KR101067153B1 - chestnut inner skin extract having neuronal cell protective effect, acetylcholinesterase inhibitory effect, and use thereof - Google Patents

Abstract

The present invention relates to a method for separating catechins, epicatechins, and garlic acids from yulpi extracts, and to novel uses of yeast extracts and catechins, epicatechins, and garlic acids isolated therefrom. More specifically, the present invention relates to a composition for preventing and treating cerebral disease and a functional health food composition for improving brain disease, which comprises at least one polyphenol component selected from yulpi extract or catechin, epicatechin, and garlic acid isolated therefrom. The functional health food composition exhibits particularly excellent effects on degenerative neurological diseases, including Alzheimer's disease.

Catechin, Epicatechin, Garlic Acid, Polyphenol Compound, Alzheimer's Disease, Yulpi, Acetylcholinesterase, Amyloid Beta Protein

Description

Chestnut inner skin extract having neuronal cell protective effect, acetylcholinesterase inhibitory effect, and use}

The present invention relates to a composition for preventing and treating cerebral disease and a functional health food composition for improving brain disease, which comprises at least one polyphenol component selected from yulpi extract or catechin, epicatechin, and garlic acid isolated therefrom.

Since the launch of the WTO system and the conclusion of the FTA with Chile and the United States, Korea's agriculture has been in crisis in many ways. Existing agricultural crops have already reached their limits, and Korea's agricultural production base is in total collapse.

In particular, chestnut is the most important item among forest products, which has greatly contributed to the income increase through exports, but in 2002 and 2003, the typhoon plunged due to the continuous typhoon, the export volume to Japan, the main export market, and the import of Chinese chestnut. The rapid increase of the entire industry is facing a significant crisis. According to a study by the Korea Rural Economic Institute, the total production of chestnuts decreases by about 21% if market conditions remain the same. It is expected to be enough. In addition, per capita consumption is expected to decline by about 24% to 0.99kg, and producer prices are expected to rise by 62%. Therefore, growers' profits are expected to increase by 122% per ha as producer prices rise.

Chestnut tree ( Castanea) crenata S.) is a deciduous tree belonging to the oak family, and is a plant that is widely grown or grown throughout Korea. Mainly, the fruit is called Yulja (栗 子), which is harvested and edible or used as processed foods such as herbal medicine and sediment. In addition to fruits, bark, roots, flowers and leaves are used to treat wounds and inflammation. The leaves were used as folk medicine for allergic diseases and asthma cough caused by sumac in Korea and Europe.

However, Yulpi has been disposed of in the past as well as the present. Therefore, it is not a very difficult process to secure Yulpi at local processing factories because chestnut yulpi is invisible. Especially, yulpi occupies about 25% of the total chestnut content during processing. Estimates of about 20, and discarded from these plants, are estimated at 15,000 tonnes based on 1996 processing volume. Therefore, if you use materialized research to develop high value-added products using Yulpi, the productive and economic value will be very high.

In addition, until now, research on chestnuts has been conducted for the purpose of processing with canned meat and processed research with starch, and the nutrition and functional substances of yulpi have not been scientifically identified. to be.

In addition, the main production sites of chestnut trees include Gyeongnam (Hadong, Sancheong, Pearl, Hapcheon, etc.), Jeonnam (Gwangyang, Suncheon, Gurye, etc.), Chungnam (Gongju, Buyeo), and Chungbuk Chungju. Accounts for 85% of national production in 2005. Among them, Gyeongnam has the largest cultivated area in the whole country, so research on Yulpi also implies regional needs.

On the other hand, recent Alzheimer's disease research has shown that brain cell destruction by amyloid β protein and the levels of acetylcholine (ACh; neurotransmitter) due to their structure, physiology and accumulation in the brain ( level) is the focus. Treatment of Alzheimer's disease is primarily aimed at lightening the patient's symptoms and slowing the progression of the disease.

The anti-Alzheimer's disease activating components reported to date are mainly plant-derived small molecules. In particular, choline esterase inhibitors (ChEIs) are widely used as drugs to improve cognitive symptoms in patients with senile dementia. As the first generation of ChEIs, tacrine, which was first approved for anti-dementia, has to be administered four times a day due to its short duration of action. The second generation of ChEIs, which are attracting attention these days, can be taken once a day with donepezil, which was developed by Eisaisa in Japan, approved by the US FDA at the end of 1996, and sold in 30 countries since 1997, and reduced peripheral side effects by selective inhibition. Rivastingmine is a drug developed by Novartis in the United States, approved in December 1997 in Switzerland, used in the EU and South American countries, and is being prepared for approval in the United States and Canada. Rivastingmine can be taken twice a day and is highly specific to the central nervous system, greatly reducing peripheral side effects, and is reported to have little liver toxicity due to metabolism in the kidney. Metrifonate is undergoing a phase 3 clinical trial in patients with dementia and has been reported to have a long duration of action as irreversible AChEIs.

However, since the drugs only temporarily relieve symptoms, there is an urgent need for a drug development technology that treats the disease fundamentally or suppresses the progression of the disease itself. In addition, studies on inhibiting or blocking amyloid beta protein production and amyloid beta protein-induced cytotoxicity continue, but there are no new materials or drugs that realize this.

Accordingly, the present inventors have searched for phytochemicals for the prevention and treatment of brain diseases including Alzheimer's disease, which are increasingly increasing in severity, and searching for methods for materializing them into pharmaceuticals, food medicines, and functional health food compositions. It was. As a result, the present invention was completed by searching for micromaterials that inhibit neurotoxicity induced by amyloid beta from Yulpi, one of the native forest products of Korea, and separating and purifying them. This could be a measure of the use of Yulpi resources that are being discarded, which is expected to contribute to the prevention of waste of resources and to increase income of chestnut farmers.

An object of the present invention is to provide a pharmaceutical composition for preventing and treating cerebral disease comprising at least one polyphenol component selected from the group consisting of yulpi extract or garlic acid, catechin, and epicatechin isolated therefrom.

Still another object of the present invention is to provide a health food composition for improving brain disease comprising at least one polyphenol component selected from the group consisting of yulpi extract or garlic acid, catechin, and epicatechin isolated therefrom.

Still another object of the present invention is to provide a method for separating garlic acid, catechin, and epicatechin from yulpi extract.

As one embodiment, the present invention relates to a pharmaceutical composition for preventing and treating brain diseases comprising the extract of Yulpi as an active ingredient.

In another aspect, the present invention relates to a composition for the prevention and treatment of brain diseases comprising at least one polyphenol component selected from the group consisting of garlic acid, catechin, and epicatechin or a pharmaceutically acceptable salt thereof.

The present inventors have found that the extract of Yulpi has antioxidant activity against oxidative stress (ROS, lipid peroxidation, protein alteration, mitochondrial DNA oxidation, etc.), and has cerebral nerve cell protective activity therefrom. In addition, it was further intended to identify the active ingredient showing the neuroprotective activity, especially among the yulpi extract. To this end, according to a specific embodiment of the present invention, the present inventors extract a yulpi with water to obtain a water extract (Example 1), and then fractionate it with ethyl acetate and perform a chromatography therefrom to obtain a polyphenol compound exhibiting activity. Isolated and purified, these polyphenol compounds were specifically garlic acid, catechin, and epicatechin (Example 2, FIG. 1).

Yulpi extract has neuroprotective activity against brain cells by antioxidant activity.

As used herein, the term "Yulpi" refers to a part of the deciduous tree of the oak family, Chestnut ( Castanea crenata S.), except for the flesh (yellow) and the skin, and is also called chestnut endothelium, which includes natural, hybrid, and varieties of plants. All are included, the yulpi extract can be extracted from natural, hybrid, and varieties chestnut.

The present inventors confirmed that oxidative stress is induced during neural cell necrosis process by amyloid beta protein, and yulpi extract effectively inhibits damage caused by amyloid beta protein, thereby ultimately exhibiting neuronal cell protective action. As a result, Yulpi extract effectively reduces the oxidative stress induced by amyloid beta protein, thereby effectively blocking the neural cell necrosis mechanism caused by excessive oxidative stress in the cell to maintain high cell viability, and consequently damage the nerve cell It was found that the effective defense.

According to a specific embodiment of the present invention, the present inventors have confirmed the neuroprotective effect of the neurofibrillary nerve cell extract of the bark water extract More specifically, as a result of measuring the DPPH radical scavenging activity, it was found that the antioxidant activity is excellent, the bark water As the concentration of the extract increases, the activity was also found to increase (Experimental Example 1).

In addition, garlic acid, catechin, and epicatechin, which are polyphenol compounds isolated from Yulpi extract, have cerebral nerve cell protective activity and acetylcholinesterase inhibitory activity.

"Gallic acid, catechin, and epicatechin" having brain neuronal cell protective activity and acetylcholinesterase inhibitory activity according to the present invention can be isolated from natural substances, preferably yulpi, and by chemical synthesis by known methods in the art. It may also be prepared.

The term "pharmaceutically acceptable salts" herein means salts prepared by conventional methods and are known to those skilled in the art. "Pharmaceutically acceptable salts" include, but are not limited to, salts derived from the following pharmacologically or physiologically acceptable inorganic and organic acids and bases. Examples of suitable acids include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid , Benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, and the like. Salts derived from suitable bases may include alkali metals such as sodium, alkali cometals such as magnesium, ammonium and the like.

As used herein, the term "brain nerve cell protective activity" refers to blocking or reducing the efficiency of brain nerve cell destruction due to oxidative stress. Amyloid beta protein induces oxidative stress, creating intra-cellular free radical status and ultimately necrosis of neuronal cells. Therefore, the elimination of oxidative stress and the survival of cerebral neurons are important indicators of neuronal cell protection against cytotoxicity induced by amyloid beta protein.

As used herein, the term “amyloid beta protein” refers to 40-42 protein substances produced from non-ideal cleavage of amyloid precursor protein (APP), which is a degenerative disease as well as the cause of neural inactivation and necrosis. It is also considered as an important pathogenetic material in Alzheimer's disease. In addition, amyloid beta protein induces intracellular free radical status and ultimately induces cell death. It is estimated that amyloid beta and brain nerve cell destruction are correlated. Production of ROS free radicals and the like by amyloid beta proteins is expected to lead to amyloid beta-induced free radical-mediated neurotoxicity.

As used herein, the term "acetylcholinesterase inhibitory activity" refers to the inhibition of acetylcholinesterase, thereby increasing the concentration of acetylcholine in synapses, restoring neuroprotective effects and nerve damage. It means to act. For example, in Alzheimer's disease, a decrease in the level of acetylcholine (ACh; neurotransmitter) caused by the accumulation of amyloid beta protein in the brain is considered as one of the causes of the acetylcholine esterase inhibitory activity It may be a key factor for the treatment of Alzheimer's disease.

In addition, the said "acetylcholinesterase" means an acetylcholine hydrolase. Acetylcholine (ACh), a neurotransmitter found in all nerve cells of brain tissue, is known as an important neurotransmitter involved in the neurotransmission between synapses and synapses. When acetylcholine (ACh) is secreted at the presynaptic end in certain parts of the cranial nervous system, it binds to postsynaptic receptors and transmits stimuli between neurons. However, acetylcholine (ACh) secreted at the first stimulus must be hydrolyzed by acetylcholinesterase (AChE) before the second stimulus transfers through the synapse. However, the content of acetylcholine (ACh) and the activity of choline acetyltransferase (ChAT) as a synthetase are known to decrease with age in most humans and rodents. It has also been found that the activity of acetylcholinesterase decreases like acetylcholine (ACh).

In a specific embodiment of the present invention, the present inventors confirmed the neuronal cell protective activity and acetylcholinesterase inhibitory activity of gallic acid, catechin, and epicatechin, polyphenol compounds isolated from Yulpi extract.

More specifically, the neuronal cell protective activity of garlic acid, catechin, and epicatechin was confirmed through the following experimental examples.

The ABTS radical scavenging activity and the vitamin C equivalent antioxidant capacity (VCEAC) of gallic acid, catechin, and epicatechin showed a tendency to decrease the absorbance of ABTS radicals with increasing compound concentration. It was found that it showed higher radical scavenging ability than ascorbic acid used as a positive control at all concentrations (Example 2, Fig. 4).

In addition, as a result of measuring the protective effect on neurons in the oxidative stress induced by amyloid beta protein, it was found that the effects of catechin and epicatechin are particularly excellent (Example 3, FIG. 5).

Neurons contain a relatively large amount of lipids, which makes them very vulnerable to oxidative stress. Therefore, in order to examine the relationship between neuronal cell protective effect and neuronal cell membrane damage, the cytoplasmic components of neurons contained in neurons were examined to confirm the protective effect against amyloid beta protein induced by garlic acid, catechin, and epicatechin. LDH (lactate dehydrogenase) release was measured. As a result, the treatment of garlic acid, catechin, and epicatechin of the present invention showed that LDH emission was decreased, which means that garlic acid, catechin, and epicatechin were superior to neuronal cell membrane damage protection as well as neuronal cell membrane damage. (Example 4, FIG. 6).

In addition, the treatment of garlic acid, catechin, and epicatechin to neurons induced by oxidative stress showed their neuronal protective effects through reduced cytotoxicity (Example 5, FIG. 7). In particular, the effects of catechin and epicatechin were excellent.

In addition, as a result of confirming the acetylcholinesterase inhibitory activity of garlic acid, catechin, and epicatechin through Experimental Examples 6 and 7, in particular, the garlic acid showed a high inhibitory activity (FIG. 8). As a result of specifying the relationship with the enzyme reaction rate, it was found that garlic acid acts as a competitive inhibitor of acylcholine esterase (FIG. 9).

As described above, by confirming that the polyphenol compounds, garlic acid, catechin, and epicatechin isolated from the yul extracts had excellent neuronal cell protective activity and acetylcholinesterase inhibitory activity, the yul extracts from which these compounds were isolated also had the same activity. It can be expected to have enough.

Thus, yulpi extract or garlic acid, catechin, and epicatechin isolated therefrom have excellent prophylactic and therapeutic activity against brain diseases for the reasons discussed above.

The yulpi extract and garlic acid, catechin, and epicatechin are not artificially synthesized compounds but are based on ingredients obtained from natural extracts, so they are safe, toxic, and have no side effects. In addition, the composition may be used not only in humans, but also in animals such as cows and dogs in which brain diseases may occur.

Therefore, the present invention can be used as a composition for preventing and treating cerebral disease, including garlic acid, catechin, and epicatechin or pharmaceutically acceptable salts thereof isolated from the extract of the extract and the extract.

For example, there are various brain diseases caused by amyloid beta protein, and brain diseases in which oxidative stress is caused by these amyloid proteins and ultimately, brain nerve cells are destroyed are the targets of treatment or prevention according to the present invention. Being a disease belongs to.

Specifically, the brain disease includes the following diseases.

Neurodegenerative diseases (Alzheimer's disease, Huntington's chorea, Parkin's disease), senile dementia, prefrontal dementia, vascular dementia, migraine, neurodegenerative pain of central origin;

Depression (endogenous, resistant, reactive or unexplained depression), nervous breakdown, schizophrenia, bipolar syndrome, general anxiety disorder, stress-related disorders, panic disorder, OCD, post-traumatic stress disorder, attention deficit hyperactivity disorder, diet Mental disorders such as disorders (especially bulimia, anorexia), phobias (especially agoraphobia), autism;

Memory, attention and arousal disorders related to nervous or mental disorders.

The cause of neural inactivation and necrosis is thought to be cytotoxicity caused by amyloid beta protein, an abnormal metabolite of amyloid precursor protein (APP). Its neurotoxicity begins with brain nerve cell destruction caused by oxidative stress (ROS, etc.).

In particular, the causes of Alzheimer's disease vary widely but the overall onset scheme is as follows; Mutation of Presenilin 1, 2 genes (PS 1, 2) → Non-ideal cleavage of amyloid precursor protein (APP) by β-secretase → Production of amyloid beta protein → Necrosis of cerebral nerve cells by amyloid beta protein → Onset of Alzheimer's Disease.

The cause of neural cell necrosis in Alzheimer's disease is still unclear, but oxidative damage such as the generation of oxygen free radicals and reactive oxygen species (ROS) is believed to be associated with cerebral neuronal cell destruction and etiology. Increased oxidative stress (active oxygen, lipid peroxidation, protein denaturation, mitochondrial DNA oxidation) in the brain of Alzheimer's patients has been demonstrated by several experiments, and these physiological metabolic abnormalities are amyloid plaques and nerve fiber enrichment. It results in clinical results specific to Alzheimer's brain, such as neurofibrillary tangles (NFT).

Accordingly, garlic acid, catechin, and epicatechin or a pharmaceutically acceptable salt thereof isolated from the yeast extract and the extract of the present invention effectively reduce the oxidative stress induced by amyloid beta protein, thereby oxidizing excessively in the cell. By effectively blocking the neural cell necrosis mechanism caused by stress to maintain a high cell survival rate, as a result, the nerve cell damage is effectively prevented, and by inhibiting acetylcholinesterase, brain nerve cells are destroyed by the amyloid beta protein. Effective for the prevention and treatment of brain diseases.

As used herein, the term "prevention" refers to any action that inhibits or delays the onset of the disease by administration of a composition comprising a yulpi extract according to the invention or a garlic acid, catechin, and epicatechin or a pharmaceutical salt thereof isolated therefrom. Say.

As used herein, the term "treatment" refers to any action in which the symptoms of the disease improve or benefit altered by administration of an extract according to the invention or the composition described above.

The composition for preventing and treating brain diseases of the present invention can be administered to mammals such as mice, mice, livestock, humans and the like by various routes. All modes of administration can be expected and can be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The dosage of the composition for preventing and treating brain diseases used in the present invention depends on the condition and weight of the patient, the extent of the disease, the drug form, the route of administration and the duration, and may be appropriately selected by those skilled in the art. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect. An effective amount is the amount of compound required to give the desired effect. Effective amounts may vary depending on the route of administration, the use of excipients, and the possibility of use with other agents, as will be appreciated by those skilled in the art.

The pharmaceutical dosage forms of the compositions for preventing and treating brain diseases of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds, as well as in a suitable collection. .

Compositions for preventing and treating brain diseases of the present invention can be prepared in pharmaceutical formulations using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. In the preparation of the formulation, it is preferred that the active ingredient is mixed or diluted with the carrier or enclosed in a carrier in the form of a container.

Therefore, the composition for preventing and treating brain diseases of the present invention, in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, external preparations, suppositories, and sterile injectable solutions according to conventional methods. It may be used in formulated, and may further comprise suitable carriers, excipients and diluents commonly used in the preparation of the composition.

For example, carriers, excipients, and diluents that may be included in the compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate , Calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used.

Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations include at least one excipient such as starch, calcium carbonate, sucrose in the compound. Or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.

Liquid preparations for oral use include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have.

Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

In another aspect, the present invention provides a functional health food composition for improving brain disease comprising the extract of Yulpi as an active ingredient.

In still another aspect, the present invention provides a functional health for improving brain disease comprising at least one polyphenol component or a pharmaceutically acceptable salt thereof selected from the group consisting of garlic acid, catechin, and epicatechin isolated from yulpi extract Provide a food composition.

The health food composition of the present invention includes the form of pills, powders, granules, acupuncture, tablets, capsules, drinks or liquids, and the food to which the composition of the present invention can be added, for example, various foods, for example For example, drinks, gum, tea, vitamin complexes, and dietary supplements.

There is no particular limitation on the other ingredients except for containing garlic acid, catechin, and epicatechin or pharmaceutically acceptable salts thereof, which are isolated from the yulp extract or the yulp extract as essential ingredients that may be included in the health food composition of the present invention. Like herbal foods, various herbal extracts, food supplements, or natural carbohydrates may be added as additional ingredients.

The health food composition is one or more herbal extracts selected from the group consisting of plum, cheonma, Schisandra chinensis, licorice, deficiency, Seokchangpo, Ohjigol, Wonji, Astragalus, Sanjoin, Shiho, Creation, Donkey, Jija, Anak, Bokryong, etc. It can be included as.

In addition, as mentioned above, the food supplement additive may further include food supplement additives, including food supplement additives conventional in the art, such as flavoring agents, flavoring agents, coloring agents, fillers, stabilizers, and the like. .

Examples of such natural carbohydrates include monosaccharides such as glucose, fructose, and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (e.g., rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. .

In addition to the above, the health food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid And salts thereof, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. Others may contain pulp for the production of natural fruit juices and fruit juice drinks and vegetable drinks. These components can be used independently or in combination.

In another aspect, the present invention provides a process for preparing a yulpi extract using water, an organic solvent or a mixed solvent thereof, b) obtaining a fraction using water or a non-polar organic solvent from the obtained extract, c) At least one polyphenol selected from the group consisting of garlic acid, catechin, and epicatechin according to the present invention, comprising separating and purifying at least one polyphenol component selected from the group consisting of garlic acid, catechin, and epicatechin in the fraction. It provides a method for producing a composition for preventing and treating brain diseases, including a phenol component.

More specifically, the method for preparing the yulpi extract The method for preparing a composition for preventing and treating brain diseases by separating the active ingredients, garlic acid, catechin, and epicatechin from it is as follows.

Yulpi extract according to the present invention can be obtained by extracting with water, an organic solvent or a mixed solvent thereof, and preferably, the yulpi extracted by heating for a predetermined time, cold extraction, heat extraction, ultrasonic wave as known in the art Extraction can be performed according to various extraction methods such as cooling extraction. The extraction method is not particularly limited and may be extracted at room temperature or warmed under conditions where the active ingredient is not destroyed or minimized. A high activity fraction is obtained from the extract of Yulpi extracted therefrom, and the polyphenol compounds according to the present invention, such as garlic acid, catechin, and epicatechin, can be separated by further separating them by methods such as chromatography.

Organic solvents that can be used for the extraction of Yulpi include ethanol, methanol, isopropanol, butanol, ethylene, acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, dichloromethane, N, N-dimethylformamide (DMF), Dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol or a mixed solvent thereof.

In a specific embodiment of the present invention, the yulpi water extract was obtained using reflux cooling extraction by adding water to the yulpi powder (Example 1).

In order to obtain a highly active fraction from the first extracted Yulpi extract, a fraction was obtained using an organic solvent. The organic solvent is preferably a nonpolar organic solvent, and particularly preferred is hexane, ether, dichloromethane, chloroform, ethyl acetate, or a mixed solvent thereof, and most preferably ethyl acetate.

The active component can be separated by performing chromatography one or more times sequentially on the nonpolar solvent fraction, that is, the nonpolar solvent soluble layer obtained as described above, and the type and developing solvent of the column of the chromatograph can be variously controlled.

In a specific embodiment of the present invention, the present inventors added and mixed ethyl acetate from Yulpi water extract to separate the ethyl acetate fraction and separated the active material by HPLC chromatography. Compounds which are active substances isolated by the above method were found to be garlic acid, catechin, and epicatechin.

As described above, Yulpi extract can be effectively used in the form of a functional health food composition that can be easily taken in daily life as well as the pharmaceutical composition because it has a neuronal cell protective activity from oxidative stress. In addition, the polyphenol components separated and purified from these yulpi extracts also have the inhibitory activity of neuronal cytotoxicity by amyloid β and acetylcholinesterase (AChE) inhibitory activity, thereby preventing brain diseases such as Alzheimer's disease. And effectively for treatment. In addition, it is expected to reduce the production cost of protective materials by recycling the waste resources of Yulpi, an uninhabitable woman who has been discarded without using it, and to help increase the income of chestnut-producing farmers and develop the local economy.

Example  1: Preparation of Yulpi Water Extract

September 2008 Purchasing chestnut cultivated in Hadong, Gyeongnam, screening and washing to remove the outer skin, remove the inner skin, and then sort and wash the peeled skin off again and dry it at a temperature of 40 ℃ using a hot air dryer to remove moisture. 10% or less. The dried inner skin was pulverized, and 500 mL of water was added to 50 g of yulpi powder. 2 filter paper (Whatman plc., Kent, UK) The filtrate was used by lyophilization with a freeze dryer.

Example  2: from garlic water extract to garlic acid ( gallic acid ), Catechin ( catechin ) And Of epicatechin  detach

100 g of distilled water was added to 10 g of yelp, and reflux-cooled for 2 hours. The same amount of ethyl acetate was added and mixed with the extract, and the ethyl acetate layer was separated and extracted three times. The extract was concentrated under reduced pressure using a vacuum rotary condenser and completely dissolved in 10 mL of the test solution {0.2 M phosphate buffer (pH 3.0): methanol: distilled water = 2: 3: 15, v / v / v} 0.45 μm syringe Filtration with a filter (syringe filter) was isolated using HPLC (Agilent 1100 series, Santa Clara, Calif., USA) (see Figure 1). HPLC analysis conditions used are as follows. The detector is a DAD (Diodarray detector) 280 nm, the column is ODS (250 mm × 4.9 mm, 5 μm), the mobile phase methanol (methanol): acetic acid (acetic acid): acetonitrile: ddH ₂ O (20: 5: 113: 862 = v / v / v / v) and the flow rate was analyzed at 1 mL / min.

The contents of garlic acid, catechin and epicatechin isolated from Yulpi water extract were 31.04, 7.54 and 6.60 mg / 100 g, respectively.

Experimental Example  1: Yulpi water extract DPPH Radical  Scavenging activity measurement

Concentrations as ethanol yulpi 1 mL water extract 1.5 × 10 ^-4 M concentration in the DPPH (1,1-diphenyl-2- picryl -hydrazyl) solution was uniformly mixed with 4 mL ssikeul mixer (vortex) at room temperature and then causes the After standing for 30 minutes, the optical density (OD) was measured at 517 nm.

The results of measuring DPPH radical scavenging activity, which is one of measuring methods of antioxidant activity, using the extract of Yulpi are shown in FIG. 3. DPPH radical scavenging activity tended to increase as the concentration of Yulpi water extract increased, and very high DPPH radical scavenging activity of 91.07% was observed in the water extract of 1 mg / mL.

Experimental Example  2: garlic acid gallic acid ), Catechin ( catechin ) And epicatechin ( epicatechin) ABTS Radical  Scavenging activity and VCEAC ( vitamin  C equivalent  antioxidant capacity ) Measure

PBS (Phosphate-buffered saline, pH 7.4; 100 mM potassium phosphate buffer containing 150 mM NaCl) was mixed with 1.0 mM 2,2'-azobis (2-methylpropionamidine) dihydrochloride (AAPH) and 2.5 mM ABTS at about 13 ° C. After reacting for a minute, the blue-green ABTS® solution was adjusted to an absorbance value of 0.650 ± 0.020 with PBS (phosphate-buffered saline) at 734 nm. 20 μL of each compound by concentration and 980 μL of ABTS solution were uniformly mixed with a vortex and left at 37 ° C. for 10 minutes, and then the optical density (OD) decrease was measured at 734 nm. Activity is expressed as VCEAC (mg / 100mL) value.

VCEAC (mg / 100mL) Values of Compounds Isolated from Yulpi Water Extract Compounds VCEAC (mg / 100 mL) ^a 2 4 6 8 10 Garlic Acid 5.54 ± 0.03 8.56 ± 0.16 13.37 ± 0.25 14.53 ± 0.04 14.38 ± 0.21 Epicatechin 4.38 ± 0.20 7.84 ± 0.35 11.40 ± 0.10 14.27 ± 0.24 14.47 ± 0.05 Catechin 4.29 ± 0.22 7.71 ± 0.34 11.81 ± 0.80 14.46 ± 0.04 14.49 ± 0.01

The results of measuring ABTS radical scavenging activity and VCEAC (vitamin C equivalent antioxidant capacity) of gallic acid, catechin and epicatechin isolated from Yulpi water extract are shown in FIG. 4. As the concentration of the compound increased, the absorbance of the ABTS radicals tended to decrease and showed higher radical scavenging ability than ascorbic acid used as a positive control at all concentrations. At the concentration of 6 mg / 100 mL, the VCEAC values of gallic acid catechin and epicatechin were 13.37, 11.81, and 11.40 (VCEAC mg / 100 mL), respectively. The difference in absorbance reduction of the compounds was similar (FIG. 4 and Table 1).

Experimental Example  3: garlic acid gallic acid ), Catechin ( catechin ) And epicatechin ( epicatechin)  Cerebral Nerve Cell Damage Inhibitory Activity

The protective effect on amyloid beta protein-induced PC12 cells was measured by MTT (3- (4,5-dimethyl- thiazol-2-yl) -2,5-diphenyltetrazolium bromide) reduction assay. Three compounds (gallic acid, catechin and epicatechin) contained in Yulpi were pre-incubated for 48 hours by treatment with PC12 cell lines by concentration, and then the amyloid beta protein was dissolved in distilled water at a concentration of 80 μM. Incubate for 3 hours after addition. Incubate the PC12 cells in this state with MTT stock solution (10 μL / well) and incubate at 37 ° C for 3 hours, then terminate the reaction by adding 100 μL of MTT solubilizing solution (10% Triton X-100 in acidic isopropanol). I was. Finally, absorbance was measured at 570 nm (determination) and 630 nm (reference wave) in a microplate reader (680, Bio-rad, Japan). Positive control used vitamin C (200 μM) and cell viability was expressed as% concentration relative to control.

In oxidative stress induced by amyloid beta protein (Aβ), the major compounds isolated from yeast, catechin, epicatechin, and gallic acid, protect the PC12 neurons against MTT reduction. The results measured using the assay method are shown in FIG. 5. As a result of measuring cell viability, the treatment with amyloid beta protein showed 20.1% survival rate compared to the control 100%, and the treatment with amyloid beta protein and vitamin C at the same time treated showed 43.2% survival rate of about 23%. Neuroprotective effect was shown. Treatment with catechin at a low concentration of 50 μM in the purified product showed a high neuronal protective effect of 93.7%, which is 50% higher than 43.2% of vitamin C used as a positive control, and distinct concentration-dependent protection with increasing catechin concentrations. Showed the effect.

Treatments with epicatechin showed similar results with vitamin C at low concentrations of 50 μM and also showed a distinct concentration-dependent protective effect as the concentration of epicatechin increased. In particular, the survival rate of catechin and epicatechin at concentration of 200 μM was 242.7% and 187.8%, respectively, indicating that catechin showed 55% higher neuroprotective effect than epicatechin.

Experimental Example  4: garlic acid gallic acid ), Catechin ( catechin ) And epicatechin ( epicatechin)  Inhibitory effect on nerve cell membrane damage

Using three compounds (gallic acid, catechin and epicatechin) contained in Yulpi, they were pre-incubated for 48 hours by treatment with PC12 cell lines by concentration, followed by incubation for 3 hours with 80 μM amyloid beta protein. After centrifugation (250 × g) to precipitate, 100 μL of the supernatant was transferred to a new well, the cell damage was measured by LDH assay kit (Sigma Chemical Co, St. Louis MO., USA), neutral red assay It was measured using a neutral red assay kit (Sigma Chemical Co, St. Louis MO., USA).

In order to confirm the protective effect of catechin, epicatechin and garlic acid tablets against amyloid beta protein-induced neuronal cell membrane damage, the amount of release of lactate dehydrogenase (LDH) of cytoplasmic components contained in neurons was measured.

In the control group, the release rate was about 30.3%, while the amyloid beta protein treated group showed 50.5% release, indicating that LDH release increased by 20.2% due to amyloid beta protein. The 200 μM vitamin C treatment group showed LDH release of 19.3% and the catechin, epicatechin and garlic acid treatments showed 56.8%, 54.8%, and 69.1% LDH release, respectively, when treated at 50 μM. When compared to 24.6%, 40.3%, and 34.1%, the concentrations of LDH emissions were decreased. In particular, catechins showed that neuronal membrane protective effects were obtained by recovering LDH release at high concentrations to control levels.

Experimental Example  5. Garlic Acid gallic acid ), Catechin ( catechin ) And epicatechin ( epicatechin)  Cerebral nerve cell protective effect

Cytotoxicity was evaluated by NRU (neutral red uptake) analysis by treating catechin, epicatechin and garlic acid in PC12 cell line induced by oxidative stress. The treatment with amyloid beta protein showed 72.5% survival rate compared to the control group 100% and the treatment with amyloid beta protein and vitamin C at the same time showed 137.9% survival rate of about 65% neurons.

Treatments with catechin and epicatechin showed a survival rate of 81-100%, which was about 20% higher than those treated with amyloid beta protein.

Experimental Example  6: Garlic Acid gallic acid ), Catechin ( catechin ) And epicatechin ( epicatechin) Acetylcholinesterase ( acetylcholinesterase , AChE Inhibitory activity

Acetylcholinesterase inhibitory activity was measured using acetylcholine iodide as a substrate. Enzyme was added to 1 mL of PC12 cell culture solution, and 5 mL of buffer for homogenization (1 M NaCl, 50 mM MgCl ₂ , adjusted to pH 7.2 with 10 mM Tris-HCl in 1% Triton X-100 mixture) Homogenized with Glass-Col homogenizer. Homogenized cell culture was centrifuged at 10,000 rpm for 30 minutes and the supernatant was used for the enzyme experiment. All extraction processes were performed at 4 ° C, and BCA kit (bicin-choninic acid; Sigma Co., St. Louis, Mo, USA) was used to measure the protein content of the extracted enzyme solution. The content was converted according to the calibration curve prepared by). 10 μL of the extract was added to 10 μL of purified enzyme (protein content: 2.38 mg / mL) and reacted at 37 ° C. for 15 minutes. The reaction mixture was dissolved in Ellman's reaction mixture in 50 mM sodium phosphate buffer (pH 8.0). After adding 70 μL of [0.5 mM acetylthiocholine, 1 mM 5,5'dithio-bis (2-nitro benzoic acid)], absorbance was measured at 405 nm for 10 minutes at 2 minute intervals.

The inhibitory activity of acetylcholinesterase was measured using three compounds isolated and identified in Yulpi as shown in FIG. 8.

The inhibitory activity of acetylcholinesterase of catechin and epicatechin was shown to be about 40% of inhibitory activity at the concentration of 6.25 ~ 400 μM, but no concentration-dependent tendency was observed. In addition, the garlic acid showed a concentration-dependent tendency to increase the inhibitory activity with increasing concentrations, especially the highest inhibitory activity of acetylcholinesterase at 94.77% at 400 μM concentration showed higher inhibitory activity than the positive control. Seemed. In addition, IC ₅₀ was measured using garlic acid, which showed the highest inhibitory activity among the major compounds of Yulpi.

Experimental Example  7: garlic acid of gallic acid  As a competitive inhibitor, acetylcholinesterase ( acetylcholinesterase , AChE Inhibitory activity

Three kinds of compounds isolated from Yulpi (gallic acid), catechin (catechin) and epicatechin (epicatechin) were used to measure acetylcholinesterase inhibitory activity. By varying the concentration of (1.25 ~ 20 μM) substrate concentration, the relationship between the initial enzyme reaction rate and the results of the Lineweaver-Burk (Lineweaver-Burk, LB) plot is shown in FIG.

The Lineweaver-Burk (LB) plot shows the correlation between the initial enzyme reaction rate and the concentration of the substrate with reciprocal values. The concentration of the substrate (acetylthiocholine iodide) is 0.025 mM to 0.5 mM. It was. K _m = 0.094 mM, V _max = 0.253 dA / min.

K _m with increasing concentration of garlic acid While the value tends to increase, the V _max value does not increase and shows a constant value. It can be seen that garlic acid isolated as the main compound of Yulpi acts as a competitive inhibitor against acetylcholinesterase. For reference, the acetylcholinesterase inhibitor tacrine, a drug used in clinical practice after being approved by the FDA as an Alzheimer's drug, has been reported to be a non-competitive inhibitor.

Figure 1 shows a flow chart for separating the active material from Yulpi.

Figure 2 shows an HPLC chromatogram of gallic acid, catechin and epicatechin isolated from Yulpi water extract, where 1 is garlic acid, 2 is catechin, and 3 is epicatechin.

Figure 3 shows the DPPH radical scavenging activity of Yulpi water extract, which is the Yulpi water extract.

Figure 4 shows the ABTS radical absorbance reduction (A) and VCEAC (B) measurements of the compounds isolated from Yulpi water extract. Representing ● is ascorbic acid, ○ is catechin, ▼ is epicatechin, and △ is gallic acid. Values are expressed in mg of VCEAC (mg / 100 mL) and mean ± SD (n = 3).

Figure 5 shows the neuronal protective effect of catechin (catechin), epicatechin (epicatechin) on amyloid beta protein-induced cytotoxicity.

Figure 6 shows the inhibitory effect of LDH release on amyloid beta protein induced cell membrane damage of gallic acid (catelic), catechin (catechin) and epicatechin (epicatechin).

Figure 7 shows the protective effect of catechin (catechin) and epicatechin (epicatechin) on amyloid beta protein induced cell death using a neutral red assay.

Figure 8 shows the inhibitory effect of acetylcholine esterase of gallic acid (catelic), catechin (catechin) and epicatechin (epicatechin) isolated from Yulpi water extract.

Figure 9 shows the results of analysis of acetylcholinesterase (AChE) inhibition pattern of gallic acid (gallic acid) isolated from Yulpi water extract.

Claims (13)

a) obtaining yulpi extract from yulpi using water, an organic solvent or a mixed solvent thereof; b) obtaining fractions from the extracts obtained using water or a non-polar organic solvent; c) separating and purifying at least one polyphenol component selected from the group consisting of garlic acid, catechin, and epicatechin in the fraction, A method of preparing a composition for preventing and treating brain diseases, comprising at least one polyphenol component selected from the group consisting of garlic acid, catechin, and epicatechin, or a pharmaceutically acceptable salt thereof. The method of claim 1, wherein the nonpolar organic solvent of step b) is ethyl acetate. The method of claim 1, wherein the separation and purification of step c) comprises a chromatography process. delete delete delete delete delete delete delete delete delete delete

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