucrose or lactose, gelatin, or the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.

Liquid formulations for oral administration include suspensions, solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, and preservatives may be included. have.

Formulations for parenteral administration may include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like. Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

The content of the hibiscus, rosemary, and grape seed extract contained in the pharmaceutical composition of the present invention is not particularly limited.

As used herein, the term “prevention” refers to any act of inhibiting or delaying the onset of a disease by the pharmaceutical composition according to the present invention.

As used herein, the term “treatment” refers to any action in which the symptoms of the disease-causing individual are improved or beneficially changed by the pharmaceutical composition.

As used herein, the term “cognitive function” refers to a series of activities and techniques, internal control processes, etc. occurring in the brain to receive and process external information.

As used herein, the term "improvement" refers to suppressing or inhibiting the symptoms of a disease or alleviating the symptoms of an already onset disease using the extract of the present invention.

The pharmaceutical composition of the present invention may be a composition for inhibiting aggregation of water-soluble or insoluble beta-amyloid (Aβ) protein.

As used herein, the term "beta-amyloid" may also be referred to as amyloid beta, which is a major component of amyloid plaques found in the brain of Alzheimer's patients, and refers to a peptide of 36 to 43 amino acids that is critically involved in Alzheimer's disease.

As used herein, the term “aggregation” refers to phase separation from a solution phase to a non-dispersible form due to the attraction of colloidal particles dispersed in the solution.

Another embodiment of the present invention for achieving the above object provides a method for preventing or treating degenerative brain disease, comprising administering a composition comprising hibiscus, rosemary, and grape seed extract as an active ingredient to an individual other than a human. .

In this case, the descriptions of “hibiscus”, “rosemary”, “grape seed”, “degenerative brain disease”, “prevention” and “treatment” are the same as described above.

As used herein, the term "administration" means introducing the composition of the present invention to a patient by any suitable method, and the administration route of the composition may be administered through any general route as long as it can reach the target tissue. . Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, may be administered intranasally, but is not limited thereto.

The composition of the present invention may be administered daily or intermittently, and the number of administrations per day may be administered once or divided into 2-3 times. It can also be used alone or in combination with other drug treatments. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, and can be easily determined by those skilled in the art.

As used herein, the term "individual" refers to all animals, such as rats, mice, and livestock, that have or can develop degenerative brain disease.

Another embodiment of the present invention for achieving the above object provides a health functional food composition for preventing or improving degenerative brain disease comprising hibiscus, rosemary, and grape seed extract as an active ingredient.

Another embodiment of the present invention for achieving the above object provides a health functional food composition for improving memory or cognitive function comprising hibiscus, rosemary, and grape seed extract as an active ingredient.

In this case, the descriptions of “hibiscus”, “rosemary”, “grape seed”, “degenerative brain disease”, “prevention”, “improvement”, and “cognitive function” are the same as described above.

Since the hibiscus, rosemary, and grape seed extracts according to the present invention exhibit excellent memory improvement and cognitive function improvement effects, they may be included in a food composition for the purpose of preventing or improving degenerative brain disease, and the food composition is not recommended for daily consumption. Because it is possible, a high effect on the prevention or improvement of degenerative brain disease can be expected.

As used herein, the term “health functional food” refers to food manufactured and processed using raw materials or ingredients useful for the human body in accordance with Act No. 6727 of the Health Functional Food Act, and 'functionality' refers to the structure of the human body. And it means to obtain a useful effect for health use, such as regulating nutrients for function or physiological action. On the other hand, health food means food that has an active health maintenance or promotion effect compared to general food, and health supplement means food for the purpose of health supplementation. In some cases, the terms health functional food, health food and health supplement food can be mixed.

The hibiscus, rosemary, and grape seed extracts of the present invention may be added as they are or used together with other foods or food ingredients, and may be appropriately used according to a conventional method.

The food of the present invention can be prepared by a method commonly used in the art, and at the time of manufacture, it can be prepared by adding raw materials and components commonly added in the art. Specifically, the food composition may further include a physiologically acceptable carrier, the type of carrier is not particularly limited and any carrier commonly used in the art may be used. In addition, the food composition contains food additives such as preservatives, disinfectants, antioxidants, colorants, coloring agents, bleaching agents, seasonings, sweeteners, flavoring agents, expanding agents, strengthening agents, emulsifying agents, thickeners, filming agents, gum base agents, foam inhibitors, solvents, and improving agents. may include The additive may be selected according to the type of food and used in an appropriate amount.

In addition, the formulation of the food may be prepared without limitation as long as it is a formulation recognized as a food. The composition for food of the present invention can be prepared in various forms, and unlike general drugs, it has the advantage of not having side effects that may occur during long-term administration of drugs using food as a raw material, and is excellent in portability, so the present invention Foods of can be consumed as a supplement to promote liver regeneration.

The hibiscus, rosemary, and grape seed extracts of the present invention may be included in various weight % in the food composition if they can exhibit efficacy for preventing or improving degenerative brain disease or improving memory/cognitive function. Specifically, it may be included in an amount of 0.00001 to 100% by weight or 0.01 to 80% by weight relative to the total weight of the food composition, but is not limited thereto. In the case of long-term ingestion for health and hygiene purposes, it may contain an amount below the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount above the above range.

Another embodiment of the present invention for achieving the above object provides a feed composition for preventing or improving degenerative brain disease comprising hibiscus, rosemary, and grape seed extract as an active ingredient.

In this case, the descriptions of “hibiscus”, “rosemary”, “grape seed”, “degenerative brain disease”, “prevention” and “improvement” are the same as described above.

As used herein, the term "feed" means any natural or artificial diet, meal, etc., or a component of said meal, intended for or suitable for being eaten, consumed, and digested by an animal.

The type of feed is not particularly limited, and feed commonly used in the art may be used. Non-limiting examples of the feed include plant feeds such as grains, root fruits, food processing by-products, algae, fibers, pharmaceutical by-products, oils and fats, starches, gourds or grain by-products; and animal feeds such as proteins, inorganic materials, oils and fats, minerals, oils and fats, single cell proteins, zooplankton, or food. These may be used alone or in mixture of two or more.

Hereinafter, the present invention will be described in more detail by way of Examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1: Preparation of extract

To prepare hibiscus, rosemary, and grape seed extracts, each plant was extracted with 80% ethanol (Ethyl alcohol) at 37° C. for 24 hours, and extracted three times. Hibiscus, rosemary, and grape seed extract were mixed in a ratio of 2:1:1 to prepare a complex extract (FIG. 1).

Example 2: Confirmation of toxicity of extract

Cellular and genotoxicity of the single and complex extracts prepared in Example 1 were confirmed.

Specifically, the RAW264.7 cell line and the BV-2 cell line were treated with a single extract of hibiscus, rosemary, or grape seed and a complex extract of the present invention at a concentration of 0 to 1000 ppm, and toxicity was measured through an MTT experiment.

As a result, as can be seen in FIG. 2 , it was confirmed that the single extract of rosemary exhibited rapid cytotoxicity at a concentration of 250 ppm, whereas the complex extract did not reduce the cell viability even at a high concentration of 1000 ppm.

In addition, a Comet assay was performed to investigate the effect of the complex extract on the genotoxicity. At this time, 500 μM of etoposide was used as a positive control.

As a result, as shown in FIG. 3 , when the positive control was treated, it was possible to observe the fluorescent tails around the cells showing genotoxicity, but in the case of the complex extract, it was confirmed that the fluorescent tails showing the genotoxicity did not appear even at a concentration of 1000 ppm. did

Through this, it was confirmed that the complex extract of the present invention does not have cytotoxicity and genotoxicity.

Example 3: Antioxidant Efficacy of Complex Extracts

In order to confirm the antioxidant efficacy of the complex extract of the present invention, an in vitro DPPH assay was performed.

More specifically, the complex extract prepared in Example 1 was prepared for each concentration, and then reacted with a DPPH (2,2-diphenyl-1-picrylhydrazyl) reagent. DPPH is a purple-colored compound that turns yellow through reduction when reacted with a substance with antioxidant activity. In this way, antioxidant efficacy can be measured. At this time, ascorbic acid was used as a positive control.

As a result, as can be seen in FIG. 4 , a significant antioxidant effect was confirmed in the complex extract of the present invention.

Example 4: Measurement of NO inhibitory activity of the complex extract

When an inflammatory reaction occurs in the body, the expression of iNOS increases by the NF-ĸB signaling process, and the production of NO (nitric oxide), a product thereof, increases. The amount of NO generated by the inflammatory reaction can be measured through Griess assay, and the complex extract of the present invention prepared above was treated in RAW264.7 cell line to measure the NO production inhibitory activity of the extract. As a result of conducting an experiment to measure NO production inhibitory activity, it can be seen that NO production is inhibited in the complex extract of the present invention in a concentration-dependent manner ( FIG. 5 ).

Example 5: Memory improvement effect using Alzheimer's induction mouse model

Example 5-1: Y-shaped maze test

The memory improvement efficacy of hibiscus, rosemary, and grape seed extracts was confirmed through a Y-shaped maze test in an Alzheimer's-induced mouse model.

More specifically, through oral administration to 7-week-old 5XFAD Alzheimer's-induced mice for 14 weeks, the complex extract of the present invention was ingested at a concentration of 200 mg/kg (FIG. 6A). After administration, the mouse's short-term memory was tested through a Y-shaped maze test.

As a result, as can be seen in FIG. 6 , the memory ability (Y-maze alteration = 51.88%) of the Alzheimer's-induced mouse fell compared to the normal control memory ability (Y-maze alteration = 63.01%), whereas the complex extract of the present invention In the group administered with Alzheimer's, it was confirmed that the decrease in memory due to induction was improved (Y-maze alteration = 61.28%) (FIG. 6B).

Through this, it was confirmed that the complex extract of the present invention has the effect of improving memory loss due to Alzheimer's.

Example 5-2: Underwater Maze Test

The memory improvement effect of the complex extract of the present invention was confirmed through an underwater maze experiment in an Alzheimer's induced mouse model.

More specifically, through oral administration to 7-week-old 5XFAD Alzheimer's-induced mice for 14 weeks, the complex extract of the present invention was ingested at a concentration of 200 mg/kg (FIG. 6A). After the administration, an experiment was conducted to see if Alzheimer's-induced mice remember the platform through the Morris water maze experiment, and the learning ability to remember the platform position was measured with 5 days of training.

As a result, as can be seen in FIG. 7 , it can be seen that the learning ability of the Alzheimer's dementia-induced mouse (red) is the lowest, and it is confirmed that the learning ability of the dementia-induced mouse (blue) administered with the complex extract of the present invention is greatly improved did

In addition, as can be seen in Figure 8, after removing the platform on the 6th day, when the frequency of mice staying in the section where the platform was located was measured, in the Alzheimer-induced mouse group administered with the extract of the present invention, the platform was located in the section It was confirmed that the frequency increased compared to the control group (FIGS. 8A, B). On the other hand, it was confirmed that the total distance moved by mouse group was similar (FIG. 8C).

Through this, it was confirmed that administration of the complex extract of the present invention relieved Alzheimer's dementia symptoms and increased memory without affecting mobility.

Example 6: Verification of synergistic effect on memory improvement of complex extracts using Alzheimer's induction mouse model

In order to confirm the synergistic effect of the complex extract of the present invention on the improvement of memory, a Y-shaped maze test was performed on an Alzheimer's induced mouse model.

The specific experimental method is the same as in Example 5-1.

As a result, as can be seen in FIG. 9 , the memory ability (Y-maze alteration = 51.88%) of the Alzheimer's-induced mice was lower than that of the normal control group (Y-maze alteration = 63.01%). As a result of administering each single extract and their complex extracts, the hibiscus single extract (Y-maze alteration = 57.76%) group showed significant memory improvement, and the complex extract administration group improved the memory ability level of the normal control group. It was confirmed that (Y-maze alteration = 61.28%) (FIG. 9).

The concentration of each extract in the complex extract is 100 mg/kg of hibiscus extract, 50 mg/kg of grape seed extract, and 50 mg/kg of rosemary extract, although the concentration is lower than that of the single extract (200 mg/kg each). and showed significant improvement in memory ability. Moreover, in the case of rosemary or grape seed extract alone, although the efficacy in improving memory was rather reduced, it was confirmed that there was a synergistic effect when they were combined.

Example 7: Memory improvement effect by concentration of complex extract using Alzheimer's induction mouse model

In order to find out the appropriate concentration of the complex extract having an effect on memory improvement, the complex extract was orally administered at concentrations of 100, 200, and 400 mg/kg for 14 weeks to 7-week-old 5XFAD Alzheimer's-induced mice. After the administration, the short-term memory of mice was tested through a Y-shaped maze test.

As a result, as can be seen in FIG. 10 , it was found that as the concentration of the complex extract increased, the memory was improved to a normal level.

Example 8: Brain tissue analysis of Alzheimer's-induced mice

Example 8-1: Immunohistochemical analysis

The degree of beta-amyloid aggregation and reactive gliosis (Gliosis) of the complex extract of the present invention were analyzed through immunohistochemistry experiments in the brain tissue of Alzheimer's-induced mice.

More specifically, the brain tissue was observed using the 6E10 antibody as an immunohistochemical test method, and when beta-amyloid aggregation decreased, the level of reactive gliosis was measured because the inflammatory activity of surrounding astrocytes decreased.

As a result, as can be seen in Figure 11, compared to the Alzheimer's-induced mouse group not administered with the complex extract of the present invention, beta-amyloid aggregation in the hippocampus and cerebral surface regions of the Alzheimer's-induced mouse group administered with it was about 1/3, respectively. And it was confirmed that about 1/4 decrease (FIGS. 11A, B).

In addition, if the beta-amyloid aggregates were reduced, the activity due to the inflammatory reaction of the surrounding astrocytes would have decreased, so the level of reactive gliosis of the surrounding astrocytes was analyzed.

More specifically, the brains of Alzheimer's-induced mice were excised and sliced, and then analyzed using an antibody against GFAP, an astrocyte-specific protein (FIGS. 12A, B).

As a result, as shown in FIG. 12 , it was confirmed that, as a result of administering the complex extract of the present invention, the antibody response indicating the reactive gliosis level of the surrounding astrocytes was significantly reduced.

Example 8-2: ELISA assay

In order to confirm the change in water-soluble and insoluble beta-amyloid protein, it was analyzed through ELISA experiment.

As a result, as can be seen in FIG. 13 , it was confirmed that the amount of water-soluble and insoluble beta-amyloid protein decreased.

Through this, it was confirmed that the complex extract of the present invention has a preventive or therapeutic effect on degenerative brain diseases such as Alzheimer's dementia by reducing beta-amyloid protein.

From the above description, those skilled in the art to which the present invention pertains will understand that the present invention may be embodied in other specific forms without changing the technical spirit or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, all changes or modifications derived from the meaning and scope of the claims to be described later and their equivalents.

Claims (9)

1. A pharmaceutical composition for preventing or treating degenerative brain disease, comprising hibiscus, rosemary, and grape seed extract as an active ingredient.
2. The pharmaceutical composition for preventing or treating degenerative brain disease according to claim 1, wherein the ratio of the hibiscus, rosemary, and grape seed extracts is 2:1:1.
3. The pharmaceutical composition for preventing or treating degenerative brain disease according to claim 1, wherein the degenerative brain disease is caused by aggregation of soluble or insoluble beta-amyloid (Aβ) protein.
4. The pharmaceutical composition for preventing or treating degenerative brain disease according to claim 1, wherein the degenerative brain disease is dementia or Alzheimer's.
5. The degenerative brain disease according to claim 1, wherein the hibiscus, rosemary, and grape seed extracts exhibit antioxidant, NO production inhibition, memory improvement, reduction of beta-amyloid aggregates, reduction of gliosis level, and reduction of beta-amyloid protein amount. A pharmaceutical composition for the prevention or treatment of
6. A method for preventing or treating degenerative brain disease, comprising administering the pharmaceutical composition of any one of claims 1 to 5 to a subject other than a human.
7. A health functional food composition for preventing or improving degenerative brain disease, comprising hibiscus, rosemary, and grape seed extract as an active ingredient.
8. A health functional food composition for improving memory or cognitive function, comprising hibiscus, rosemary, and grape seed extract as an active ingredient.
9. A feed composition for preventing or improving degenerative brain disease, comprising hibiscus, rosemary, and grape seed extract as an active ingredient.

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