KR102282883B1 - Composition for preventing and/or treating a degenerative brain disease comprising as an active ingredient an extract of Cimicifuga dahurica, a fraction thereof, or a compound derived from Cimicifuga dahurica - Google Patents

Abstract

The present invention provides a pharmaceutical composition for the prevention or treatment of degenerative brain disease, comprising an equestrian extract or a compound isolated therefrom as an active ingredient, a method for preventing or treating degenerative brain disease using the pharmaceutical composition, preventing or treating degenerative brain disease It relates to a food composition for improvement and a feed composition.
The equestrian extract or the compound isolated therefrom of the present invention is characterized in that it specifically promotes the amount of protein involved in autophagy, and specifically, the equestrian extract or the compound isolated therefrom is an autophagosome It increases the expression of LC3 protein, a membrane protein, and has an excellent effect of suppressing the expression of mTOR protein, which suppresses autophagy, and furthermore, it has an excellent effect of inhibiting the production of beta-amyloid, the causative agent of dementia, and memory damage. It can be effectively used in the treatment of degenerative brain diseases such as diseases, Alzheimer's disease and dementia, and improvement of cognitive impairment.

Description

Equestrian extract, its fraction or a composition for preventing or treating degenerative brain disease comprising a compound derived from horseback riding as an active ingredient, the composition for preventing and/or treating a degenerative brain disease comprising as an active ingredient an extract of Cimicifuga dahurica, a fraction thereof, or a compound derived from Cimicifuga dahurica}

The present invention relates to a horse riding (Cimicifuga dahurica) extract, a composition for preventing or treating a degenerative brain disease comprising a compound derived from a fraction thereof as an active ingredient or riding.

Recently, as the aging phenomenon due to the increase in life expectancy has emerged as a social problem, the importance of research on degenerative brain disease is being emphasized along with the increase in the elderly population. Degenerative brain disease is known to be caused by neurodegeneration due to aging and the death of nerve cells due to aggregation of proteins due to genetic or environmental factors. Degenerative brain diseases include Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis, and Lou Gehrig's disease in consideration of the main symptoms and brain regions affected.

Alzheimer's disease, known as the most common degenerative brain disease, is a disease that progressively causes severe impairments such as memory, concentration, thinking, learning ability, language ability and judgment ability due to damage and loss of nerve cells, leading to loss of mental ability and social activity ability. . A major neuropathological finding of Alzheimer's disease is the aggregation and deposition of beta-amyloid (Aβ) and hyperphosphorylated tau protein. Outside the brain neurons, beta-amyloid accumulates and becomes entangled, forming a senile plaque, which causes neurotoxicity. Alzheimer's disease is known to develop (Probst et al., Brain Pathol. 1, 229-239, 1991).

Decreased function in acetylcholine, glutamic acid, neuropeptides and monoamine is also known to affect the onset of Alzheimer's disease (Coyle et al. Science, 219, 1184-1190, 1983; Gottfries et al. Psychopharmacology, 86, 245-252, 1985). Accordingly, acetylcholinesterase activity inhibitors (tacrine, donepezil, etc.) that improve symptoms while slowing the removal rate of acetylcholine from the synapse are used as therapeutic agents, but hepatotoxicity, Problems with serious side effects such as vomiting, nausea and diarrhea are known. NMDA receptor antagonist (Memantine), which inhibits neuronal cell death by Ca ²⁺ , is also used as a therapeutic agent, but all are used for the purpose of symptom relief.

Accordingly, various studies are being conducted to develop a therapeutic agent for Alzheimer's disease that can be causally treated, and one of them is to develop a substance capable of inhibiting the production of beta-amyloid.

Parkinson's disease is the second most common disease after Alzheimer's disease, and it is chronic and progressive. Research has been conducted for the past several decades to determine the cause of the disease, but the exact cause and mechanism have not been elucidated. However, as a pathological factor that appears in patients with Parkinson's disease, the substantia nigra par compacta of the midbrain is damaged and abnormal death of nerve cells that secrete the neurotransmitter dopamine is known. Abnormal death of dopaminergic neurons occurs when abnormal proteins and organelles cannot be decomposed and accumulate due to a decrease in mitochondrial function and a disorder of the proteolytic system due to various factors such as heredity, aging, and environment. Therefore, the importance of the proteolytic system is emphasized.

Drugs for the treatment of Parkinson's disease include levodopa (L-dopa), dopamine agonists, and anticholinergics, and among them, levodopa is the most effective drug. This is a treatment that increases the dopamine concentration in the blood by directly injecting levodopa, a precursor of dopamine, and brings substantial symptom relief to patients with Parkinson's disease. It is known to cause side effects such as dyskinesia (Rock and Peterson, J Neuroimmune Pharmacol. 1, 117-126, 2006). Therefore, for the fundamental treatment of Parkinson's disease, the development of drugs capable of inhibiting the protection and death of nerve cells is urgently needed.

Autophagy, or autophagy, is a major intracellular proteolytic system along with the ubiquitin-proteasome system, which decomposes aging or dysfunctional organelles and damaged proteins to restore cell homeostasis and genetic stability. Autophagocytosis occurs when the edge of pagopole, the precursor of autophagosome, is gradually extended to surround unnecessary proteins to form autophagosome, an endoplasmic reticulum, and autophagosome moves along microtubules and fuses with intracellular lysosomes. While forming lysosomes, it is made while decomposing proteins by proteolytic enzymes secreted from the inside of lysosomes. Therefore, when autophagy is lowered, it is known that the accumulation of denatured proteins causes neurodegenerative diseases (Rubinsztein et al., Nature reviews 6, 304-312, 2007; Dehay et al., The Journal of neuroscience 30). , 12535-12544, 2010).

Research to activate autophagy for the treatment of degenerative brain diseases is ongoing. In an animal model of Alzheimer's disease, the production of beta-amyloid and tau was decreased and cognition was enhanced by rapamycin, an mTOR inhibitor that inhibits autophagy (Caccamo et al., Mol. Neurodegener. 5, 51, 2010). ), it was confirmed that the overexpressed mutant alpha-synuclein protein coagulated in an animal model of Parkinson's disease was removed (Webb et al., J. Biol. Chem., 278, 25009-25013, 2003). In addition, alpha-synuclein protein coagulation was reduced in an animal model of Parkinson's disease by induction of autophagy by lithium (Sarkar et al., J. Cell Biol. 170, 1101-1111, 2005).

Horseback riding ( Cimicifuga dahurica ) is a plant belonging to the genus Ranunculaceae, and there are about 10 species of horse riding, snow riding, and dog riding in Korea. It grows in the bushes of forests or mountain slopes, and its rhizomes are thick nodes, 6-8 cm long and 10-25 cm in diameter, used for medicinal purposes. Horseradish extract is known to regulate insulin secretion and promote stem cell proliferation or differentiation. Visnagin, known as an active ingredient in horseback riding, has been reported to inhibit vascular smooth muscle contraction while enhancing blood pressure and inhibiting calcium inflow. However, there have been no studies specifically related to the activation of autophagy by equestrian extracts or compounds, Alzheimer's disease, Parkinson's disease, or memory.

Accordingly, the present inventors found that LC3, an autophagosome membrane protein involved in autophagy, which is one of the causes of Parkinson's disease, is a dopaminergic cell line and a compound derived from Cimicifuga dahurica extract, a fraction thereof, or horse riding in an animal model of Parkinson's disease. This study confirmed that it has the effect of suppressing the expression of mTOR protein, which specifically promotes the production of -II and suppresses autophagy, the effect of inhibiting the production of beta-amyloid, the causative agent of dementia, and the effect of suppressing memory impairment. The invention was completed.

One object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of degenerative brain disease comprising a horse riding extract, a fraction thereof, or a compound derived from horse riding 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 food composition for the prevention or improvement of degenerative brain disease comprising a horse riding extract, a fraction thereof, or a compound derived from horse riding as an active ingredient.

Another object of the present invention is to provide a food composition for improving learning or memory or improving cognitive function, comprising as an active ingredient a horse riding extract, a fraction thereof, or a compound derived from horse riding.

Another object of the present invention is to provide a feed composition for the prevention or improvement of degenerative brain disease comprising a horse riding extract, a fraction thereof, or a compound derived from horse riding as an active ingredient.

Each description and embodiment disclosed in the present invention is also applicable 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 aspect of the present invention for achieving the above object is horseback riding ( Cimicifuga dahurica ) It provides a pharmaceutical composition for preventing or treating degenerative brain disease comprising an extract, a fraction thereof, or a compound derived from horseback riding as an active ingredient.

As used herein, the term "equestrian" is scientifically named Cimicifuga dahurica (Cimicifuga dahurica) In the present invention, the above-ground part of the plant can be extracted and used, and specifically, the leaves and stems of the plant can be used, and more specifically, the rhizome, which is a stem that has a root shape and extends into the ground, can be used. , as long as it has a preventive or therapeutic effect on degenerative brain disease, it is not particularly limited thereto.

The horseback riding may be purchased commercially sold, or harvested or grown in nature.

As used herein, the term "extract" refers to an extract obtained by extracting horseback riding, a diluted or concentrated liquid of the extract, a dried product obtained by drying the extract, a prepared or purified product of the extract, or a mixture thereof, such as the extract itself and It includes extracts of all formulations that can be formed using the extract.

The method of extracting the horseback riding is not particularly limited, and may be extracted according to a method commonly used in the art. Non-limiting examples of the extraction method include a hot water 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.

In the present invention, the type of extraction solvent used for extracting the horseback riding is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the extraction solvent include water, C1 to C4 alcohol, and a mixed solvent thereof, and these may be used alone or in combination of one or more.

In addition, the extract may be prepared and used in the form of a dry powder after extraction, but is not limited thereto.

As used herein, the term "fraction" refers to a result obtained by performing fractionation in order to separate a specific component or a specific group of components from a mixture including various components.

The fractionation method for obtaining the fraction in the present invention is not particularly limited, and may be performed according to a method commonly used in the art. Non-limiting examples of the fractionation method include a solvent fractionation method performed by treating various solvents, an ultrafiltration fractionation method performed by passing an ultrafiltration membrane having a constant molecular weight cut-off value, and various chromatography (size, charge, hydrophobicity) or a chromatographic fractionation method for performing separation according to affinity), and combinations thereof. Specifically, there may be mentioned a method of obtaining a fraction from the extract by treating the extract obtained by extracting the lice of the present invention with a predetermined solvent.

In the present invention, the type of the fractionation solvent used to obtain the fraction is not particularly limited, and any solvent known in the art may be used. Non-limiting examples of the fractionation solvent include water, a polar solvent such as an alcohol having 1 to 4 carbon atoms; hexane (Hexan), ethyl acetate (Ethyl acetate), chloroform (Chloroform), a non-polar solvent such as dichloromethane (Dichloromethane); or a mixed solvent thereof. These may be used alone or in combination of one or more, but are not limited thereto, and specifically, hexane, ethyl acetate, butanol, or water may be used alone or in combination of one or more, but limited thereto it's not going to be

In addition, the extract or fraction may be prepared and used in the form of a dry powder after extraction, but is not limited thereto.

As used herein, the term "compound" refers to acerionol (compound 1), cimiricaside F (compound 2), cimiricaside B (compound 3), 24-epi-24-O -Acetyl-7,8-didehydrohydroshengmanol 3-O-β-D-xylopyranoside (24-Epi-24-O-acetyl-7,8-didehydrohydroshengmanol 3-O-β-D- xylopyranoside; compound 4), cimiciphenone (compound 5), cimiracemate A (compound 6), (E)-3-(3'-methyl-2'-butenylidene)-1- Methyl-2-indolinone ((E)-3-(3'-methyl-2'-butenylidene)-1-methyl-2-indolinone; compound 7), (E)-3-(3'-methyl-2 '-Butenylidene)-2-indolinone ((E)-3-(3'-methyl-2'-butenylidene)-2-indolinone; compound 8), 3'-O-acetyl-24-epi-7 ,8-didehydrosimijenol-3-O-β-D-xylopyranoside (3'-O-acetyl-24-epi-7,8-didehydrocimigenol-3-O-β-D-xylopyranoside ; Compound 9), Cimiaceroside B (Compound 10), and are represented by the following Chemical Formulas 1 to 10. The compounds are derived from horseback riding and have an autophagy activation inducing effect, and pharmaceutical compositions, food compositions, and feed compositions containing them as an active ingredient are effective in preventing or treating degenerative brain diseases and improving learning or memory or improving cognitive function. indicates

The compound is a compound having the structure of Formulas 1 to 10, and may include those derived from other plants, specifically, may be derived from horseback riding, but is not limited thereto.

As used herein, the term "degenerative brain disease" refers to a disease occurring in the brain among degenerative diseases that occur with aging, and can be classified into Parkinson's disease, Alzheimer's disease, dementia, etc. According to one embodiment of the present invention, the degenerative brain disease may be Parkinson's disease or Alzheimer's disease.

As used herein, the term "dementia" refers to a disease in which a person who has been living normally suffers from brain function damage due to various causes, and the cognitive function is continuously and generally deteriorated compared to before, which significantly interferes with daily life, Alzheimer's It is a broad concept that includes disease, vascular dementia and senile dementia.

As used herein, the term "alzheimer's disease" is the most common degenerative brain disease, characterized by a very slow onset and a gradual progression. It is a disease that is accompanied by abnormalities in various other cognitive functions, such as judgment, and eventually loses all functions of daily living.

As used herein, the term “vascular dementia” refers to dementia that occurs when brain tissue is damaged by a cerebrovascular disease. Decreased cognitive function such as memory loss, language ability, temporal and spatial understanding, judgment and daily living performance, apathy, depression, anxiety, delusion, hallucinations, wandering, aggression, irritability, abnormal behavior, dietary changes, sleep In addition to mental and behavioral abnormalities such as disability, various neurological abnormalities such as unilateral motor paralysis, unilateral loss or loss of sensation, visual field disturbance, facial paralysis, pronunciation abnormality, difficulty swallowing, gait disturbance, and stiffness of extremities are accompanied from a relatively early stage.

As used herein, the term "senile dementia" refers to a person who has been living a normal life since the age of 65, as brain function is impaired due to various causes, and cognitive function is continuously and generally deteriorated compared to before, which significantly interferes with daily life. It is a disease that gives

As used herein, the term "Parkinson's disease" is caused by the gradual loss of dopamine neurons distributed in the substantia nigra of the brain, and is characterized by stability tremor, rigidity, laxity (slow movement) and postural instability. It is a chronic progressive degenerative disease of the nervous system.

As used herein, the term "prevention" refers to any action of inhibiting or delaying the progression of degenerative brain disease by administration of the pharmaceutical composition of the present invention comprising the horse horse extract or a compound isolated therefrom.

As used herein, the term “treatment” refers to any action in which the degenerative brain disease is improved or beneficially changed by administration of the pharmaceutical composition.

The "pharmaceutical composition" of the present invention may further include a pharmaceutically acceptable carrier, excipient or diluent commonly used in the preparation of a pharmaceutical composition, wherein the carrier is a non-naturally occurring carrier. may include.

The pharmaceutical composition may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., oral dosage forms, external preparations, suppositories, and sterile injection solutions according to conventional methods, respectively. .

The "pharmaceutically acceptable" means exhibiting properties that are not toxic to cells or humans exposed to the composition.

Specifically, the type of the carrier is not particularly limited, and any carrier commonly used in the art and pharmaceutically acceptable may be used. Non-limiting examples of the carrier include saline, sterile water, Ringer's solution, buffered saline, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and the like. These may be used alone or in mixture of two or more. In addition, if necessary, other conventional additives such as antioxidants, buffers and/or bacteriostats can be added and used, and diluents, dispersants, surfactants, binders, lubricants, etc. It can be used by formulating it into a dosage form, pill, capsule, granule, or tablet.

The method of administering the pharmaceutical composition for preventing or treating degenerative brain disease according to the present invention is not particularly limited, and may follow a method commonly used in the art. As a non-limiting example of the administration method, the composition may be administered by oral administration or parenteral administration. In addition, the composition for preventing or treating degenerative brain disease of the present invention may be prepared in various formulations according to the desired administration method.

Another aspect of the present invention provides a method for preventing or treating degenerative brain disease comprising administering the pharmaceutical composition to an individual.

In this case, the definition of the degenerative brain disease, prevention and treatment is the same as described above.

As used herein, "administration" means introducing a given substance into a subject by an appropriate method.

As used herein, the term "individual" refers to all animals, such as rats, mice, livestock, including humans, that have or can develop Parkinson's disease. As a specific example, it may be a mammal including a human.

More specifically, the “individual” may include companion animals. The companion animal means an animal that lives together with humans, and specific types include mammals such as dogs, cats, hamsters, and guinea pigs, and birds such as parrots and canaries, but is not limited thereto.

Specifically, the method for preventing or treating degenerative brain disease of the present invention may include administering to an individual a pharmaceutical composition comprising an extract or a compound isolated therefrom in a pharmaceutically effective amount.

The "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment and not to cause side effects, and the effective dose level is determined by the patient's gender, age, weight, Health status, disease type, severity, drug activity, drug sensitivity, administration method, administration time, administration route, and excretion rate, treatment duration, factors including drugs used in combination or concomitantly, and other factors well known in the medical field It can be easily determined by a person skilled in the art depending on the factors.

The pharmaceutical composition of the present invention may be administered at a dosage of 0.0001 to 500 mg/kg of body weight per day, more specifically 0.01 to 500 mg/kg of body weight, based on the solid content. For administration, the recommended dosage may be administered once a day, or divided into several administrations.

The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. It may also be administered single or multiple. 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 causing side effects, which can be easily determined by those skilled in the art.

In the method for preventing or treating degenerative brain disease of the present invention, the route and mode of administration for administering the composition are not particularly limited, and any route of administration as long as the composition including the composition can reach the desired site. and the mode of administration. Specifically, the composition may be administered through a variety of oral or parenteral routes, and non-limiting examples of the route of administration include oral, rectal, topical, intravenous, intraperitoneal, intramuscular, intraarterial, transdermal, and nasal and those administered intralaterally or through inhalation.

Another aspect of the present invention provides a food composition for preventing or improving degenerative brain disease comprising a horse riding extract, a fraction thereof, or a compound derived from horse riding as an active ingredient.

In this case, the definition of the horseback riding, extract, fraction, compound, degenerative brain disease and prevention is the same as described above.

As used herein, the term "improvement" refers to any action that at least reduces the severity of a parameter, eg, a symptom, associated with a condition to be treated by administration of the composition of the present invention.

As used herein, the term "food" refers to meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, beverages, tea, drinks, alcoholic beverages , vitamin complexes, health functional foods, and health foods, and includes all foods in a conventional sense.

Since the food composition of the present invention can be consumed on a daily basis, a high Parkinson's disease improvement effect can be expected, and thus it can be very usefully used for health promotion purposes.

The functional food (functional food) is the same term as food for special health use (FoSHU), and in addition to supplying nutrients, it is processed to efficiently exhibit bioregulatory functions and has high medical effects. means food. Here, 'function (sex)' refers to obtaining useful effects for health purposes, such as regulating nutrients or physiological effects on the structure and function of the human body. 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. 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 that there are no side effects that may occur when taking the drug for a long period of time by using a natural product as a raw material, and it is excellent in portability. The food of the invention can be ingested as an adjuvant to enhance the effect of improving Parkinson's disease.

The health food means a food having an active health maintenance or promotion effect compared to general food, and the health supplement food means a food for the purpose of health supplementation. In some cases, the terms health functional food, health food, and dietary supplement are used interchangeably.

Specifically, the health functional food is a food prepared by adding the extract of the present invention to food materials such as beverages, teas, spices, gum, and confectionery, or encapsulating, powdering, suspension, etc., and when ingested, It means to bring an effect, but unlike general drugs, it has the advantage that there are no side effects that may occur when taking the drug for a long time using food as a raw material.

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 may include additional ingredients that are commonly used in food compositions to improve odor, taste, vision, and the like. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, and the like may be included. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), chromium (Cr); and amino acids such as lysine, tryptophan, cysteine, and valine.

In addition, the food composition includes a preservative (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), a disinfectant (bleaching powder and high bleaching powder, sodium hypochlorite, etc.), an antioxidant (butylhydroxyanisole (BHA), butyl hydro Loxytoluene (BHT), etc.), coloring agents (tar pigments, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleach (sodium sulfite), seasonings (MSG sodium glutamate, etc.), sweeteners (dulcin, cyclamate, saccharin, etc.) , sodium, etc.), flavorings (vanillin, lactones, etc.), swelling agents (alum, potassium hydrogen tartrate, etc.), strengthening agents, emulsifiers, thickeners (flavors), film agents, gum base agents, foam inhibitors, solvents, improvers, etc. It may contain food additives. The additive may be selected according to the type of food and used in an appropriate amount.

Another aspect of the present invention provides a feed composition for the prevention or improvement of degenerative brain disease comprising a horse riding extract, a fraction thereof, or a compound derived from horse riding as an active ingredient.

The horse riding, extract, fraction, compound, prevention and improvement are the same as described above.

The composition comprising the equestrian extract, fraction, or compound derived from horseback riding of the present invention can be used for preventing or treating the onset of degenerative brain disease in individuals other than humans, such as livestock or companion animals, and a functional feed additive, or composition for feed can be used as

The content of the equestrian extract, fraction or compound derived from horseback riding in the feed composition according to the present invention can be appropriately adjusted according to the type and age of the applied livestock, the application form, the desired effect, etc., for example, 1 to 99% by weight, preferably may be used in an amount of 10 to 90% by weight, more preferably 20 to 80% by weight, but is not limited thereto.

For administration, the feed composition of the present invention may further include organic acids such as citric acid, fumaric acid, adipic acid, and lactic acid in addition to horseback riding extracts, fractions, or compounds derived from horseback riding; phosphates such as potassium phosphate, sodium phosphate, and polymerized phosphate; One or more of natural antioxidants such as polyphenol, catechin, tocopherol, vitamin C, green tea extract, chitosan, and tannic acid may be mixed and used. In addition, diluents, dispersants, surfactants, binders or lubricants may be additionally added to form an injectable formulation such as an aqueous solution, suspension, emulsion, etc., capsules, granules or tablets.

In addition, the feed composition of the present invention includes various auxiliary agents such as amino acids, inorganic salts, vitamins, antioxidants, antifungals, and antibacterial agents as auxiliary components, and plant protein feed such as milled or crushed wheat, barley, and corn, blood meal, meat meal, fish meal, etc. In addition to the main ingredients such as animal protein feed, animal fat and vegetable fat, it can be used together with nutritional supplements, growth promoters, digestion and absorption promoters, and disease prevention agents.

When the feed composition of the present invention is used as a feed additive, the feed composition may be added as it is or used together with other ingredients, and may be appropriately used according to a conventional method. The dosage form of the feed composition may be prepared as an immediate release or sustained release formulation in combination with a non-toxic pharmaceutically acceptable carrier. Such edible carriers may be corn starch, lactose, sucrose, propylene glycol. In the case of a solid carrier, it may be in dosage forms such as tablets, powders, and pills, and in the case of a liquid carrier, it may be in dosage forms such as syrups, liquid suspensions, emulsions, and solutions. In addition, the administration agent may contain a preservative, an emollient, a solution accelerator, a stabilizer, and may contain other inflammatory disease improving agents and substances useful for virus prevention.

The feed composition of the present invention can be applied to a number of animal diets, ie, feed, including mammals, poultry, fish and crustaceans. It can be used for commercially important mammals such as pigs, cattle and goats, zoo animals such as elephants and camels, and livestock such as dogs and cats. Poultry of commercial importance includes chickens, ducks and geese, and may include commercially raised fish and crustaceans such as trout and shrimp.

The feed composition according to the present invention may be mixed in an amount of about 10 to 500 g, specifically, 10 to 100 g per 1 kg by dry weight to livestock feed, and supplied as a mash after complete mixing, or additional processing process It can be through the process of palletizing, expanding, and extrusion.

Another aspect of the present invention provides a food composition or feed composition for improving learning or memory, or improving cognitive function, comprising a horse riding extract, a fraction thereof, or a compound derived from horse riding as an active ingredient.

The horse riding, extract, fraction, compound, prevention and improvement, food or feed is the same as described above.

As used herein, the term “learning or memory improvement” refers to any action in which a decrease in learning function or a decrease in memory is improved or beneficially changed by the horse riding extract, fraction, or compound derived from horse riding according to the present invention. In addition, as used herein, the term "improving cognitive function" refers to any action in which a decrease in cognitive ability is improved or advantageously changed by the horse riding extract, fraction, or compound derived from horse riding according to the present invention.

In one embodiment of the present invention, it was confirmed that the expression of LC3 protein, a membrane protein of autophagosomes, was increased, as a result of treating the horse riding extract and fractions in the SH-SY5Y cell line, which is dopaminergic cells, through which Compound 4 isolated therefrom. , it was confirmed that LC3 protein expression was increased by 5, 6, 7, 8, 10 (see Example 2).

In a specific example of the present invention, as a result of treating the compound (Compound 4) derived from horseback riding in the SH-SY5Y cell line, which is a dopaminergic cell, it was confirmed that the expression of LC3 protein, a membrane protein of autophagosomes, was increased (Example 3- see 1).

In another specific embodiment of the present invention, it was confirmed by western blotting through cell experiments that the compound (Compound 4) derived from horseback riding reduced the expression of mTOR protein (see Example 3-2).

In another specific embodiment of the present invention, it was confirmed that the compound (Compound 4) derived from horseback riding showed cytotoxicity in the SH-SY5Y cell line at a concentration of 25 μM or more (see Example 3-3).

In one specific embodiment of the present invention, as a result of administration of a compound derived from horseback riding (Compound 4) to an animal model induced by Parkinson's disease through MPTP, an activity capable of effectively inhibiting brain cell death through induction of autophagy was confirmed by western blotting (see Example 4).

In another specific embodiment of the present invention, as a result of processing the horse riding extract and fractions on a HeLa cell line transfected with amyloid precursor protein (APP), it was found that beta-amyloid 42 production was inhibited by the extract, EA fraction, and BuOH fraction. Confirmed. In addition, it was confirmed that beta-amyloid 42 production was inhibited by compounds 2, 3, 4, 9, and 10 derived therefrom (see Example 5).

In another embodiment of the present invention, as a result of treatment with a CHO cell line transfected with amyloid precursor protein (APP) with a compound derived from horseback riding, beta-amyloid 40, sAPPβ by compounds 1, 2, 3, 4 , it was confirmed that the production of β-secretase (BACE1) was inhibited (see Example 6-1).

In another embodiment of the present invention, it was confirmed that beta-amyloid 42 production was inhibited in a concentration-dependent manner by the compound (Compound 4) derived from horseback riding. In addition, it was confirmed that beta-amyloid 40 production was inhibited (see Example 6-2).

In another embodiment of the present invention, it was confirmed that the HeLa cell line transfected with amyloid precursor protein (APP) by the compound (Compound 4) derived from horseback riding did not show cytotoxicity (see Example 6-3). ).

In another embodiment of the present invention, a compound derived from horseback riding (Compound 4) is used to treat memory and cognitive decline, which is one of the symptoms of dementia, in a dementia-induced animal model. Water maze test, Y-shaped maze test, new It was confirmed through an object recognition test and a passive avoidance test (see Example 7).

As such, the composition comprising an equestrian extract, fraction or a compound derived from horseback riding of the present invention as an active ingredient specifically promotes the production of LC3-II, an autophagosome membrane protein, and suppresses mTOR protein expression to promote autophagy. It is a composition that can be effectively used for the prevention or treatment and improvement of degenerative brain diseases because it is very effective in inhibiting the generation and memory damage of beta-amyloid, which is known as the ultimate cause of degenerative brain disease. Confirmed.

The equestrian extract, fraction, or compound derived from horseback riding of the present invention increases the expression of LC3 protein, which is a membrane protein of autophagosomes, and has an excellent effect of reducing mTOR, which inhibits autophagy, and is a causative agent of Alzheimer's disease. Known beta- has an excellent effect of inhibiting the production of amyloid and memory impairment, and a composition comprising the same can be effectively used for preventing or treating degenerative brain diseases such as Parkinson's disease, Alzheimer's disease, dementia, and improving cognitive impairment.

1 is a result confirming the effect of increasing the expression level of LC3, an autophagosome membrane protein, of horse horse extract (60 μg/ml) and fraction (30 μg/ml) in SH-SY5Y cell line by Western blot. In addition, the effect of increasing the expression level of LC3 of the compound (5 μM) derived from horseback riding was confirmed by Western blot.
FIG. 2 is a result confirming the effect of increasing the expression level of LC3, an autophagosome membrane protein, according to the concentration of compound 4 in the SH-SY5Y cell line by Western blot (Control: control). ** indicates P<0.01 for control.
3 is a result confirming the effect of reducing the expression level of mTOR protein according to the concentration of compound 4 by Western blot (Control: control). ** indicates P<0.01 for control.
Figure 4 is the result of confirming the cytotoxicity according to the concentration of compound 4 in the SH-SY5Y cell line (Control: control). ** indicates P<0.01 for control.
5 is a result confirming the effect of Tyrosine hydroxylase (TH) and mTOR protein expression levels according to the administration of Compound 4 in the substantia nigra of C57BL/6 mice induced by MPTP administration by Western blotting ( Control: control group; MPTP: MPTP alone treatment group; ropinirole: ropinirole + MPTP treatment group). *** indicates P<0.001 compared to the control group, ## indicates P<0.01 for the MPTP-only treatment group, and ### indicates P<0.001 for the MPTP-only treatment group.
Figure 6 is the result of confirming the effect of increasing the expression level of Beclin1 and LC3 protein according to the administration of Compound 4 in the substantia nigra of Parkinson's disease-induced animals by western blotting (Control: Control; MPTP: MPTP alone treatment group; Ropinirole: Ropinirole) pinirol + MPTP treatment group). *** indicates P<0.001 compared to the control group, # indicates P<0.05 for the MPTP-only treatment group, ## indicates P<0.01 for the MPTP-only treatment group.
7 is a result confirming the increase in the expression level of phosphorylated ULK1 (s757) protein according to the administration of compound 4 in the substantia nigra of Parkinson's disease-induced animals by Western blotting (Control: Control; MPTP: MPTP alone treated group; Lopini) Roll: Ropinirole + MPTP treatment group). ## indicates P<0.01 for the MPTP-only treatment group, and ### indicates P<0.001 for the MPTP-only treatment group.
8 is a result confirming the increase in phosphorylated AMPK and alpha-synuclein protein expression levels according to the administration of Compound 4 in the substantia nigra of Parkinson's disease-induced animals by Western blotting (Control: control; MPTP: MPTP alone treated group; Ropinirole: Ropinirole + MPTP treatment group). * indicates P<0.05 compared to the control group, ** indicates P<0.01 compared to the control group, # indicates P<0.05 for the MPTP-only treatment group, and ## indicates P<0.01 for the MPTP-only treatment group.
9 is a Swedish mutated (Swedish) mutated HeLa cell line overexpressing APP, beta-amyloid (Aβ42) production inhibitory effect of horseback riding extract (60 μg / ml) and fraction (30 μg / ml) was measured with an Aβ42 quantification kit. It is the result. Also, it is the result of confirming the inhibitory effect of the compound (5 μM) derived from horseback riding on the production of beta-amyloid (Aβ42) (Control: control). * indicates P<0.05 compared to control, *** indicates P<0.001 for control.
10 is a result confirming the inhibitory effect of the compound on the production of beta-amyloid (Aβ40) and sAPPβ in a CHO cell line overexpressing APP. In addition, the expression level of β-secretase (BACE1) was confirmed by western blotting (CTR: control). * indicates P<0.05 compared to the control group, ** indicates P<0.01 compared to the control group, and *** indicates P<0.001 compared to the control group.
11 is a result of measuring the inhibitory effect on the production of beta-amyloid (Aβ42, Aβ40) according to the concentration of compound 4 added with the Aβ42 and Aβ40 quantitative kit (Control: control). *** indicates P<0.001 for control.
12 is a result of measuring the cytotoxicity according to the concentration of compound 4 added in HeLa cell line overexpressing APP (Control: control).
13 is an acquisition test result of comparing and measuring the time to find a platform in order to examine the memory improvement effect of compound 4 in an underwater maze test of a dementia-inducing animal, and after removing the platform, the platform stayed in the quadrant during training This is the result of a retention test measuring time (Escape latency: time taken to find the platform; Time in target quadrant: time spent in the quadrant where the platform was; Scop: scopolamine treated group; Don: donepezil treated group) . ** indicates P<0.01 for the saline-administered control group, *** indicates P<0.001 for the saline-administered control group. # indicates P<0.05 for scopolamine control, ### indicates P<0.001 for scopolamine control.
14 is a result of comparing and measuring voluntary alternating behavior (alternation behavior) in order to examine the memory improvement effect of compound 4 in a Y-shaped maze test in dementia-inducing animals (Spontaneous alteration: amount of cross behavior). *** indicates P<0.001 for the saline-administered control group. # indicates P<0.05 for scopolamine control.
15 is a result of comparing and measuring the search time for a new object in order to examine the memory improvement effect of compound 4 in a novel object recognition test in a dementia-induced animal. * indicates that P<0.05 for the saline administration control group. # indicates P<0.05 for scopolamine control.
16 is a result of measuring the time taken to enter a dark room to examine the memory improvement effect of Compound 4 in a passive avoidance test in dementia-inducing animals (retention latency time: time taken to enter a dark room). * indicates that P<0.05 for the saline administration control group. ## indicates P<0.01 for scopolamine control.

Hereinafter, the present invention will be described in more detail through Examples and Experimental Examples. However, these Examples and Experimental Examples are for illustrative purposes of the present invention, and the scope of the present invention is not limited to these Examples and Experimental Examples.

Example 1: Preparation of Equestrian Extract and Fractions and Isolation of Compounds

60 L ethanol was added to 5 kg of horseback riding, and the aqueous phase was ultrasonically extracted for 4 hours using a sonicator, and then filtered to obtain an extract. The extraction process was repeated 3 times. The obtained extract was concentrated under reduced pressure at 40° C. or lower and then freeze-dried to obtain 636.5 g of a powdery extract, 12.7%. The horse horse extract was turbid in distilled water, and n-hexane was added thereto, shaken sufficiently, and then separated for a certain period of time. The hexane layer obtained by repeating this process twice or more was filtered and concentrated under reduced pressure to obtain a final hexane fraction. In this way, ethyl acetate (EA), n-butanol (BuOH), and water were sequentially fractionated to obtain a final fraction. Horse riding gave a hexane fraction (31.5 g, 5%), an EA fraction (182 g, 28.6%), a BuOH fraction (52 g, 8.2%), and a water fraction (293 g, 46%).

The EA fraction was repeated silica gel column chromatography using n-hexane-EtOAc (95:5, 80:20, 40:60, v/v) as a developing solvent to obtain seven fractions (B1 to B7). Sephadex LH-20 column chromatography and YMC-RP18 column chromatography were repeatedly performed for fraction B-3 to obtain acerionol ( compound 1 , 4.8 mg), cimiciphenone ( compound 5 , 6.0 mg), simi A compound of racemite A (cimiracemate A; compound 6, 22.3) was obtained.

Subsequently, fraction B7 (1.1 g) was added to Sephadex?? LH-20 column chromatography was performed to obtain small fractions (B7.1 to B7.3). Small fraction B7.1 (0.4 g) was repeatedly subjected to YMC RP-C18 column chromatography and silica gel column chromatography to (E)-3-(3'-methyl-2'-butenylidene)-1-methyl- 2-indolinone ((E)-3-(3'-methyl-2'-butenylidene)-1-methyl-2-indolinone; compound 7 , 5.0 mg) and (E)-3-(3'-methyl- 2'-butenylidene)-2-indolinone ((E)-3-(3'-methyl-2'-butenylidene)-2-indolinone; Compound 8 , 20.3 mg) was obtained.

The BuOH fraction was divided into 4 fractions (W- 1 to W-4) was obtained. The fraction W-2 (2.1 g) was subjected to silica gel column chromatography using CHCl3-MeOH-H2O, 6.5:1:0.1 as a developing solvent, followed by YMC RP using water-methanol (65→100%) as a developing solvent. -C18 column chromatography and Sephadex LH-20 column chromatography were repeatedly performed to obtain cimiricaside F (compound 2 , 7.5 mg).

Fraction W-3 (1.7 g) was subjected to YMC RP-C18 column chromatography using H2O-MeOH (5:1, 3:1, 1:1, and 100% MeOH) as a developing solvent to obtain a small fraction (W-3.1 to W-3.4) were obtained. Small fraction W-3.1 (0.42 g) was subjected to silica gel column chromatography using CH2Cl2-—MeOH (10:1) as a developing solvent to obtain 3'-O-acetyl-24-epi-7,8-didihydrosimase nol-3-O-β-D-xylopyranoside (3'-O-acetyl-24-epi-7,8-didehydrocimigenol-3-O-β-D-xylopyranoside; compound 9 , 5.6 mg) was obtained, and small fraction W-3.3 (0.07 g) was repeatedly performed with YMC RP-C18 column chromatography and silica gel column chromatography with a developing solvent of water-methanol (1:1) to cimiricaside B (cimiricaside B; compound) 3 , 5.1 mg) was obtained.

Small fractions W-4 (1.3 g) were subjected to silica gel column chromatography using CHCl3-EtOAc (8:1, 4:1, and 2:1) as a developing solvent to obtain 5 small fractions (W-4.1 to W-4.5). ) was obtained. Sephadex LH-20 column chromatography and silica gel column chromatography were repeatedly performed for sub-fraction W-4.1 to 24-epi-24-O-acetyl-7,8-didihydrosepmanol 3-O-β-D- Xylopyranoside (24-Epi-24-O-acetyl-7,8-didehydrohydroshengmanol 3-O-β-D-xylopyranoside; Compound 4 , 6.5 mg) was isolated and purified, and a small fraction W-4.5 was used in acetone- YMC RP-C18 column chromatography was performed with water (1:2) as a developing solvent to obtain Cimiaceroside B (Cimiaceroside B; Compound 10 , 4.56 mg).

Example 2: Autophagy-inducing effect of equestrian extract and fractions

In order to measure the effect of increasing the LC3 protein expression level of the horse riding extract and fractions obtained in Example 1, the SH-SY5Y cell line, which is a dopaminergic cell line, is a 10% heat-inactivated fetus from Gibco (Grand Island, NY, USA). Dulbecco's modified eagle medium nutrient mixture F-12 (Ham) 1x containing serum (hereinafter referred to as FBS) and 1% penicillin/streptomycin (hereinafter referred to as P/S, Gibco); It was used by culturing in DMEM/F12 (Gibco) medium. This cell line was purchased from ATCC: The Global Bioresource Center (CRL-2266??) (Manassas, Virgina 20108, USA).

Specifically, to the cell line (SH-SY5Y), 60 μg/ml of the equestrian extract obtained in Example 1 and 30 μg/ml of the fraction were added, and then _{incubated at 5% CO 2} , 37° C. for 24 hours, and collected. and centrifuged at 13,000 rpm for 6 minutes and washed twice with DPBS (Dulbecco's phosphate buffered saline). A lysis solution (1X lysis buffer, 1X protease inhibitor cocktail, 1x phenylmethyl sulfonyl fluoride) was added to the obtained cells and reacted at 4°C for 30 minutes, and then completely lysed while shaking at the same 4°C condition for 30 minutes After centrifugation at 13,000 rpm for 20 minutes, the supernatant from which the protein was released was taken. Protein assay dye Reagent concentrate (Bio-Rad) was used for the supernatant and the protein was quantified by the Bradford method. The expression levels of LC3 and GAPDH proteins in the cell lysate were measured by Western blot using an LC3, GAPDH (Cell signaling technology) antibody. GAPDH was used as a loading control.

Thereafter, the result of confirming the rate of transfer from the LC3I protein to the LC3II protein is shown in FIG. 1 . A case in which horseback riding extract was not treated was used as a control group. As a result, as can be seen in FIG. 1 , it was confirmed that the protein expression level of LC3 was remarkably increased by the equestrian extract, the EA fraction, and the BuOH fraction. In addition, it was confirmed that LC3 protein expression was increased by the compounds 4, 5, 6, 7, 8, and 10 isolated therefrom. Through this, the expression level of LC3 protein, one of the autophagic pulmonary membrane proteins, when the horse horse extract and fractions or compounds isolated therefrom (compounds 4, 5, 6, 7, 8, 10) were treated with dopaminergic cell lines. and increased the transition from LC3 I to LC3 II, which is an indicator of autophagy activity, and as a result, it was confirmed that the autophagy-inducing effect was exhibited.

Example 3: Autophagy Inducing Effect of Compounds Derived from Horse Riding

Example 3-1: Effect of increasing LC3 protein expression level of compounds derived from horse riding

In order to measure the concentration-dependent effect of increasing the LC3 protein expression level of the compound isolated from the equestrian extract obtained in Example 1 (hereinafter, equestrian compound; compound 4), the equestrian compound described in FIG. 2 was added to the cultured SH-SY5Y cell line. After the addition amount, 5% CO ₂ , and incubated at 37° C. for 24 hours, the experiment was performed in the same manner as in Example 2-1. Primary antibodies used in this experiment include LC3 and GAPDH (Cell signaling technology), and GAPDH was used as a loading control.

Thereafter, the result of confirming the rate of transfer from the LC3I protein to the LC3II protein is shown in FIG. 2 . A case in which no horse riding compound (Compound 4) was treated was used as a control group. As a result, as can be seen in FIG. 2 , it was confirmed that the expression level of LC3 protein was significantly increased in a concentration-dependent manner by the addition of equestrian compound 4 compared to the control group. When treated, it was confirmed that the rate of conversion from LC3I protein to LC3II protein was 2.55. Through this, when the equestrian compound (Compound 4) was treated with dopaminergic cell lines, the expression level of LC3 protein, one of the autophagic lung membrane proteins, was increased, and the transition from LC3 I to LC3 II, which is an indicator of autophagy activity, was inhibited. increased, and as a result, it was confirmed that it exhibits an autophagy inducing effect.

Example 3-2: Effect of compounds derived from horseback riding on mTOR protein expression inhibition

In order to confirm the effect of the equestrian compound (Compound 4) on inhibiting the expression level of mTOR protein, it was added to the cultured SH-SY5Y cell line at the added amount of the equestrian compound described in FIG. 3, and then _{incubated at 5% CO 2} , 37° C. for 24 hours. Experiments were carried out in the same manner as in Example 2. Primary antibodies used in this experiment include mTOR and GAPDH (Cell signaling technology), and GAPDH was used as a loading control.

Thereafter, the results showing the relative density of mTOR were described in FIG. 3 , and the case where the horse riding compound was not treated was used as a control group. As a result, as can be seen in FIG. 3 , it was confirmed that the expression of mTOR protein was decreased in a concentration-dependent manner by the riding compound compared to the control group. Through this, when the equestrian compound was treated in dopaminergic cell lines, it was confirmed that the expression level of the mTOR protein, which inhibits autophagy, was reduced, resulting in an autophagy-inducing effect.

Example 3-3: Confirmation of cytotoxicity of compounds derived from horse riding

In order to confirm the cytotoxicity of equestrian compound 4, it was added to the cultured SH-SY5Y cell line at the amount of equestrian compound added in FIG. 3 , and then _{cultured at 5% CO 2} , 37° C. for 24 hours, and EZ-Cytox kit (DAEILLAB Co, Ltd, Republic of Korea). The absorbance was measured after incubating the EZ-Cytox solution at 37° C. for 2 hours.

Thereafter, the results showing the relative cell viability compared to the control were described in FIG. 4 , and the case where the horse riding compound 4 was not treated was used as a control. As can be seen in FIG. 4 , it was confirmed that the SH-SY5Y cell line showed cytotoxicity at a concentration of 25 μM or more compared to the control. Through this, it was confirmed that there is no cytotoxicity even at a concentration 5 times higher (50 μM) than the concentration (5 μM) showing autophagy-inducing activity, so it can be safely used by including the equestrian compound as an active ingredient.

Example 4: Effect of a compound derived from horseback riding on brain cell death through autophagy in an animal model of Parkinson's disease by administration of MPTP

C57BL/6 male mice (8 weeks old) were used, and feed and water were ad libitum. Mice were divided into 5 groups, 7 mice in each group. The control group and the MPTP single treatment group were orally administered with physiological saline once for 3 days, and the horse riding compound (5 mg/kg or 15 mg/kg) group obtained in Example 1 was dissolved in physiological saline and then administered orally once for 3 days. did. For the positive control group, ropinirole was dissolved in physiological saline and administered orally once for 3 days. From the 4th to the 8th day of drug administration, MPTP (30 mg/kg) dissolved in physiological saline was administered orally once to all groups except the control group 1 hour after drug administration, and on the 9th to 10th days after drug administration, all groups except the control group The same amount of drug was administered orally once. After killing the mice in each group on the 7th day after the end of drug administration, brain tissues (substantia nigra and striatum) were isolated. Brain tissue was homogenized with PRO-PREPTM lysis buffer (iNtRON, Gyeonggi, Korea) supplemented with phosphatase inhibitor cocktail set I (Sigma-Aldrich, MO, USA). The homogenate was left at 4°C for 30 minutes, and then centrifuged at 13,000 rpm, 4°C, for 30 minutes to obtain a supernatant. The supernatant was quantified for protein by the Bradford method using Protein assay dye reagent concentrate (Bio-Rad). After mixing with a certain amount of SDS loading buffer and heating at 99° C. for 5 minutes, protein expression was measured by western blotting in the same manner as in Example 2. The primary antibodies used in this experiment were TH, Beclin-1, p-mTOR, mTOR, p-ULK1(s757), ULK1, p-AMPK, AMPK, LC3, alpha-synuclein, GAPDH (Cell signaling technology). and GAPDH was used as a loading control.

Thereafter, the results showing the relative densities of the proteins are described in FIGS. 5 to 8, the case in which the horse riding compound and MPTP are not treated as a control group, and the case in which ropinirole and MPTP are administered as a positive control group. At this time, MPTP is a substance used in an animal model of Parkinson's disease, a dopamine antagonist, and ropinirole is a substance used in drug treatment for Parkinson's disease, and is a dopamine agonist.

As a result, as can be seen in FIG. 5 , as a result of confirming the expression of phosphorylated mTOR protein that inhibits autophagy, it was confirmed that it was significantly reduced in the group administered with the horse riding compound 15 mg/kg. In addition, while the expression of TH, an enzyme regulating the dopamine precursor (L-DOPA) in the MPTP-only group, was decreased compared to the control group, it was confirmed that the expression of TH protein was significantly increased in the group administered with the horse riding compound. there was.

As a result, as can be seen in FIG. 6 , in the case of Beclin-1 protein, which is a representative protein inducing autophagy, the MPTP alone treatment group decreased compared to the control group, whereas the horse riding compound 15 mg/kg was administered. It was confirmed that there was a significant increase in one group. In addition, in the case of LC3 protein, which is an autophagosome membrane protein, it was confirmed that the LC3 protein expression was significantly increased in the group administered with the horse riding compound compared to the control group.

As a result, as can be seen in FIG. 7 , the expression of phosphorylated ULK1 protein inducing autophagy in the MPTP-only treatment group was decreased compared to the control group, whereas the expression of phosphorylated ULK1 protein in the group administered with the horse riding compound was reduced. It can be seen that this increases.

As a result, as can be seen in FIG. 8 , in the case of the MPTP-only treatment group, the expression of phosphorylated AMPK protein inducing autophagy was reduced compared to the control group, whereas in the group administered with the horse riding compound 15 mg/kg It was confirmed that there was a significant increase. In addition, in the case of the MPTP-only treatment group, the abnormal aggregation of α-synuclein protein, known as the causative protein of Parkinson's disease, was significantly increased compared to the control group, whereas in the group administered with the horse riding compound, alpha- It can be seen that the synuclein protein expression is reduced.

From the above results, it can be confirmed that the equestrian compound exhibits significant concentration-dependent activity in the treatment and improvement of Parkinson's disease through the inducing effect of autophagy in brain cells in an animal model induced with MPTP toxicant. there was.

Example 5: Alzheimer's disease treatment and improvement effect of horse riding extract and fractions

In order to measure the Aβ42 production inhibitory effect of the horse riding extract and fractions obtained in Example 1, the HeLa cell line transfected with Swedish mutants of human-derived amyloid precursor protein (APP) was treated with DMEM culture medium ( Gibco), 10% FBS, 1% P/S, 260 μg/ml Zeocin, and 400 μg/ml G418 were used. This cell line was provided by Professor Tae-Wan Kim (Prof. Tae-Wan Kim, Department of Pathology, Columbia University Medical Center, New York, NY10032, USA).

Specifically, to the cell line (swAPP HeLa cell), 60 μg/ml of the equestrian extract and the fraction obtained in Example 1 were added in an amount of 30 μg/ml, followed by incubation at 5% CO2, 37° C. for 8 hours, and as a culture medium. Secreted Aβ42 was measured using human beta-amyloid [1-42](Aβ42) Colorimetric ELISA kit (Thermo Fisher scientific).

The results of measuring the amount of Aβ42 are described in FIG. 9, and the case in which horseback riding extract was not added was used as a control group. As a result, as can be seen in FIG. 9 , it was confirmed that the production of Aβ42 was significantly reduced by the horse riding extract, EA fraction, and BuOH fraction. In addition, it was confirmed that beta-amyloid 42 production was inhibited by compounds 2, 3, 4, 9, and 10 isolated therefrom. Through this, when horse horse extract, fraction or a compound isolated therefrom is treated, beta-amyloid protein, which is known as the cause of Alzheimer's disease, is reduced, thereby significantly showing activity in the treatment and improvement of Alzheimer's disease. could

Example 6: Alzheimer's disease treatment and improvement effect of compounds derived from horseback riding

Example 6-1: sAPPβ, β-secretase production inhibitory effect of compounds derived from horse riding

In order to measure the Aβ40 production inhibitory effect of the equestrian compound obtained in Example 1, a CHO cell line transfected with a human-derived amyloid precursor protein (APP) was used.

Specifically, the cell line (APP CHO cell) was treated with the equestrian compound obtained in Example 1 to 2.5, 5, and 10 uM, and then cultured at 5% CO2, 37°C for 24 hours, and Aβ40 secreted into the culture medium was converted into human Beta-amyloid [1-40] (Aβ40) was measured using a Colorimetric ELISA kit (Thermo Fisher scientific). In addition, the IC50 was calculated by confirming the cytotoxicity using the MTT assay, and the result of measuring the IC50 is shown in FIG. 10A. The production of beta-amyloid 40 was inhibited by 50% at a concentration of 15 uM or less.

sAPPβ secreted into the culture medium was confirmed by western blotting using a water-soluble beta amyloid precursor protein (sAPPβ) (IBL) antibody. The expression level of β-secretase (BACE1) was confirmed in the same manner as in Example 2 at all times by culturing the horse riding compound for 24 hours. The primary antibody used in this experiment was β-secretase (Cell signaling technology), and β-tubulin was used as a loading control.

Thereafter, the results of measuring the expression levels of sAPPβ and BACE1 were described in FIG. 10B, and the case where the horse riding compound was not treated was used as a control group. As a result, as can be seen in FIG. 10 , it was confirmed that sAPPβ production and β-secretase (BACE1) protein expression were decreased in a concentration-dependent manner by the riding compound 1-4 compared to the control group. Through this, it can be confirmed that the equestrian compound reduces the production of beta-amyloid soluble precursor protein (sAPPβ), known as the causative agent of dementia, and the expression of BACE1, an enzyme that makes beta-amyloid, thereby exhibiting therapeutic and ameliorating activity of Alzheimer's disease. there is.

* Example 6-2: beta-amyloid (Aβ) production inhibitory effect of compounds derived from horseback riding

In order to measure the Aβ42 production inhibitory effect of the horse riding compound (Compound 4) obtained in Example 1, a HeLa cell line transfected with Swedish mutants of human-derived amyloid precursor protein (APP) was treated with DMEM. Culture solution (Gibco), 10% FBS, 1% P/S, 260 μg/ml Zeocin and 400 μg/ml G418 were used. This cell line was provided by Professor Tae-Wan Kim (Prof. Tae-Wan Kim, Department of Pathology, Columbia University Medical Center, New York, NY10032, USA).

Specifically, the equestrian compound (Compound 4) obtained in Example 1 was added to the cell line (swAPP HeLa cell) in the amount described in FIG. 11 , and then _{incubated at 5% CO 2} , 37° C. for 8 hours, and secreted into the culture medium. Aβ42 was measured using human beta-amyloid [1-42] (Aβ42) Colorimetric ELISA kit (Thermo Fisher scientific). To quantify Aβ40, human beta-amyloid [1-40] (Aβ40) Colorimetric ELISA kit (Thermo Fisher scientific) was used.

Thereafter, the results of measuring the relative amounts of Aβ42 and Aβ40 to the control were described in FIG. 11 , and the case where the horse riding compound was not treated was used as a control. As a result, as can be seen in FIG. 11 , the production of Aβ42 and Aβ40 was inhibited in a concentration-dependent manner by the equestrian compound, and specifically, the production of Aβ42 secreted in the medium containing 2.5 μM and 5 μM of the equestrian compound was 69% and It was confirmed that the decrease was 77%. In addition, it was confirmed that the production of Aβ40 secreted in the medium containing 2.5 μM and 5 μM of the equestrian compound was reduced by 52% and 60%, respectively. Through this, when the equestrian compound was treated, it was confirmed that the production of beta-amyloid protein, known as the cause of Alzheimer's disease, was reduced, and as a result, the equestrian compound was significantly concentration-dependently active in the treatment and improvement of Alzheimer's disease. could

Example 6-3: Confirmation of cytotoxicity of compounds derived from horse riding

In order to confirm the cytotoxicity of the equestrian compound 4 obtained in Example 1, the equestrian compound obtained in Example 1 was added to the cell line (swAPP HeLa cell) in the amount described in FIG. 11, and then 5% CO ₂ , After incubation at 37°C for 8 hours, EZ-Cytox solution was incubated at 37°C for 1 hour, and then absorbance was measured.

Thereafter, the results showing the relative cell viability compared to the control group were described in FIG. 12 , and the case where the horse riding compound 4 was not treated was used as a control group. As a result, as can be seen in FIG. 12 , it was confirmed that the HeLa cell line overexpressing APP did not show cytotoxicity by the riding compound 4 compared to the control group. Through this, there is no cytotoxicity even at a concentration 10 times higher (50 μM) than the concentration (5 μM) showing beta-amyloid production inhibitory activity. was able to confirm that

Example 7: Effect of compounds derived from horseback riding on improving memory and cognitive function

ICR male mice (8 weeks old) were used, and feed and water were ad libitum. The equestrian compound was orally administered once a week at 1.25 mg/kg, 2.5 mg/kg or 5 mg/kg. The positive control group was orally administered with donepezil (donepezil) 4 mg/kg, and the control group was orally administered with saline. In addition, 1 mg/kg of scopolamine was subcutaneously administered 30 minutes before the test to induce brain function memory and cognitive impairment in mice. By conducting an experiment, it was analyzed whether there was an effect of improving memory compared to the scopolamine control group.

Example 7-1: Underwater Maze Test

Acquisition of the underwater maze test was performed by measuring the time (escape latency, unit: seconds) until finding a platform submerged in water at 30-minute intervals 3 times a day for 5 days. The maximum allowable time was limited to 90 seconds, and once they got on the platform, they were allowed to stay there for at least 5 seconds.

The holding test was performed by measuring the time (Time in target quadrant, unit: seconds) to stay in the quadrant where the platform was by allowing them to swim freely in the tank where the platform was removed 24 hours after the acquisition trial was completed.

Thereafter, the results of the acquisition test and the grip test of the water maze test were described in FIG. 13, and the experimental group in which cognitive dysfunction was induced by scopolamine administration was used as a control group. As a result, as can be seen in FIG. 13 , the equestrian compound 2.5 mg/kg or 5 mg/kg administration group found the platform faster than the control group from the 4th day of the test and showed a significant difference. In addition, as a result of the holding test, the time spent in the quadrant where the platform of the riding compound administration group was located showed a tendency to increase compared to the control group. Through this, it was confirmed that when the horse riding compound was treated, it exhibited an effect of improving cognitive function.

Example 7-2: Y-shaped maze test

Three arms were in the shape of a letter Y, each of which had a length of 30 cm, a height of 12 cm, and a width of 5 cm, and was performed using a Y-shaped maze measuring device connected at an angle of 120 degrees. The mouse was placed at the end of one passage of the maze, and the passage was freely explored for 5 minutes, and the movement was recorded as the number of crossings (alternation) by recognizing that the passage through the rear foot of the mouse was regarded as an arm entry. When passing through three different passageways in succession, 1 point was given as the actual number of crossings. %) was obtained.

Then, the results of the spontaneous cross-behavior of the Y-shaped maze test were described in FIG. 14, and the scopolamine-administered group was used as a control group. As a result, as can be seen in FIG. 14 , the amount of spontaneous crossover in the 5 mg/kg group of riding compound was 61.8±3% and 50.1±2.6% of the scopolamine control group, which was significantly 23.4 by the administration of the riding compound. % of the memory improvement effect was confirmed. Through this, it was confirmed that when the horse riding compound was treated, it exhibited an effect of improving memory.

Example 7-3: Novel object recognition test

For the new object recognition test, a 50 cm Х 50 cm Х 50 cm box was used as an experimental device, and it was freely acclimatized to the environment in the experimental device for 5 minutes once a day for 2 days. After installing two objects (A and B) in the experimental apparatus for the next day's acquisition phase, the mouse approached within 1 cm of each object and the search time was measured for 5 minutes. On the 4th day, for the retention phase, one of the two objects was replaced with a new object (new object) (A and C), and the time the mouse approached and explored within 1 cm of each object was measured for 5 minutes. did. The ratio of the search time for each object (Novel Objecting Exploring Index) was obtained.

Afterwards, the result of the search time ratio for the object of the new object recognition test was described in FIG. 15 , and the scopolamine administration group was used as a control group. As a result , as can be seen in FIG. 15 , the new object search time ratio of the horse riding compound 5 mg/kg administration group was 68 ± 2.4% and that of the scopolamine control group was 60.1 ± 2.1%, which was significantly increased by the administration of the horse riding compound. increased. Through this, it was confirmed that when the horse riding compound was treated, it exhibited an effect of improving cognitive ability.

Example 7-4: passive avoidance test

For the passive avoidance test, a shuttle box divided into two compartments by a partition door was used as an experimental device. One compartment was illuminated by lighting and the other compartment was unlit and darkened with a black cloth. On the first day, mice were allowed to stay in a bright room for 30 seconds to explore, and then open a partition door to enter the dark room. The time until entering the dark room (acquisition latency time) was measured, and as soon as entering the dark room, the partition door was closed and an electric shock of 0.3 mA was applied through the grid floor for 3 seconds to make the mouse remember the electric stimulation. After 24 hours, the mouse was placed in a bright room, the partition door was opened, and the retention latency time was measured for all the mice's feet to enter the dark room, and the maximum time was limited to 180 seconds. It was judged that the larger the retention latency time, the better the memory of passive avoidance was maintained.

Thereafter, the retention latency time results of the passive avoidance test are shown in FIG. 16 . As a result, as can be seen in FIG. 16 , the time to enter the dark room after learning the electric stimulation of the horse riding compound 2.5 mg/kg and 5 mg/kg administration group was 28.8 ± 4.3 seconds and 46.9 ± 11.4 seconds, respectively, and scopolamine It was confirmed that the memory for passive avoidance was significantly improved by administration of the riding compound in 10.8 ± 4.1 seconds of the control group. Through this, it was confirmed that when the horse riding compound was treated, it exhibited an effect of improving memory.

From the above results, it was confirmed that the horse riding extract of the present invention or a compound isolated therefrom has an excellent effect on improving memory and cognitive function for dementia.

From the above results, treatment with equestrian extracts, fractions thereof or compounds derived from equestrian horses increased the expression of autophagic pulmonary membrane protein LC3 (Figs. 1 and 2), and decreased the expression of m-TOR, which inhibits autophagy ( 3) It was confirmed that the autophagy-inducing effect was exhibited. In addition, in an animal model of Parkinson's disease induced with MPTP toxicant, treatment with a compound derived from horseback riding promotes the amount of TH, an enzyme that regulates a precursor of dopamine (L-DOPA), and p-mTOR, which inhibits autophagy. By reducing the expression of the protein and increasing the expression of p-AMPK, p-ULK1, and Beclin-1 proteins that induce autophagy ( FIGS. 5-8 ), it was confirmed that it exhibits activity in the treatment and improvement of Parkinson's disease. Also, equestrian extract. It was confirmed that treatment with a fraction thereof or a compound derived from horseback riding inhibits the expression of beta-amyloid (Aβ42 and Aβ40), which is a causative protein of Alzheimer's disease (FIGS. 9-11), thereby showing activity in the treatment and improvement of Alzheimer's disease. In addition, it was confirmed that the treatment of compounds derived from horseback riding showed improvement effects in various cognitive ability and memory tests, such as a water maze test and a Y-shaped maze test ( FIGS. 13-16 ). Through this, it can be confirmed that the equestrian extract of the present invention, a fraction thereof, or a compound derived from horse riding has an excellent effect in the treatment and prevention of degenerative brain disease, and improvement of memory and cognitive function.

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 described below and their equivalents.

Claims (13)

A pharmaceutical composition for preventing or treating Parkinson's disease, comprising an ethyl acetate or butanol fraction of horseback riding ethanol extract as an active ingredient. delete delete delete delete The pharmaceutical composition according to claim 1, wherein the composition activates autophagy function. The pharmaceutical composition of claim 1, wherein the composition increases the expression of LC3 protein and inhibits the expression of mTOR protein. delete A method for preventing or treating Parkinson's disease, comprising administering the pharmaceutical composition according to any one of claims 1 to 7 to a subject other than a human. A food composition for preventing or improving Parkinson's disease, comprising the ethyl acetate or butanol fraction of horseback riding ethanol extract as an active ingredient. delete A feed composition for preventing or improving Parkinson's disease, comprising an ethyl acetate or butanol fraction of horseback riding ethanol extract as an active ingredient. delete

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