Composition for preventing or treating degenerative brain diseases containing rosa roxburghii tratt extract as active ingredient

Abstract

The present invention relates to a composition for preventing, alleviating, or treating dementia diseases, the composition containing a Rosa roxburghii Tratt extract as an active ingredient. It was confirmed that the composition according to the present invention has a high polyphenol content and excellent anti-inflammatory efficacy. Therefore, the composition can be effectively used in various industrial fields for the purpose of preventing, alleviating, or treating degenerative brain diseases.

Description

Composition for preventing or treating degenerative brain diseases containing extract of the fruit of Sosahwa as an active ingredient

[0001] The present invention relates to a pharmaceutical composition for preventing and treating degenerative brain diseases, and more particularly, to a pharmaceutical composition, a health functional food composition and a feed composition for preventing, improving or treating degenerative brain diseases, which contain an extract of Rosa roxburghii Tratt fruit as an active ingredient.

[0002] With the rapid modernization and aging of the population, the incidence of degenerative brain diseases is increasing among the elderly, particularly dementia. South Korea has had the highest rate of aging among OECD countries over the past 40 years, and the number of dementia patients is also increasing. Due to the increasing number of dementia patients, the socioeconomic cost of dementia treatment already reached 10 trillion won in 2012 and is projected to reach 134.6 trillion won by 2050. The dementia treatment market is also growing.

[0003] Alzheimer's disease (AD), a common neurodegenerative disease that poses a major threat to the health of the elderly, is a neurodegenerative disorder characterized by the formation of beta-amyloid (Aβ) plaques and neurofibrillary tangles, which are produced by chronic inflammation. It is characterized by memory deficits and cognitive impairment. Aggregation of soluble beta-amyloid (Aβ) oligomers / fibrils is a key pathological feature of AD. Since neuroinflammation is a key factor contributing to the development of neurodegenerative diseases, including AD, targeting inflammation and applying agents with anti-inflammatory properties may have beneficial effects on AD.

[0004] Reactive oxygen species (ROS) are generated by various stresses or metabolic processes in the body. Reactive oxygen species, including singlet oxygen, peroxide radicals, hydroxyl radicals, superoxide, nitrite, and peroxynitrite, cause oxidative stress, damaging cells and oxidizing body components. Damaged cells and body components are primarily removed by macrophages, and the inflammation induced by this process can contribute to diseases such as cancer, atherosclerosis, neurodegenerative diseases, infections, chronic inflammatory diseases, diabetes, and autoimmune diseases. To neutralize these ROS, the body functions with various antioxidant enzymes, such as glutathione, thioredoxin, and glutaredoxin. However, ROS generated solely by these enzymes cannot be eliminated, so external antioxidants are a valuable tool for suppressing various diseases. Polyphenols and carotenoids, found in plants, along with antioxidant vitamins, are reported to have the most potent antioxidant effects. Therefore, measuring antioxidant activity in the search for bioactive materials becomes an important tool in terms of disease suppression.

[0005] Microglia in the brain contribute to neuroinflammation. Excessive microglial activation can cause neuronal damage and induce increased levels of amyloid beta protein. The brains of Alzheimer's disease patients exhibit increased oxidative stress, elevated levels of iNOS, and inflammatory molecules. Neurons are reported to be more sensitive to amyloid beta toxicity in the presence of astrocytes, including microglia, and conditioned media from astrocytes treated with the inflammatory agent LPS or amyloid beta induces oxidative stress-induced cell death in neurons.

[0006] Pro-inflammatory cytokines, including TNF-α, are produced through NF-kB-dependent signaling pathways, and TNF-α produced by microglia cells activated by inflammatory stimuli causes damage to surrounding brain tissue.

[0007] Recent studies have reported that neuroinflammation plays a crucial role in the pathophysiology of Alzheimer's disease, which is characterized by the activation of microglia (BV-2 cells). Increased IL-10 levels have been found in the brains of Alzheimer's patients, suggesting that while the body attempts to limit inflammation by producing the anti-inflammatory factor IL-10, inflammation ultimately prevails in Alzheimer's disease.

[0008] Macrophages, which play a crucial role in regulating inflammation, are generally polarized into two types: the classical M1 phenotype and the alternative M2 phenotype. M1 microglial cells are reported to promote inflammatory damage in Alzheimer's disease, while the M2 phenotype exerts neuroprotective effects. Therefore, numerous studies have reported that the inflammation-induced conversion of microglial cells from the M1 to the M2 phenotype suppresses the pathological damage of AD.

[0009] AMPK is a master sensor of energy balance, phosphorylated and activated when energy balance is low, thereby conserving cellular energy. Because AMPK is highly expressed in cortical and hippocampal neurons under both normal and ischemic conditions, pharmacological inhibition of AMPK has been reported to reduce stroke damage, whereas AICAR-induced AMPK activation exacerbates this damage.

[0010] BCL-2 proteins are proteins that regulate apoptosis. BCL-2 does not promote cell proliferation but rather promotes cell survival. Therefore, BCL-2 prevents apoptosis rather than increasing the rate of cell division, thereby increasing the total cell number. Unlike BCL-2 proteins, Bax, Bak, and Bid proteins are proapoptotic proteins. Bax was first identified as an inhibitory binding partner of BCL-2, and its proapoptotic function is known to be activated in response to various harmful events inside or outside the cell, inducing apoptosis. The role of antiapoptotic proteins such as BCL-2 proteins is to inhibit the function of proapoptotic Bax, and therefore, the balance between antiapoptotic and proapoptotic proteins is a factor that determines cell survival.

[0011] Under the above-described technical background, the inventor of the present invention completed the present invention by confirming the enhanced polyphenol content and strong antioxidant action of the extract of the fruit of the pomegranate.

[0012] One object of the present invention is to provide a pharmaceutical composition for preventing or treating degenerative brain diseases, which comprises an extract of the fruit of Sosahwa as an active ingredient.

[0013] Another object of the present invention is to provide a method for preventing or treating a degenerative brain disease, comprising a step of administering the pharmaceutical composition to a subject other than a human.

[0014] Another object of the present invention is to provide a health functional food composition for preventing or improving degenerative brain diseases, which contains an extract of the fruit of Sosahwa as an active ingredient.

[0015] Another object of the present invention is to provide a feed composition for preventing or improving degenerative brain diseases, which contains an extract of the fruit of Sosahwa as an effective ingredient.

[0016] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by a person having ordinary skill in the technical field to which the present invention belongs from the description below.

[0017] To achieve the above purpose, the present invention provides a pharmaceutical composition for preventing or treating degenerative brain diseases, comprising a Sosahwa extract as an active ingredient.

[0018] In addition, the present invention provides a method for preventing or treating a degenerative brain disease, which comprises directly administering to a subject a pharmaceutical composition for preventing or treating a degenerative brain disease containing the extract of Sosahwa according to the present invention as an active ingredient.

[0019] In addition, the present invention provides a health functional food composition for preventing or improving degenerative brain diseases, which comprises a Sosahwa extract as an active ingredient.

[0020] In addition, the present invention provides a feed composition for preventing or improving degenerative brain diseases, which comprises a sosahwa extract as an effective ingredient.

[0021] The extract of the fruit of the present invention has a high polyphenol content and effectively exhibits anti-inflammatory action, and thus can be usefully utilized in various industrial fields for the prevention, improvement, or treatment of degenerative brain diseases.

[0022] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the detailed description of the present invention or the composition of the invention described in the claims.

[0023] Figure 1 is a diagram showing the total polyphenol content of samples A, B, and A+B.

[0024] Figure 2 is a diagram showing specific results of the total polyphenol content of samples A, B, and A+B using tannic acid as a standard substance.

[0025] Figure 3 is a diagram showing the antioxidant activity (ABTS) level of sample A.

[0026] Figure 4 is a diagram showing the antioxidant activity (DPPH free radical) level of sample A.

[0027] Figure 5 is a diagram showing direct toxic activity against the Raw264.7 cell line.

[0028] Figure 6 is a diagram showing direct toxic activity against the HT-22 cell line.

[0029] Figure 7 is a diagram showing direct toxic activity against Raw cell lines.

[0030] Figure 8 is a diagram showing direct toxic activity against the BV-2 cell line.

[0031] Figure 9 is a diagram showing the inhibitory effect of sample A on NO production in macrophages stimulated with LPS.

[0032] Figure 10 is a diagram showing the inhibitory effect of sample B on NO production in macrophages stimulated with LPS.

[0033] Figure 11 is a diagram showing the inhibitory effect of samples A+B on NO production in macrophages stimulated with LPS.

[0034] Figure 12 is a diagram showing the inhibitory effect of sample A on TNF-a production in macrophages stimulated with LPS.

[0035] Figure 13 is a diagram showing the inhibitory effect of sample B on TNF-a production in macrophages stimulated with LPS.

[0036] Figure 14 is a diagram showing the inhibitory effect of samples A+B on TNF-a production in macrophages stimulated with LPS.

[0037] Figure 15 is a diagram showing the effect of sample A on IL-10 production.

[0038] Figure 16 is a diagram showing the effect of sample B on IL-10 production.

[0039] Figure 17 is a diagram showing the effect of samples A+B on IL-10 production.

[0040] Figure 18 is a diagram showing the effect of microglia cell conditioned media treated with sample A on HT-22 cell proliferation.

[0041] Figure 19 is a diagram showing the effect of microglia cell conditioned media treated with sample B on HT-22 cell proliferation.

[0042] Figure 20 is a diagram showing the effect of microglia cell conditioned media treated with samples A+B on HT-22 cell proliferation.

[0043] Figure 21 is a diagram showing the effect of cell culture solution treated with sample A on HT-22 cell proliferation in a microglial cell model stimulated with LPS.

[0044] Figure 22 is a diagram showing the effect of cell culture solution treated with sample B on HT-22 cell proliferation in a microglial cell model stimulated with LPS.

[0045] Figure 23 is a diagram showing the effect of cell culture solution treated with sample A+B on HT-22 cell proliferation in a microglial cell model stimulated with LPS.

[0046] Figure 24 is a diagram showing the cytotoxic protective effect of sample A in HT-22 cells stimulated with beta-amyloid.

[0047] Figure 25 is a diagram showing the cytotoxic protective effect of sample A in HT-22 cells stimulated with beta-amyloid.

[0048] Figure 26 is a diagram showing the effect of the sample on the expression of apoptosis-regulating proteins in HT-22 cells.

[0049] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and will not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0050] In addition, when describing the present invention, if it is determined that a detailed description of a related known technology may unnecessarily obscure the gist of the present invention, the detailed description is omitted.

[0051] Hereinafter, the present invention will be described in more detail.

[0052] The present invention provides a pharmaceutical composition for preventing or treating degenerative brain diseases, comprising a Sosahwa extract.

[0053] In the present invention, the extract of the sosahwa is preferably an extract of the sosahwa fruit.

[0054] Preferably, the degenerative brain disease may be one selected from the group consisting of dementia, Alzheimer's disease, stroke, cerebral infarct, head trauma, cerebral arteriosclerosis, or Parkinson's disease, but is not limited thereto.

[0055] Preferably, the pharmaceutical composition can suppress brain inflammation by alleviating microglial activation, but is not limited thereto.

[0056] Preferably, the pharmaceutical composition can have the function of proliferating macrophages and expressing GF for nerve cells, but is not limited thereto.

[0057] Preferably, the pharmaceutical composition can suppress brain inflammation caused by cell damage by beta-amyloid by increasing the expression level of Bcl-2 protein and decreasing the expression level of Bax protein, thereby suppressing brain neuronal cell death, but is not limited thereto.

[0058] Preferably, the extraction solvent of the above-mentioned sosahwa extract may be water, C1 to C4 alcohol, ethanol or a mixture thereof, but is not limited thereto.

[0059] Preferably, the ethanol extract of the Prunus mume fruit may be an extract obtained by extracting the Prunus mume fruit with 50 to 50% ethanol, and more preferably, it may be an extract obtained by extracting the Prunus mume fruit with 50% ethanol, but is not limited thereto.

[0060] The pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. The excipients may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, moisturizers, film-coating materials, and controlled-release additives.

[0061] The pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric-coated granules, liquids, eye drops, ellipsoids, emulsions, suspensions, alcohols, troches, aromatic waters, limonades, tablets, sustained-release tablets, enteric-coated tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigation solutions, ointments, lotions, pastes, sprays, inhalants, patches, sterile injection solutions, or aerosols, according to a conventional method, and the external preparations may have formulations such as creams, gels, patches, sprays, ointments, ointments, lotions, liniments, pastes, or cataplasmas.

[0062] Carriers, excipients and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharides, 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 and mineral oil.

[0063] When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0064] The additives of the tablets, powders, granules, capsules, pills, and troches according to the present invention include excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, and binders such as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Disintegrants such as carboxymethylcellulose calcium, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, baking soda, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium dentata, kaolin, petrolatum, sodium stearate, cacao butter, sodium salicylate, magnesium salicylate, polyethylene glycol 4000, polyethylene glycol 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid can be used.

[0065] As additives of the liquid formulation according to the present invention, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc. can be used.

[0066] The syrup according to the present invention may include a solution of white sugar, other sugars, or sweeteners, and may also include a fragrance, a coloring agent, a preservative, a stabilizer, a suspending agent, an emulsifier, a viscosity modifier, and the like, as needed.

[0067] Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0068] The suspension according to the present invention may include suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, and HPMC 2910, and surfactants, preservatives, stabilizers, colorants, and fragrances may be used as needed.

[0069] The injection according to the present invention includes a solvent such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; a solubilizing agent such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tween, nitrile acid amide, hexamine, and dimethylacetamide; a buffer such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gums; It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; oxidizing agents such as sodium bisulfite 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid disodium, and acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium cis-methylenediamine, sodium alginate, Tween 80, and aluminum monostearate.

[0070] The suppository according to the present invention comprises cocoa butter, lanolin, withepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, ranet wax, glycerol monostearate, Tween or Span, Imhausen, monolene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydroxocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Mechanisms such as Hydrokote 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Masupol, Masupol-15, Neosupostal-N, Paramound-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), Suppository type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wecovi (W, R, S, M, Fs), Tezester triglyceride basis (TG-95, MA, 57) can be used.

[0071] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing the extract with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

[0072] Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups. In addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0073] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level can be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field.

[0074] The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents. It can be administered sequentially or simultaneously with conventional therapeutic agents, or in single or multiple doses. Taking all of the above factors into account, it is important to administer an amount that achieves maximum efficacy with minimal side effects. This amount can be readily determined by those skilled in the art to which the present invention pertains.

[0075] The pharmaceutical composition of the present invention can be administered to a subject via various routes. All modes of administration are conceivable, including oral ingestion, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosa administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, oral or nasal spraying, dermal administration, and transdermal administration.

[0076] The pharmaceutical composition of the present invention is determined according to the type of drug as an active ingredient along with various related factors such as the disease to be treated, route of administration, age, sex, weight of the patient, and severity of the disease.

[0077] In the present invention, the term “subject” means a subject requiring treatment for a disease, and more specifically, means a mammal such as a human or non-human primate, mouse, rat, dog, cat, horse, and cow.

[0078] In the present invention, “administration” means providing a predetermined composition of the present invention to a subject by any suitable method. In the present invention, “prevention” means any act of inhibiting or delaying the onset of a target disease, “treatment” means any act of improving or beneficially changing a target disease and its resulting metabolic abnormality symptoms by administering a pharmaceutical composition according to the present invention, and “improvement” means any act of reducing a parameter related to a target disease, for example, the severity of a symptom, by administering a composition according to the present invention.

[0079] The present invention provides a health functional food composition for preventing or improving degenerative brain diseases, containing a sosahwa extract as an active ingredient.

[0080] In the present invention, “food” means a natural product or processed product containing one or more nutrients, preferably a product that has gone through a certain degree of processing to become directly edible, and in its general meaning includes all health functional foods, beverages, food additives, and beverage additives.

[0081] In the present invention, the term “functional food” is the same as food for special health use (FoSHU), and refers to a food with high medical or healthcare effects that is processed to efficiently exhibit a bioregulatory function in addition to providing nutrition, and may be manufactured in the form of tablets, capsules, pills, granules, powders, liquids, flakes, pastes, syrups, gels, jellies, bars, or films. Here, “function” means regulating nutrients for the structure and functions of the human body or obtaining a useful effect for health purposes such as physiological actions.

[0082] When the extract of the fruit of the present invention is used as a food additive, it can be added as is or used together with other foods or food ingredients, and can be used appropriately according to a conventional method. The amount of the active ingredient mixed can be appropriately determined depending on the purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing a food or beverage, the fruit of the present invention can be added in an amount of 15% by weight or less, or 10% by weight or less, based on the raw material. However, in the case of long-term intake for the purpose of health and hygiene or health control, the amount can be below the above range, and since there is no problem in terms of safety, the active ingredient can also be used in an amount above the above range.

[0083] There are no specific restrictions on the types of the above foods. Examples of foods to which the above substances 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 all health functional foods in the conventional sense are included.

[0084] The health beverage composition according to the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients, similar to conventional beverages. The natural carbohydrates mentioned above include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As a sweetener, natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame may be used. The proportion of the natural carbohydrate is generally about 0.01-0.20 g, or about 0.04-0.10 g, per 100 mL of the composition of the present invention.

[0085] In addition to the above, the composition of the present invention may 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 adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. In addition, the composition of the present invention may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients may be used independently or in combination. The proportion of these additives is not particularly critical, but is typically selected within the range of 0.01 to 0.20 parts by weight per 100 parts by weight of the composition of the present invention.

[0086] The present invention provides a feed composition for preventing or improving degenerative brain diseases, comprising the extract of the present invention as an effective ingredient.

[0087] The term "feed" in the present invention means any natural or artificial diet, meal, etc. or a component of said meal for eating, ingesting, and digesting by an animal or suitable therefor.

[0088] The type of the above feed is not particularly limited, and feed commonly used in the relevant technical field can be used. Non-limiting examples of the above feed include plant-based feed such as grains, nuts, food processing by-products, algae, fiber, pharmaceutical by-products, oils, starches, meal, or grain by-products; and animal-based feed such as proteins, inorganic substances, oils, minerals, oils, single-cell proteins, zooplankton, or food. These may be used alone or in combination of two or more.

[0089] The present invention confirmed that the extract of the fruit of the present invention, Sosahwa, is excellent in preventing and improving degenerative brain diseases, and identified a mechanism by which it alleviates inflammation by reducing the expression of TNF-α. Therefore, the present invention can be utilized as a pharmaceutical composition or health functional food composition for the prevention, improvement, and treatment of degenerative brain diseases.

[0090] The present invention will be described in detail through the following examples. These examples are intended solely to illustrate the present invention, and therefore the scope of the present invention is not to be construed as being limited by these examples.

[0091] [Experiment Preparation]

[0092] The experimental material was prepared as a 10 mg / ml solution of the extract in PBS solution, but as it was not completely dissolved, a 100 mg / ml solution was prepared using DMSO solution and used as a sample. At this time, the final concentration of DMSO applied to the cells was less than 0.1%. Sample A and sample B are extracts of Rosa roxburghii Tratt and Jasminum nudiflorum Linn, respectively.

[0093] cell lines

[0094] Mouse hippocampal neural cell line (HT22), which is widely used in in vitro Alzheimer's disease (AD) research, was used as the neural cell, and BV2 microglia cells were used as the microglia. RAW2647 macrophage cell line was used to measure anti-inflammatory activity, and each cell was cultured in DMEM medium containing 10% fetal bovine serum (FBS) with penicillin (100 U / ml) and streptomycin (100 mg / ml) at 37°C and 5% CO2.

[0095]

[0096] Example 1: Analysis of total polyphenol content of samples

[0097] Total polyphenol content was measured using the Folin-Denis method. The standard material for quantifying the polyphenol compound content was prepared as a standard solution using tannic acid (Sigma, St. Louis, MO, USA). 0.2 ml of 10% Folin-Ciocalteu's phenol reagent (Sigma, St. Louis, MO, USA) and 0.7 ml of 700 mM Na2CO3 solution were added to 0.1 ml of the sample, mixed well, and reacted for 30 min at room temperature under light-shielding conditions. The absorbance was measured at 765 nm, and the total polyphenol content (mg / g) was calculated by substituting it into the standard curve (Fig. 1).

[0098] Looking at Figure 2, the total polyphenol content was detected as 0280±0014 mg / g in the extract of the fruit of Sosahwa (sample A), 0148±0035 mg / g in the extract of the Newddeung (sample B), and 0225±0011 mg / g in the sample mixture extract when measured using tannic acid as a standard substance.

[0099]

[0100] Example 2: Free radical scavenging activity

[0101] Antioxidant activity using ABTS (3-ethyl-benzothiazoline-6-sulfonic acid) and DPPH (1,1-Diphenyl-2-picrylhydrazyl) radicals was measured using their respective assay kits. As a control, a widely known synthetic antioxidant, tetramethylchromanecarboxylic acid [Trolox, ((±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid)], was used. The radical scavenging activity of each sample was expressed as a percentage of the radical scavenging activity of the control group, using PBS as a solvent.

[0102] The ABTS and DPPH free radical scavenging activities of each extract were investigated, and as shown in Figures 3 and 4, it was confirmed that the extract of the fruit of Sosahwa (Sample A) had the best antioxidant effect. In particular, the DPPH scavenging activity was higher than that of Trolox, which was used as a control, suggesting that the water-soluble component contained in Sample A has a strong antioxidant function.

[0103]

[0104] Example 3: Direct toxicity of the sample against cell lines

[0105] Each extract was applied to macrophages (RAW2647 macrophages), microglia (BV-2 microglia cells), and HT-22 mouse hippocampal neurons, and the effects on cell proliferation were investigated. After seeding each cell in a 96-well plate, the sample was treated for 24 hours. At this time, the cells were treated with various concentrations starting from 100 μg / ml, the highest concentration at which the cells were not affected by DMSO. (At this time, DMSO was treated alone at 0.1% to confirm that it did not affect the cells, and a DMSO alone control group was selected, and it was confirmed that there was no toxicity.) After completing the culture, the culture medium was removed, and EZ cytox (DoGENBIO, Korea) solution using WST-8 was added (1 / 10 diluted in DMEM media and added to 100 ml), and the absorbance was measured at 450 nm after 1 hour. The absorbance for each sample concentration was measured as cell viability (% of control, % compared to the control group) compared to the control group (PBS treatment), and the cell activity experiment was performed based on the highest concentration at which no cytotoxicity was observed.

[0106] For macrophages (RAW2647 macrophages), all three samples showed no significant toxic effects at concentrations below 100 μg / mL, and showed proliferative activity in macrophages at concentrations of 6.25 μg / mL to 25 μg / mL (Fig. 5). However, no significant proliferative activity was induced in BV-2 microglia (Fig. 8). On the other hand, in HT-22 cells, which are neural cells, some proliferative inhibition was observed at concentrations of 100-50 μg / mL, and some proliferative activity was induced at concentrations of 0.1-6.25 μg / mL (Fig. 6).

[0107]

[0108] Example 4: Inhibitory effect of samples on production of inflammatory substances in LPS-stimulated macrophages

[0109] BV-2 Microglia cells or RAW264.7 cells were pretreated with each extract sample for 2 hours, then treated with lipopolysaccharide (LPS, 0.1 ug / ml), an inflammatory substance, and cultured for 24 hours before collecting the supernatant. The amount of NO, an inflammatory substance produced in the supernatant, was measured using a Nitric Oxide assay kit (DoGENBIO, Korea, capable of measuring 0–200 μM). After adding Solution A to the sample, the reaction was incubated for 10 minutes in a light-shielded location. Then, Solution B was added, the reaction was incubated for 10 minutes in a light-shielded location, and the absorbance was measured at 540 nm. Simultaneously, the inflammatory cytokines TNF-α, IL-6, and IL-10 produced in the culture supernatant were measured using their respective ELISA assay kits (BD OptEIA Kit). Coating antibodies were treated in 96-well immunoplates and incubated overnight at 4°C. The next day, the samples were washed five times with PBS-T, blocked for 1 hour, and reacted with the standards and samples at RT for 2 hours. After washing six times, the samples were treated with detection antibodies and HRP, reacted for 1 hour, washed six times, and developed with TMB. After color development, the stop solution was used and the absorbance was measured at 450 nm.

[0110]

[0111] 4.1. Effect on NO (Nitric oxide) production

[0112] About 30 mM of NO was measured in the positive control for macrophages (Raw 264.7 macrophages), and all three samples showed a concentration-dependent effect of suppressing inflammatory substances at concentrations of 6.25 to 100 μg / mL compared to the positive control (Figs. 9 to 11).

[0113]

[0114] 4.2. Effect on TNF-α production

[0115] In this study, we investigated the effects of samples on the production of inflammatory cytokines in LPS-treated microglia. Pretreatment with sample A (Sosaehwa extract) dose-dependently inhibited LPS-stimulated TNF-α production at concentrations below 25 μg / mL (Figure 12). Meanwhile, IL-6 production did not show any significant changes.

[0116]

[0117] 4.3. Effect on IL-10 production

[0118] Anti-inflammatory cytokines play a crucial role in neutralizing neuroinflammation in the brain of Alzheimer's disease. Specifically, in an Alzheimer's disease model induced using activated microglia, a decrease in the production of anti-inflammatory factors such as IL-10 was reported to promote neuroinflammation and cognitive impairment.

[0119] The experimental results showed that all three samples increased the production of IL-10 compared to LPS treatment alone, and the extract of the fruit of Sosahwa (sample A) showed the best results, suggesting that it would have the function of suppressing brain diseases caused by inflammation (Figs. 15 to 17).

[0120] In conclusion, all three samples were thought to have the ability to induce anti-inflammatory activity. Therefore, to further confirm these results, culture supernatants from LPS-stimulated macrophages treated with the samples were applied to HT-22 cells, a neuronal cell line, and their activity on HT-22 cell viability was examined.

[0121]

[0122] Example 5: Inhibitory effect of microglial culture supernatant-mediated neurotoxicity

[0123] Excessive production of pro-inflammatory mediators such as NO, TNF-α, and IL-6 from activated microglia causes damage to peripheral neurons. 2Х10 4Conditioned medium was prepared by treating various concentrations of extracts 30 minutes before stimulating BV-2 microglia cultured in a 96-well plate at a concentration of 10 cells / ml with LPS (1 μg / ml).

[0124] HT-22 neurons were dispensed into each well of a 96-well plate (2Х103 4 cells / 100 ㎕) and 100 ㎕ of the cell culture medium (conditioned medium) of BV-2 cells were treated and cultured for 24 hours. After completion of culture, the viability of HT-22 neural cells was measured using the Ez-cytox kit.

[0125]

[0126] 5.1. Analysis of the effect of sample treatment on the proliferation of HT-22 cells

[0127] When the macrophage culture medium containing only the sample was applied to HT-22 cells, significant neuronal proliferation activity was observed only in the case of the Sosahwa fruit extract (sample A) at a concentration of 6.25-25 μg / mL (Fig. 18). These results are consistent with the macrophage proliferation activity induced by the sample alone, and it is thought that the Sosahwa fruit extract (sample A) produces growth factors that promote the proliferation of HT-22 cells from macrophages, including the activity of self-proliferating macrophages.

[0128]

[0129] 5.2. Analysis of HT-22 cell cytotoxicity by sample treatment

[0130] After treating macrophages with the sample, the conditioned media added with LPS and cultured for 24 hours was applied to HT-22 cells, and the result of 24-hour culture showed that the control group had approximately 50% of the cytotoxic effect compared to the LPS-only group. However, in the case of the culture supernatant containing the Sosahwa fruit extract (sample A), significant HT-22 cell proliferation activity was observed at a concentration of 6.25-25 μg / mL (Fig. 21). Macrophage culture media contain various factors that affect cell differentiation, proliferation, or survival, including various cytokines, prostaglandins, and chemokines. The significant increase in HT-22 cell survival in the culture supernatant simultaneously treated with the Sosahwa fruit extract (sample A) strongly suggests that the sample itself can suppress nerve cell damage caused by strong inflammatory stimuli or the production of substances that induce other inflammatory-mediated cell damage (e.g., NO or TNF-α).

[0131]

[0132] Example 6: Inhibitory effect of sample on beta-amyloid-induced cell neurotoxicity

[0133] The therapeutic effect of Sosahwa fruit extract on beta-amyloid-induced neurotoxicity was investigated using the HT22 hippocampal neuronal cell line. Beta-amyloid treatment was administered at a concentration of 40 μg / mL, which showed a neuronal cell death effect in a preliminary experiment, and the protective effect of Sosahwa fruit (sample A) extract (6.25-50 μg / mL) on this toxicity was measured. The sample concentration of 50 μg / mL did not significantly affect Aβ-induced cytotoxicity, but the sample treatment at concentrations of 6.25-25 μg / mL showed a protective effect against beta-amyloid-induced cell damage (Figs. 24-25).

[0134]

[0135] Example 7: Western Blot

[0136] Beta-amyloid induces toxicity in neurons, and to confirm the neuronal cell death pathway, the expression patterns of beta-amyloid, Bax, Bcl-2, and β-actin proteins in neurons were investigated using Western blot.

[0137] HT-22 cells (5х105 cells / well) were seeded in each well of a 6-well plate, treated with 200 μM mouse beta-amyloid (10 μL) and a concentration of Pseudostachys japonica fruit extract that has been shown to inhibit cytotoxicity, and cultured for 48 hours. After completion of culture, HT-22 cells were washed twice with PBS (phosphate-buffered saline), and proteins were extracted by adding lysis buffer. The extracted proteins were centrifuged (12,500 rpm / 15 min) to collect the supernatant, and the protein concentration was measured using a BCA kit to prepare samples for electrophoresis. 20 μL of the prepared protein samples were loaded onto a 10-12% SDS-PAGE gel, and electrophoresis was performed. Proteins separated by electrophoresis were transferred to nitrocellulose membrane (NC membrane) (Millipore Corporation, Burlington, MA, USA) using a Transfer System (Bio-Rad Laboratories, Hercules, CA, USA). After blocking nonspecific binding sites with 3% skim milk for 1 h at room temperature, the membrane was incubated with primary antibody overnight at 4°C.

[0138] The immune complexes reacted with secondary-HRP were reacted with the substrate using an enhanced chemiluminescence (ECL) kit, and then the protein expression pattern was analyzed using an image analysis system (Davinch-Chemi FluoroTM Imaging System).

[0139] To explain the molecular biological mechanism of the inhibitory effect of the sample on beta-amyloid-induced HT-22 cell cytotoxicity, the effect on the expression of Bcl-2 and Bax was examined. The expression pattern of Bcl-2, an anti-apoptotic protein, was examined, and while it was suppressed in the Aβ-treated group, its expression level was significantly increased in the group treated with the extract of the fruit of Sosahwa, and the concentration of the sample that induced maximum expression was 0.4 μg / mL.

[0140] Meanwhile, the expression pattern of the 20 kDa Bax protein, which promotes apoptosis and inhibits neurogenesis in the hippocampus and cerebellum of the brain, was investigated. Compared to the Aβ treatment group, the concentration of the Sosahwa fruit extract (sample A) treatment group (10-0.4 μg / mL) showed an inversely increased expression of Bax, with the lowest protein expression at 0.4 μg / mL (Fig. 26). Therefore, it was suggested that the treatment with the Sosahwa fruit extract (sample A) is a component that helps perform the functions of nerve cells, including the function of inhibiting the development of AD caused by beta-amyloid.

[0141]

[0142] In summary, sample A (Pseudomonas amurense extract) contained polyphenols that inhibited oxidation by free radicals, a inflammatory component, and exhibited potent antioxidant activity. It also suppressed the production of inflammatory mediators from stimulated macrophages and inhibited neuronal damage induced by inflammatory substances. Simultaneously, it exhibited a protective effect against neuronal damage induced by B-amyloid produced by damaged neurons. This protective effect was attributed to its ability to regulate the expression of anti-apoptotic and pro-apoptotic proteins, Bcl-2 and Bax.

Claims

1. A pharmaceutical composition for preventing or treating degenerative brain diseases, comprising an extract of Sosahwa as an active ingredient.

2. In paragraph 1, A pharmaceutical composition for preventing or treating degenerative brain diseases, characterized in that the above-mentioned extract of Sosahwa is an extract of the fruit of Sosahwa.

3. In paragraph 1, A pharmaceutical composition for preventing or treating a degenerative brain disease, wherein the degenerative brain disease is any one selected from the group consisting of dementia, Alzheimer's disease, stroke, cerebral infarct, head trauma, cerebral arteriosclerosis, and Parkinson's disease.

4. In paragraph 1, A pharmaceutical composition for preventing or treating degenerative brain diseases, wherein the extraction solvent of the above-mentioned Sosahwa extract is water, C1 to C4 alcohol, ethanol or a mixture thereof.

5. A pharmaceutical composition for preventing or treating a degenerative brain disease, wherein the extract exhibits effects of antioxidant, NO production inhibition, memory improvement, reduction of beta-amyloid aggregates, reduction of gliosis level, or reduction of beta-amyloid protein amount in the first paragraph.

6. A pharmaceutical composition for preventing or treating a degenerative brain disease, wherein the extract, in paragraph 1, exhibits the effect of increasing the proliferation of macrophages, increasing the expression level of Bcl-2 protein, or decreasing the expression level of Bax protein.

7. A method for preventing or treating a degenerative brain disease, comprising directly administering to a subject a pharmaceutical composition according to any one of claims 1 to 6.

8. A health functional food composition for preventing or improving degenerative brain diseases, comprising a sosahwa extract as an active ingredient.

9. A feed composition for preventing or improving degenerative brain diseases, comprising a sosahwa extract as an active ingredient.

Images