KR102176184B1 - Composition for Protection of Brain Neuronal Cells Using the Compound Isolated from the Extract of Ribes fasciculatum - Google Patents

Abstract

The present invention discloses a compound isolated and identified from the extract of Ribes fasciculatum and a composition for protecting cranial nerve cells using the same.

Description

Composition for Protection of Brain Neuronal Cells Using the Compound Isolated from the Extract of Ribes fasciculatum}

The present invention relates to a composition for protecting cranial nerve cells using a compound isolated and identified from Chilhaemok ( Ribes fasciculatum ).

As many countries around the world gradually enter the aging society, people suffering from various degenerative brain diseases such as stroke, Parkinson's disease (PD), Alzheimer's (dementia) are increasing rapidly, and the cost is astronomical. . Looking at the causes of death in Korea according to data from the National Statistical Office in 2015, the mortality rate from brain diseases such as malignant neoplasm (cancer), heart disease, cerebrovascular disease, and pneumonia occupies third place. The brain, which is in charge of information essential for life maintenance, causes various neurological disorders that have a fatal effect on life when an abnormality in nerve signals occurs. In the current situation where there is no effective way to treat brain diseases, the quality of life is degraded and the expenditure of enormous medical expenses is putting a considerable mental burden on the surrounding family. Recognizing the seriousness of these problems, countries around the world are seeking solutions focused on the public interest.

Neurons are constantly apoptosis in the process of development and restructuring of synapses, and apoptosis by stress and cytotoxic drugs is one of the major factors of degenerative brain disease. Degenerative brain disease is a brain disease that occurs with age, and is known to occur due to accumulation of environmental and genetic factors. A specific group of neurons in the brain and spinal cord slowly loses its function, and the death of the cranial nerve cells, which is the most important for information transmission of the cranial nerve system, formation of synapses or functional problems that transmit information between the cranial nerve cells and cranial nerve cells, ideal for electrical activity of the cranial nerve It is caused by an increase or decrease. Neurons in the brain and spinal cord perform a wide variety of functions depending on their location.Therefore, depending on which part of the nerve cells are damaged and lose their function first, and how such a dysfunction progresses, a wide variety of clinical aspects can be seen. . Degenerative brain disease can be classified by considering the main symptoms that appear and the area of the brain affected, and Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), multiple sclerosis ( Multiple sclerosis (MS) and amyotrophic lateral sclerosis (ALS) known as Lou Gehrig's disease. Among several factors, oxidative stress is known to have a lot of correlation with the cause of neurodegenerative diseases. The brain is only 2% of the body weight, but it consumes about 20% of the total oxygen consumption, so it is the part in the body where the most active oxygen is generated due to the high oxygen utilization rate. Recent studies have shown that chronic stress and oxidative stress induce oxidative stress in the hypothalamus-pituitary-adrenal cortex, hippocampus, striatum, black matter, and forebrain cortex to increase apoptosis and decrease neurons and growth factors. (Floyd RA. Proc Soc Exp Biol Med. 1999 Dec;222(3):236-45.; Wang JY et al., Curr Pharm Des. 2006;12(27): 3521-33).

In addition, among the brain diseases, cerebrovascular disease is a brain disease caused by abnormal blood vessel function such as cerebral hemorrhage and stroke, and is caused by a decrease in supply of oxygen and energy sources. Neurons use only glucose as a source of oxygen and energy. However, because the brain does not have a storage function such as glycogen, the energy source is completely blocked just by blocking blood flow. In general, the blood sugar level of a normal person is maintained at 80mg/, but when the blood sugar level reaches 20mg/, a coma is formed. In addition, since various reactive oxygen species are induced in the hypoxic state, when the supply of glucose together with oxygen from the cranial nerve cells is blocked, the function of the nerve cells is paralyzed, leading to the death of the cranial nerve cells (Muresan A et al., Med Food. 2013 Sep;16 (9):831-8.; Simran S et al., Nature Reviews Cancer 2014 14, 709721; Manzanero S et al., Neurochem Int. 2013 Apr;62(5):712-8.) Due to cerebrovascular disease. Brain tissue damage proceeds to diseases such as vascular dementia.

In addition, ground state triplet oxygen, which is a stable molecular state, is superdxide radical (O2-) and hydroxyl due to various physical and chemical and environmental factors such as enzymatic system, reduction metabolism, chemicals, pollutants, and photochemical reactions. When it is converted into highly reactive active oxygen, such as radical (HO), hydrogen peroxide (H2O2), and singlet oxigen (1O2), oxygen toxicity, which is fatal to the body, is caused to nerves.

Oxidative stress of nerve cells causes the release of cytochrome C and caspase-3 activation in mitochondria, causing apoptosis. In addition, reactive oxygen species activate glutamate, especially NMDA receptors, causing an increase in Ca2+ ions by metabotrophical cascade, and increase in intracellular Ca2+ activates caspase-2 and damages DNA. (Bonini P et al., J Neurosci Res. 2004 Jan 1;75(1):83-95.; Polster BM and Fiskum G. J Neurochem. 2004 Sep;90(6):1281-9.; Gorman AM et al. al., Neuroreport. 1998 Jul 13;9(10):R49-55). Apoptosis occurs due to excitatory neurotoxicity due to destruction of Ca2+ ion homeostasis, inability of endoplasmic reticulum and mitochondria, DNA damage due to oxidative stress, etc.(Wei et al., 1999, Toxicology 134:117-26. ).

As such, free radicals have a great influence on the onset and progression of degenerative brain diseases and cerebrovascular diseases. Therefore, experiments to confirm the protective effect of neurons under ROS-producing apoptosis conditions such as hydrogen peroxide treatment on SH-SY5Y cells or PC12 cells are performed on stroke and It is being used as an important research method in inhibiting neurological damage in degenerative brain diseases and exploring the treatment.

In the present invention, it was confirmed the neuronal protective effect of the compound isolated from the extract of Chilhaemok.

An object of the present invention is to provide a composition for protecting cranial nerve cells using a compound derived from Chilhaemok.

Other or specific objects of the present invention will be presented below.

In the present invention, as confirmed in the following Examples and Experimental Examples, the compounds of the following <Formula 1> to <Formula 4>, which are the active ingredients isolated from the extract of Chilliwood, are human neuroblastoma SH-SY5Y cell line and a wide range of neurodegenerative properties. It was completed by confirming that the PC12 cell line (pheochromocytoma-12 cell), which is being used in disease research, exhibits an inhibitory effect on cranial nerve cell death by hydrogen peroxide. The compounds were obtained by suspending the 30% ethanol extract of Red Sea Mok in water and then sequentially fractionating with hexane, dichloromethane, ethyl acetate and butanol (n-butanol), and dividing the obtained ethyl acetate fraction into 11 fractions, followed by silica gel column chromatography. Among the compounds obtained by the experiment, compounds having excellent neuroprotective activity were isolated and identified.

In consideration of the foregoing, the present invention relates to a separation compound represented by the following <Chemical Formula 1> to <Chemical Formula 4> in one aspect, a prodrug thereof, a hydrate thereof, or a solvate thereof, In another aspect, (i) the ethyl acetate fraction of the extract of Red Seaweed or (ii) the separation compound represented by the following <Formula 1> to <Formula 4>, a prodrug thereof, a hydrate thereof, or a solvate thereof as an active ingredient. It relates to a composition for protecting cranial nerve cells, and in another aspect, (i) an ethyl acetate fraction or (ii) an isolation compound represented by the following <Formula 1> to <Formula 4>, a prodrug thereof, It relates to a composition for the improvement of diseases accompanying the death of cranial nerve cells comprising a hydrate thereof or a solvate thereof as an active ingredient.

<Formula 1>

<Formula 2>

<Formula 3>

<Formula 4>

In the present specification, "prodrug" refers to a drug in which physical and chemical properties are controlled by chemically changing a drug, and itself does not exhibit physiological activity, but after administration by chemical or enzyme action in the body It refers to a drug that can be changed to the original drug and exert its efficacy.

In addition, in the present specification, "hydrate" refers to a compound to which water is bound, and includes a compound containing no chemical bonding force between water and the compound.

In addition, in the present specification, "solvate" refers to a compound formed between molecules or ions of a solute and molecules or ions of a solvent.

In addition, in the present specification, the term "chill haea extract" refers to the stem, leaves, fruits, flowers, roots, etc. of the extract of the chimeric tree as water, lower alcohols having 1 to 4 carbon atoms (methanol, ethanol, butanol, etc.), methylene chloride, ethylene, Using acetone, hexane, ether, chloroform, ethyl acetate, butyl acetate, N,N-dimethylformamide (DMF), dimethyl sulfoxide (DMSO), 1,3-butylene glycol, propylene glycol, or a mixed solvent thereof It refers to an extract obtained by leaching, an extract obtained by using a supercritical extraction solvent such as carbon dioxide, pentane, or a fraction obtained by fractionating the extract, and the extraction method includes cold sedimentation, reflux, and Any method, such as heating, ultrasonic radiation, and supercritical extraction, can be applied. In the case of a fractionated extract, a fraction obtained by suspending the extract in a specific solvent and then mixing and policing it with a solvent having a different polarity, and adsorbing the crude extract on a column filled with silica gel, etc., and then adding a hydrophobic solvent, a hydrophilic solvent, or a mixed solvent thereof. It is meant to include the fraction obtained as a mobile phase. In addition, the meaning of the extract includes a concentrated liquid extract or a solid extract from which the extraction solvent has been removed by a method such as freeze drying, vacuum drying, hot air drying, or spray drying. Preferably, it means obtained by extraction (immersion or distillation) with water, ethanol, or a mixed solvent thereof, particularly 10% to 70% of an aqueous ethanol solution as an extraction solvent. More preferably, the 10% to 70% ethanol aqueous solution extract is suspended in water (distilled water) and sequentially fractionated with hexane, dichloromethane, ethyl acetate, and butanol. Most preferably, it means a fraction of the ethyl acetate layer, which was found to exhibit high cranial nerve cell protective activity in the following experimental examples among the sequential fractions. The meaning of "sequential fractionation" here means that the residual water layer after fractionation is continuously used and fractionated with the solvent in the order listed above.

In addition, in the present specification, the term "active ingredient" refers to an ingredient capable of exhibiting a desired activity alone or with a carrier that is not itself active.

In addition, in the present specification, "protection of cranial nerve cells" includes the meaning of inhibiting the death of cranial nerve cells due to aging in addition to the meaning of inhibiting cranial nerve cell death in diseases accompanying the death of cranial nerve cells as defined below. Since inhibition of the death of cranial nerve cells leads to improvement in cognitive ability, the "protection of cranial nerve cells" can also be understood as a meaning of improving cognitive ability. For reference, the SH-SY5Y cells and PC12 cells used to confirm the protective effect of cranial nerve cells in the experimental examples of the present invention can help improve cognitive ability, "The Health Functional Food Functional Evaluation Guide issued in 2014 by the Ministry of Food and Drug Safety. It is a cell described as an example of use under

In addition, in the present specification, "improvement of diseases accompanying the death of cranial nerve cells" means symptomatic relief, treatment, and prevention (inhibition or delay of onset) of diseases accompanying the death of cranial nerve cells as defined below to be.

In addition, in the present specification, "diseases accompanying the death of cranial nerve cells" refers to Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), multiple sclerosis (MS). ), degenerative brain diseases such as amyotrophic lateral sclerosis (ALS), known as Lou Gehrig's disease, and ischemic brain diseases such as vascular dementia.

In the composition of the present invention, the active ingredient may be included in an arbitrary amount (effective amount) depending on the use, formulation, and purpose of mixing, as long as it can exhibit a cranial nerve cell protective effect, etc., and a typical effective amount should be based on the total weight of the composition. When it will be determined within the range of 0.001% to 20.0% by weight. Herein, "effective amount" refers to the intended functional and pharmacological effect, such as a protective effect on cranial nerve cells, when the composition of the present invention is administered to a mammal, preferably a human, during the administration period according to the advice of a medical professional, etc. , Refers to the amount of the active ingredient contained in the composition of the present invention. Such effective amounts can be determined empirically within the range of ordinary skill in the art.

In addition to the active ingredient, the composition of the present invention has been used in the art to increase or reinforce the protective effect of cranial nerve cells or to enhance the convenience of taking or ingestion through the addition of similar activities such as anti-stress activity and fatigue improvement activity. Any compound or natural extract that has been tested for safety and known to have the corresponding activity may be further included. These compounds or extracts include compounds or extracts, pharmaceuticals, etc. listed in the pharmacopoeia of each country ("Korean Pharmacopoeia" in Korea), health functional food code of each country ("health functional food standards and standards" notified by the Ministry of Food and Drug Safety in Korea), etc. In accordance with the laws of each country governing the manufacture and sale of products (the “Pharmaceutical Affairs Act” in Korea), laws governing the manufacture and sale of compounds, extracts, and health functional foods that have been approved for items (in Korea, the “Health Functional Food Act” ”), the functional compound or extract is included. For example, Lactobacillus Helveticus fermented product, bellflower extract (DRJ-AD), which has been recognized for its functionality as'improving cognitive ability,' according to the Korean Health Functional Food Act, These compounds include fermentation-produced amino acid complexes recognized for their functionality as'improvement', extracts from dulcis dulcis, honggyeongcheon extracts, and L-theanine, which are recognized for their functionality as'anti-stress', asiagandan extracts, hydrolyzates of milk protein, and extracts of stone leaves. Or it would correspond to an extract.

One or more of these compounds or natural extracts may be included in the composition of the present invention together with the active ingredient.

The composition of the present invention can be grasped as a food composition in a specific aspect.

The food composition of the present invention may be prepared in any form, such as beverages such as tea, juice, carbonated beverages, ionized beverages, processed oils such as milk and yogurt, gums, rice cakes, Korean confectionery, bread, Foods such as confectionery and noodles, tablets, capsules, pills, granules, liquids, powders, flakes, pastes, syrups, gels, jelly, and health functional food preparations such as bars can be prepared.

In addition, the food composition of the present invention can be classified as a product as long as it conforms to the enforcement regulations at the time of manufacture and distribution in terms of legal and functional classification. For example, it is a health functional food according to the Korean 「Health Functional Food Act」, or confectionery, bean, tea, and beverages according to each food type according to the food code of the Korean 「Food Sanitation Act」 (``Food Standards and Standards'' notified by the Ministry of Food and Drug Safety). , Special use food, etc.

The food composition of the present invention may contain food additives in addition to the active ingredients. Food additives can generally be understood as substances that are added to food and mixed or infiltrated in manufacturing, processing, or preserving food. Since they are consumed daily and for a long time with food, their safety must be ensured. In the Food Additive Code (“Food Sanitation Act” in Korea) governing the manufacture and distribution of food, food additives with guaranteed safety are limited in terms of ingredients or functions. In the Korean Food Additives Code (“Food Additive Standards and Standards” notified by the Ministry of Food and Drug Safety), food additives are classified into chemical synthetic products, natural additives and mixed preparations in terms of ingredients.These food additives are sweeteners and flavors in terms of function. It is divided into agent, preservative, emulsifier, acidulant, thickener, etc.

Sweeteners are used to impart a suitable sweetness to food, and natural or synthetic ones may be used. Preferably, a natural sweetener is used, and examples of the natural sweetener include sugar sweeteners such as corn syrup solids, honey, sucrose, fructose, lactose, and maltose.

Flavoring agents can be used to improve taste or flavor, and both natural and synthetic can be used. Preferably, it is the case of using a natural one. In the case of using natural ingredients, the purpose of nutrient enhancement can be combined in addition to flavor. As a natural flavoring agent, it may be obtained from apples, lemons, tangerines, grapes, strawberries, peaches, etc., or may be obtained from green tea leaves, green leaves, large leaves, cinnamon, chrysanthemum leaves, jasmine, and the like. In addition, you can use those obtained from ginseng (red ginseng), bamboo shoots, aloe vera, and ginkgo. The natural flavoring agent may be a liquid concentrate or a solid extract. In some cases, synthetic flavoring agents may be used. As the synthetic flavoring agents, esters, alcohols, aldehydes, terpenes, and the like may be used.

As a preservative, calcium sorbate, sodium sorbate, potassium sorbate, calcium benzoate, sodium benzoate, potassium benzoate, EDTA (ethylenediaminetetraacetic acid), etc. can be used, and as an emulsifier, acacia gum, carboxymethylcellulose, xanthan gum, pectin Etc. may be used, and as the acidulant, arithmetic, malic acid, fumaric acid, adipic acid, phosphoric acid, gluconic acid, tartaric acid, ascorbic acid, acetic acid, phosphoric acid, and the like can be used. The acidulant may be added so that the food composition has an appropriate acidity for the purpose of suppressing the growth of microorganisms in addition to the purpose of enhancing taste.

As the thickening agent, a suspending agent, a settling agent, a gel-forming agent, a swelling agent, and the like may be used.

In addition to the food additives described above, the food composition of the present invention may include a physiologically active substance or minerals known in the art for the purpose of supplementing and reinforcing functionality and nutritional properties and ensuring stability as a food additive.

Examples of such physiologically active substances include catechins contained in green tea, vitamins such as vitamin B1, vitamin C, vitamin E, and vitamin B12, tocopherol, dibenzoyl thiamine, etc., and minerals include calcium preparations such as calcium citrate, magnesium stearate. Magnesium preparations, such as magnesium preparations, iron preparations such as iron citrate, chromium chloride, potassium iodide, selenium, germanium, vanadium, and zinc.

In the food composition of the present invention, the food additive as described above may be included in an appropriate amount to achieve the purpose of addition according to the product type.

With respect to other food additives that may be included in the food composition of the present invention, reference may be made to the food code of each country or the food additive code.

The composition of the present invention may be understood as a pharmaceutical composition in other specific embodiments.

The pharmaceutical composition of the present invention may be prepared in an oral dosage form or a parenteral dosage form according to an administration route by a conventional method known in the art, including a pharmaceutically acceptable carrier in addition to the active ingredient. Here, the route of administration may be any suitable route including a local route, an oral route, an intravenous route, an intramuscular route, and direct absorption through mucosal tissue, and two or more routes may be used in combination. An example of a combination of two or more routes is a case in which two or more formulations of drugs are combined according to the route of administration, for example, when one drug is firstly administered by an intravenous route and secondly, the other drug is administered by a local route.

Pharmaceutically acceptable carriers are well known in the art depending on the route of administration or formulation, and specifically, reference may be made to the pharmacopeias of each country, including the "Korean Pharmacopoeia."

When the pharmaceutical composition of the present invention is prepared in an oral dosage form, powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, suspensions, wafers according to a method known in the art together with a suitable carrier It can be prepared in such a formulation. Examples of suitable carriers at this time include sugars such as lactose, glucose, sucrose, dextrose, sorbitol, mannitol, xylitol, starches such as corn starch, potato starch, wheat starch, cellulose, methylcellulose, ethylcellulose, sodium carboxymethylcellulose, Celluloses such as hydroxypropylmethylcellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, magnesium stearate, mineral oil, malt, gelatin, talc, polyol, vegetable oil, ethanol, grease Serol, etc. are mentioned. In the case of formulation, appropriate binders, lubricants, disintegrants, colorants, and diluents may be included as needed. Suitable binders include starch, magnesium aluminum silicate, starch ferryst, gelatin, methylcellulose, sodium carboxymethylcellulose, polyvinylpyrrolidone, glucose, corn sweetener, sodium alginate, polyethylene glycol, wax, etc., and oleic acid as a lubricant Sodium, sodium stearate, magnesium stearate, sodium benzoate, sodium acetate, sodium chloride, silica, talcum, stearic acid, magnesium salts and calcium salts thereof, polydecylene glycol, etc., and as disintegrating agents starch, methyl cellulose , Agar, bentonite, xanthan gum, starch, alginic acid, or a sodium salt thereof. Moreover, lactose, dextrose, sucrose, mannitol, sorbitol, cellulose, glycine, etc. are mentioned as a diluent.

When the pharmaceutical composition of the present invention is prepared in a parenteral formulation, it may be formulated in the form of an injection, a transdermal administration, a nasal inhalation and a suppository according to a method known in the art together with a suitable carrier. When formulated as an injection, an aqueous isotonic solution or suspension may be used as a suitable carrier, and specifically, triethanol amine-containing PBS (phosphate buffered saline), sterile water for injection, or an isotonic solution such as 5% dextrose may be used. . When formulated into a transdermal dosage form, it can be formulated in the form of an ointment, cream, lotion, gel, external solution, pasta, liniment, air roll, and the like. Nasal inhalants can be formulated in the form of an aerosol spray using a suitable propellant such as dichlorofluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, and carbon dioxide, and when formulated as a suppository, the carrier is Withepsol ( witepsol), tween 61, polyethylene glycols, cacao butter, laurin paper, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene stearates, sorbitan fatty acid esters, and the like.

Regarding the specific formulation of the pharmaceutical composition is known in the art, for example, Remington's Pharmaceutical Sciences (19th ed., 1995) and the like can be referred to. These documents are considered as part of this specification.

The preferred dosage of the pharmaceutical composition of the present invention is in the range of 0.001 mg/kg to 10 g/kg per day, preferably 0.001 mg/kg to 1 g, depending on the patient's condition, weight, sex, age, patient severity, and route of administration. May be in the /kg range. Administration can be made once a day or divided into several times. These dosages should not be construed as limiting the scope of the invention in any aspect.

As described above, according to the present invention, it is possible to provide a compound isolated and identified from the extract of Ribes fasciculatum , and a composition for protecting cranial nerve cells using the same.

The composition for protecting cranial nerve cells of the present invention may be commercialized as a food or drug.

1 is an experimental result confirming the protective activity of cranial nerve cells by extraction solvents of Chilhaemok.
Figure 2 is a schematic diagram of the separation of the compound.
3 is a 13C NMR spectrum result of a compound corresponding to <Formula 1>.
4 is a 1H NMR spectrum result of a compound corresponding to <Formula 1>.
5 is a 13C NMR spectrum result of a compound corresponding to <Formula 2>.
6 is a 1H NMR spectrum result of a compound corresponding to <Formula 2>.
7 is a DEPT90 spectrum result of a compound corresponding to <Formula 2>.
8 is a DEPT135 spectrum result of a compound corresponding to <Formula 2>.
9 is an HSQC spectrum result of a compound corresponding to <Formula 2>.
10 is a 13C NMR spectrum result of a compound corresponding to <Formula 3>.
11 is a 1H NMR spectrum result of a compound corresponding to <Formula 3>.
12 is a COSY spectrum result of a compound corresponding to <Formula 3>.
13 is a DEPT90 spectrum result of a compound corresponding to <Formula 3>.
14 is a DEPT135 spectrum result of a compound corresponding to <Formula 3>.
15 is an HSQC spectrum result of a compound corresponding to <Formula 3>.
16 is a 13C NMR spectrum result of a compound corresponding to <Formula 4>.
17 is a 1H NMR spectrum result of a compound corresponding to <Formula 4>.
18 is a COSY spectrum result of a compound corresponding to <Formula 4>.
19 is a DEPT90 spectrum result of a compound corresponding to <Formula 4>.
20 is a DEPT135 spectrum result of a compound corresponding to <Formula 4>.
21 is a result of the protective activity test of the cranial nerve cells (SH-SY5Y) of the compounds corresponding to <Formula 1> to <Formula 4>.
22 is a result of the protective activity test of the compounds corresponding to <Formula 1> to <Formula 4> on the brain nerve cells (PC12).
23 is an experimental result confirming the protective activity of the cranial nerve cells of a fraction of Chilhaemok (EA: ethyl acetate fraction).

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these Examples and Experimental Examples.

<Example 1> Isolation and identification of a novel compound as an active ingredient from the extract of Chilliwood

<Example 1-1> Isolation of the active compound from the extract of Chilhae mok

Extracts were prepared on the leaves of the dried Ribes fasciculatum with solvents (water or ethanol 30%, 70%, 100%) under various conditions and treated with hydrogen peroxide using the same method as in the experimental examples below. Brain neuron protective activity was confirmed in SY5Y cells and PC12 cells (Fig. 1). As can be seen in Figure 1, when the extract was prepared using a 30% ethanol solvent showed the best neuroprotective activity.

1.2Kg of Red Seaweed was extracted with 30% ethanol, which showed the best activity as a result of the above experiment, to obtain 303.5g of the extract, which was fractionated with hexane, dichloromethane, ethyl acetate, and butanol, respectively, to 1.5g, 4.0g, 28g, and 23.5g. g, to obtain a fraction of 246.5 g. Among them, silica gel column chromatography was performed on the ethyl acetate fraction (FIG. 23) that showed excellent cranial nerve cell protective activity by performing the experiment in the same manner as in the experimental example below, and divided into 11 small fractions. The compound was isolated from the small fraction and the corresponding process is schematically shown in FIG. 2.

As shown in Figure 2, fraction 7 of the small fraction was subjected to RP18 column chromatography to obtain a compound of <Formula 1> (hereinafter, Compound 1 ), and fraction 8 was subjected to RP18 column chromatography to obtain a compound of <Formula 2>. A compound (hereinafter, compound 2 ) was obtained. In addition, fraction 9 was subjected to RP18 column chromatography to obtain the compound of Formula 3 (hereinafter, Compound 3 ), and fraction 11 was subjected to RP18 column chromatography to obtain the compound of Chemical Formula 4 (hereinafter, compound 4 ). The structure of the isolated compound was determined through NMR spectral data and literature analysis, and are shown in FIGS. 3 to 20.

<Example 1-2> Identification of isolated compound

Nuclear magnetic resonance (NMR, Bruker Co., Ascend III 700 MHz) analysis, COSY-NMR analysis, mass spectrometry (MS), etc. to identify the structures of compounds 1 to 4 separated and purified according to Example 1-2. Was performed. The compound was dissolved in DMSO- d ₆ in NMR-dedicated Merck Co., placed in a 5 mm NMR tube, and measured for hydrogen and carbon nuclear magnetic resonance, and the peak of each solvent was used as an internal standard material or the peak of TMS (tetramethylsilane) was used as a reference. And measured.

Based on the results of the NMR analysis of FIGS. 3 and 4 and the like, the separated and purified compound 1 was identified as quercetin-3-rhamnoside, which is a compound of the <Formula 1>, and separated and purified based on the analysis results shown in FIGS. 5 to 9 The compound 2 was identified as rugosaflavonoid C, which is a compound of <Formula 2>. In addition, compound 3 was identified as kaempferol-3-rhamnoside, which is a compound of <Chemical Formula 3>, based on the NMR analysis results of FIGS. 10 to 15, and based on the analysis results of FIG. 16 to FIG. The compound kaempferol was identified.

<Experimental Example> Confirmation of the protective activity of cranial nerve cells of the compound isolated from the extract of Chilliwood

In order to confirm the protective activity of the compounds 1 to 4 isolated in the above examples, the MTT method was performed on the hydrogen peroxide-treated neurons.

SH-SY5Y cells, a human neuroblastoma, contain 10% (v/v) fetal bovine serum (FBS), 1% penicillin/streptomycin, NaHCO ₃ (2 mg/ml) and 15 mM. It was cultured at 37°C in a 5% CO ₂ incubator in an animal cell culture medium (DMEM medium) containing hepes. After seeding the cultured SH-SY5Y cells in a 96-well plate with 5×10 ⁴ cells/well (cell/well), the compound samples were treated by concentration (2, 10, 50 μg/ml) and 37 Incubated at °C for 24 hours. PC12 cells were cultured in a medium (GIBCO® RPMI Media 1640) supplemented with 10% fetal bovine serum, 1 mM sodium pyruvate, 100 unit/L penicillin, and 100 mg/L streptomycin at 37° C. supplied with 5% CO2 (Thermo Forma Direct Heat CO2 Incubator 311 model) and additionally cultured with the sample in the same conditions as the SH-SY5Y cells in a 96-well plate. Then, in order to induce the death of cranial nerve cells, about 50% of apoptosis was induced by treating a 96-well plate in which SH-SY5Y cells or PC12 cells were cultured with 100 μM of hydrogen peroxide for 1 hour.

In the cells treated with hydrogen peroxide, the protective effect of compounds 1 to 4 on cranial nerve cells was measured by the MTT method. The MTT method uses the principle that MTT changes to formazan by succinate-dehydrogenase, an enzyme in the cell's mitochondria, and decreases the production of formazan in dead cells, and shows the cell viability against external toxicity using the absorbance measurement results. Specifically, MTT (Sigma-Aldrich, cat.# M2128, USA) solution was added to each well so that the final concentration was 0.5 mg/ml in a 96-well plate. After reacting in the incubator for 3 hours, the medium and the MTT solution were removed, and then DMSO was added and stirred. When completely dissolved, the UV absorbance was measured at 540 nm using a microreader. Using the measured absorbance, the cell viability (%) was expressed as a percentage with respect to the control group (group treated with only hydrogen peroxide) (FIGS. 21 and 22 ).

Referring to FIG. 21, in SHSY5Y cells, all of Compounds 1 to 4 showed superior cranial nerve cell protective activity than the control group, and in particular Compound 1 and Compound 4 were found to be the most excellent.

In addition, as shown in FIG. 22, all of the PC12 cells also showed superior activity than the control group, of which Compound 4 showed a cell survival rate of about 150% compared to the control (100%).

Claims (9)

A composition for improving cognitive ability, comprising, as an active ingredient, an ethyl acetate fraction of a 30% ethanol extract of Chilliwood leaves or a mixed solvent extract of water and ethanol from Chilliwood leaves having a cranial nerve cell protective activity.
The method of claim 1,
A composition characterized in that the 30% ethanol extract of the leaves of the Red Sea leaves contains any one or more of the compounds of the following <Chemical Formula 1> to <Chemical Formula 3> having cognitive ability improving activity.
<Formula 1>

<Formula 2>

<Formula 3>

The method of claim 1,
The ethyl acetate fraction, a composition characterized in that it comprises any one or more of the compounds of the following <Formula 1> to <Formula 3> having cognitive ability improving activity.
<Formula 1>

<Formula 2>

<Formula 3>

The method according to any one of claims 1 to 3,
The composition is a composition, characterized in that the food composition.
The method according to any one of claims 1 to 3,
The composition is a composition, characterized in that the pharmaceutical composition.
A composition for improving diseases accompanying the death of cranial nerve cells, comprising as an active ingredient an ethyl acetate fraction of a 30% ethanol extract of Chilliwood leaves or a mixed solvent extract of water and ethanol from Chilliwood leaves, having a cranial nerve cell protective activity,
The disease accompanying the death of the cranial nerve cells is a degenerative brain disease or an ischemic brain disease,
The degenerative brain disease is Alzheimer's disease, Parkinson's disease, Huntington's disease, multiple sclerosis or amyotrophic lateral sclerosis,
The composition of the ischemic brain disease is vascular dementia.
The method of claim 6,
The 30% ethanol extract of the leaves of the red pepper tree is characterized in that it contains any one or more of the compounds of the following <Formula 1> to <Formula 3> having an activity to improve diseases accompanying the death of cranial nerve cells Composition.
<Formula 1>

<Formula 2>

<Formula 3>

The method of claim 6,
The ethyl acetate fraction, a composition comprising any one or more of the compounds of the following <Formula 1> to <Formula 3> having an activity to improve diseases accompanying the death of cranial nerve cells.
<Formula 1>

<Formula 2>

<Formula 3>

The method according to any one of claims 6 to 8,
The composition is a composition, characterized in that the food composition.

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