WO2018074793A1 - Cognitive function improving composition comprising novel post fermented tea-derived kaempferol-based compound - Google Patents

Abstract

The present specification relates to a cognitive function decrease improving composition comprising a novel compound separated from post fermented tea, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof or a solvate thereof, the composition being capable of being widely used in fields related to cognitive functions and nerve cell protection.

Description

A cognitive function improving composition comprising a novel camphorol-based compound derived from post-fermented tea

The present specification relates to a composition for improving cognitive function comprising a novel camphorol-based compound.

As the standard of living and the development of the medical industry made it possible to treat incurable diseases such as cancer, human lifespan increased, but the aging of the population caused cognitive decline and increased neurodegenerative diseases. The quality of the water is falling relatively. Neuronal dysfunction and damage can be caused by certain proteins that are prone to aggregation, and many neurological diseases are characterized by such conditions. Such nervous system diseases include diseases such as Alzheimer's disease.

As the elderly population increases, the necessity for the treatment and prevention of aging, cognitive deterioration, neurodegenerative diseases and brain diseases is increasing. Accordingly, studies on the prevention, treatment, alleviation, and improvement of such aging and diseases have been continuously conducted. However, existing materials have problems such as indeterminate effects or side effects, and thus, there is a need for development of therapeutic products derived from natural products to solve these problems.

Green tea is brewed in the form of leaf tea, or fermented tea for a deeper flavor. Fermented green tea means that the green tea leaves are subjected to oxidation treatment, including fermented tea oxidized by the oxidase present in the tea leaves, and post-fermented tea fermented by a separate microorganism other than the enzyme present in the tea leaves. . Depending on the degree of fermentation, it can be classified into weakly fermented tea, semi-fermented tea, and fully fermented tea. For example, fermented green tea is called by various names, such as green tea, oolong tea, black tea, black tea, etc., depending on the type and extent of fermentation.

The fermented tea may not only exhibit a difference in flavor compared to the green tea, but may also show a large difference in the type and content of the active ingredient depending on the specific fermentation process and the type of microorganism. Since various compounds can be produced and separated as described above, various efforts for separating and identifying unknown new compounds using green tea have been continued.

In one aspect, an object of the present invention is to discover a novel compound derived from post-fermented tea and use it for improving cognitive function and protecting neurons.

It provides a compound of Formula 1, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, or a post-fermented tea extract comprising the same as an active ingredient, a composition for improving cognitive decline.

[Formula 1]

In Formula 1, R ₁ may be C ₁₅ H ₉ O ₆ , R ₂ may be C ₆ H ₁₁ O ₅ , and R ₃ may be C ₉ H ₇ O ₂ .

In another aspect, the present invention, nerve cells for protection or neurological diseases comprising the compound, isomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, solvates thereof, or post-fermented tea extracts comprising the same as an active ingredient. A therapeutic composition is provided.

In another aspect, the present invention relates to administering to a subject in need thereof an effective amount of the compound, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, or an after-fermented tea extract comprising the same. Provided are a method for improving cognitive decline, a method for treating cognitive decline, a method for protecting neurons, or a method for treating neurological diseases.

In another aspect, the present invention, the compound, isomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, solvates thereof, or post-fermented tea extracts comprising the same for improving cognitive function, treatment for cognitive decline It provides a use for the production of a composition for protecting neurons, or treating neurological diseases.

In another aspect, the present invention, the compounds, isomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof as an active ingredient for use in improving cognitive decline, cognitive decline treatment, neuronal cell protection, neurological disease treatment It provides a solvate, or a post-fermented tea extract comprising the same.

In addition, the present invention, in another aspect, to improve the cognitive function, nerve cell protection as an active ingredient for the compound, isomers thereof, pharmaceutically acceptable salts thereof, hydrates, solvates thereof, or post-fermentation comprising the same Provides non-therapeutic use of tea extracts.

In one aspect, the present invention can be widely used in post-fermented tea-related industries, cognitive functions, and neuroscience by allowing the new compounds isolated from post-fermented tea to be used in the field of cognitive improvement and neuronal protection. have.

1 shows an MS spectrum of a compound according to one aspect of the invention.

Figure 2 shows the ¹ H-NMR (nuclear magnetic resonance) spectrum of the compound according to an aspect of the present invention.

Figure 3 shows the ¹³ C-NMR spectrum of the compound according to an aspect of the present invention.

Figure 4 shows the ¹ H- ¹³ C Heteronuclear Single Quantum Coherence (HSQC) spectrum of the compound according to an aspect of the present invention.

Figure 5 shows the ¹ H- ¹³ C Heteronuclear Multiple-Bond Coherence (HMBC) spectrum of the compound according to an aspect of the present invention.

Figure 6 confirms the effect of the compound on the aggregation of beta amyloid according to an aspect of the present invention.

As used herein, "post-fermentation" includes fermentation by a separate microorganism or substance other than the enzyme present in tea leaves. Post-fermented tea includes fermented green tea by the above method.

As used herein, the term "extract" includes any material obtained by extracting a component therefrom from a natural product, regardless of the method of extraction or the kind of the component. For example, extracting a component that is dissolved in a solvent from a natural product using water or an organic solvent, extracting only a specific component such as oil, such as oil, and fraction obtained by using a specific solvent again It is a broad concept that includes all of one fraction.

As used herein, the term "fraction" includes fractionating or extracting a specific substance or extract using a certain solvent, and extracting them again with a specific solvent. Fractionation methods and extraction methods can be any method known to those skilled in the art.

As used herein, “isomers” in particular are not only optical isomers (eg, essentially pure enantiomers, essentially pure diastereomers or mixtures thereof), but also form isomers ( conformation isomers (ie, isomers that differ only by their angles of one or more chemical bonds), position isomers (especially tautomers) or geometric isomers (eg, cis-trans isomers) do.

As used herein, "essentially pure" means at least about 90%, preferably at least about 95%, of a specific compound, for example enantiomers or diastereomers, when used in connection with an enantiomer or diastereomer. More preferably at least about 97% or at least about 98%, even more preferably at least about 99%, even more preferably at least about 99.5% (w / w).

As used herein, "pharmaceutically acceptable" refers to the approval of a government or equivalent regulatory body for use in animals, more specifically in humans, by avoiding significant toxic effects when used in conventional medicinal dosages. It is meant to be recognized or approved, or recognized as listed in a pharmacopoeia or other general pharmacopoeia.

As used herein, "pharmaceutically acceptable salts" means salts according to one aspect of the invention that are pharmaceutically acceptable and have the desired pharmacological activity of the parent compound. The salt is formed from (1) an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, or the like; Or acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, 3- (4-hydroxybenzoyl) Benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethane-disulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo [2,2,2] -oct-2-ene-1-carboxylic acid, glucoheptonic acid, 3-phenylpropionic acid, trimethylacetic acid, tert Acid addition salts formed with organic acids such as butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid; Or (2) salts formed when the acidic protons present in the parent compound are substituted.

As used herein, “hydrate” refers to a compound to which water is bound, and is a broad concept including an inclusion compound having no chemical bonding force between water and the compound.

As used herein, "solvate" means a higher order compound produced between molecules or ions of a solute and molecules or ions of a solvent.

The present invention, in one aspect, for improving the cognitive function of a compound of formula 1, its isomers, pharmaceutically acceptable salts thereof, hydrates thereof, solvates thereof, or post-fermented tea extracts comprising the same as an active ingredient To provide a composition.

[Formula 1]

In Formula 1, in Formula 1, R ₁ may be C ₁₅ H ₉ O ₆ , R ₂ may be C ₆ H ₁₁ O ₅ , and R ₃ may be C ₉ H ₇ O ₂ .

In some embodiments, R ₁ may be a compound of Formula 2 below.

[Formula 2]

According to another embodiment, R ₂ may be a compound of Formula 3 below.

[Formula 3]

R ₃ may be a compound of Formula 4 below.

[Formula 4]

According to another embodiment, the compound is camphorol3-O- [2-O ''-(E) -p-coumaroyl] [beta-D-glucopyranosyl- (1 → 3) -O-alpha -L-Ranmopyranosyl- (1 → 6) -O-beta-D-glucopyranoside] (Kaempferol3-O- [2-O ''-(E) -p-coumaroyl] [beta-D-glucopyranosyl -(1 → 3) -O-alpha-L-rhamnopyranosyl- (1 → 6) -O-beta-D-glucopyranoside]). The compound may be represented by the following Formula 5.

[Formula 5]

According to one aspect of the present invention, the compound, an isomer thereof, a pharmaceutically acceptable salt thereof, a method of preparing a hydrate thereof or a solvate thereof may include synthesis, separation from natural products, and the like.

According to another embodiment, the post-fermentation may be by strain inoculation, the strain may be Saccharomyces sp., Bacillus sp. Lactobacillus sp. And lucono Saccharomyces cerevisiae , Lactobacillus casei , Bacillus subtlis , Lactobacillus Bulgarian ( Lactobacillus bulgarius ) and Leuconostoc mesenteroides ). According to another embodiment, the post-fermented tea may be after fermenting green tea.

In one aspect, the compound is a compound discovered by the present inventors after the continuous study of the post-fermented tea, the beta-amyloid aggregation assay (beta-Amyloid aggregation assay) using the compound, as known It was confirmed that the inhibitory effect of beta amyloid aggregation and plaque formation was better than the inhibitors such as morphine and phenol red. Therefore, it has been found that the compound according to one aspect of the present invention can be used to prevent, treat, and ameliorate cognitive decline related to beta-amyloid, and also prevent neurons from damage and death due to beta-amyloid aggregation. It was demonstrated that the compound can be used for the purpose of protecting (see FIG. 6).

In addition, in one aspect of the present invention, the compound enhanced BDNF expression in neurons and decreased the expression of DNMT1. That is, the present invention was found to be useful for the prevention and treatment of neurodegenerative diseases such as cognitive decline, dementia, and Alzheimer's disease associated with decreased BDNF expression or enhanced DNMT1 expression.

In another aspect, the present invention relates to administering to a subject in need thereof an effective amount of the compound, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, or an after-fermented tea extract comprising the same. It may be a method for improving cognitive decline, a method for treating cognitive decline, a method for protecting neurons, or a method for treating neurological diseases.

In another aspect, the present invention, the compound, isomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, solvates thereof, or post-fermented tea extracts comprising the same for improving cognitive function, treatment for cognitive decline It may be related to the use for the production of a composition for the protection of neurons, or the treatment of neurological diseases.

In another aspect, the present invention, the compounds, isomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof as an active ingredient for use in improving cognitive decline, cognitive decline treatment, neuronal cell protection, neurological disease treatment It may be a solvate, or a post-fermented tea extract containing the same.

In addition, the present invention, in another aspect, to improve the cognitive function, nerve cell protection as an active ingredient for the compound, isomers thereof, pharmaceutically acceptable salts thereof, hydrates, solvates thereof, or post-fermentation comprising the same It may be related to the non-therapeutic use of tea extract.

In one embodiment, the extraction may be extraction with one or more solvents selected from water, hydrothermal water, C ₁ to C ₆ lower alcohols, and mixed solvents thereof, and in other embodiments, the lower alcohols are It may be an alcohol alone or a mixture which can be generally used in, preferably ethanol.

According to another aspect of the invention, the extract may be a fraction fractionated into ketones after extraction.

According to another embodiment, the ketone is acetone, carbon, pulegone, isolongifolanone, 2-heptanone, 2-pentanone, 3-hexanone, 3-heptanone , 4-heptanone, 2-octanone, 3-octanone, 2-nonanone, 3-nonanone, 2-undecanone, 2-tridecanone, methyl isopropyl ketone, ethyl isoamyl ketone, butyl Dencetone, methylheptenone, dimethyloctenone, geranyl acetone, farnesyl acetone, 2,3-pentadione, 2,3-hexadione, 3,4-hexadione, 2,3-heptadione, amylcyclopenta Paddy, amylcyclopentenone, 2-cyclopentyl cyclopentanone, hexylcyclopentanone, 2-n-heptylcyclopentanone, cis-jasmon, dihydrojasmon, methylcorylone, 2-tert-butylcyclo Hexanone, p-tert-butylcyclohexanone, 2-sec-butylcyclohexanone, celery ketone, krypton, p-tert-pentylcyclohexanone, methylcyclocitron, neron, 4-cyclohexyl-4-methyl- 2-pentanone, oxide ketone, emoxypron, Methylnaphthyl ketone, α-methylanisal acetone, anyl acetone, p-methoxy phenyl acetone, benzylidene acetone, p-methoxyacetophenone, p-methylacetophenone, propiophenone, acetophenone, α-dyna Dynascone, Iritone, Ionone, Pseudoionone, Methylionone, Methylyltone, 2,4-di-tert-butylcyclohexanone, Allylionone, 2 Ketones and solvents thereof, which may include acetyl-3,3-dimethylnorbornane, verbenone, fenchon, cyclopentadecanone, cyclohexadecenone, and the like, and are commonly used in the art. It may include all mixtures, preferably acetone.

According to an aspect of the present invention, the content of the compound of formula 1, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof in the composition may be 0.00001% to 10% by weight relative to the total weight of the composition. Can be. The content is 0.00001% by weight, 0.00005% by weight, 0.0001% by weight, 0.0005% by weight, 0.001% by weight, 0.005% by weight, 0.01% by weight, 0.05% by weight, 0.1% by weight based on the total weight of the composition At least 0.5%, at least 0.5%, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, or 9 It may be at least weight percent. 10 wt% or less, 9 wt% or less, 8 wt% or less, 7 wt% or less, 6 wt% or less, 5 wt% or less, 4 wt% or less, 3 wt% or less, 2 wt% or less, 1 wt% 0.5% or less, 0.1% or less, 0.05% or less, 0.01% or less, 0.005% or less, 0.001% or less, 0.0005% or less, 0.0001% or less, 0.00005% or less, or 0.00003% or less It may be less than or equal to%.

According to another aspect of the present invention, the content of the post-fermented tea extract in the composition may be 0.1% to 90% by weight relative to the total weight of the composition. The content is 0.1% by weight, 1% by weight, 5% by weight, 10% by weight, 15% by weight, 20% by weight, 25% by weight, 30% by weight, 35% by weight relative to the total weight of the composition. At least 40%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, or 85 It may be at least weight percent. 90 wt% or less, 85 wt% or less, 80 wt% or less, 75 wt% or less, 70 wt% or less, 65 wt% or less, 60 wt% or less, 55 wt% or less, 50 wt% or less, 45 wt% Up to 40 weight percent, up to 35 weight percent, up to 30 weight percent, up to 25 weight percent, up to 20 weight percent, up to 15 weight percent, up to 10 weight percent, up to 5 weight percent, up to 1 weight percent, or 0.5 weight It may be less than or equal to%.

According to another aspect of the invention, the extract is a compound of Formula 1, isomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvates thereof is at least 0.0001% by weight, 0.0005% by weight based on the total weight of the extract Or more, 0.001 or more, 0.005 or more, 0.01 or more, 0.05 or more, 0.1 or more, 0.5 or more, 1 or more, 3 or more, 5 or more, 7 or more % Or more, 10% or more, 12% or more, 15% or more, or 18% or more by weight may be included. 20 wt% or less, 15 wt% or less, 12 wt% or less, 10 wt% or less, 7 wt% or less, 5 wt% or less, 3 wt% or less, 1 wt% or less, 0.5 wt% or less, 0.1 wt% Or less, 0.05 wt% or less, 0.01 wt% or less, 0.005 wt% or less, 0.001 wt% or less, 0.0005 wt% or less, or 0.0003 wt% or less. Preferably, the extract may include 0.0001 wt% to 20 wt% of the compound of Formula 1, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof based on the total weight of the extract. .

According to another aspect of the present invention, the dosage of the compound of Formula 1, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof by administration of the composition is from 0.001 mg / kg / day to May be 100 mg / kg / day. The dosage is at least 0.001 mg / kg / day, at least 0.005 mg / kg / day, at least 0.01 mg / kg / day, at least 0.05 mg / kg / day, at least 0.1 mg / kg / day, 0.5 mg / kg / day Or more, 1 mg / kg / day or more, 5 mg / kg / day or more, 10 mg / kg / day or more, 15 mg / kg / day or more, 20 mg / kg / day or more, 25 mg / kg / day or more, 30 mg / kg / day or more , At least 35 mg / kg / day, at least 40 mg / kg / day, at least 45 mg / kg / day, at least 50 mg / kg / day, at least 55 mg / kg / day, at least 60 mg / kg / day, at least 65 mg / kg / day, At least 7 mg / kg / day, at least 75 mg / kg / day, at least 80 mg / kg / day, at least 85 mg / kg / day, at least 90 mg / kg / day, or at least 95 mg / kg / day. In addition, the dosage is 100 mg / kg / day or less, 95 mg / kg / day or less, 90 mg / kg / day or less, 85 mg / kg / day or less, 80 mg / kg / day or less, 75 mg / kg / day or less, 70 mg / kg / day or less, 65 mg / kg / day or less, 60 mg / kg / day or less, 55 mg / kg / day or less, 50 mg / kg / day or less, 45 mg / kg / day or less, 40 mg / kg / day or less, 35 mg / kg / Day or less, 30mg / kg / day or less, 25mg / kg / day or less, 20mg / kg / day or less, 15mg / kg / day or less, 10mg / kg / day or less, 5mg / kg / day or less, 1mg / kg / Days or less, 0.5 mg / kg / day or less, 0.1 mg / kg / day or less, 0.05 mg / kg / day or less, 0.01 mg / kg / day or less, 0.005 mg / kg / day or less, or 0.003 mg / kg / day It may be:

According to one embodiment, the cognitive decline is beta amyloid (β-amyloid) aggregation, beta amyloid plaque formation, or brain-derived neurotrophic factor (BDNF) expression and DNMT1 (DNA (cytosine-5) -methyltransferase 1) may be due to any one or more selected from the group consisting of increased expression.

According to another embodiment, the cognitive decline may include one or more selected from the group consisting of memory loss, cognitive decline, discrimination decline, depression, and forgetfulness.

According to another embodiment, the improvement consists of inhibiting beta-amyloid aggregation, inhibiting beta-amyloid plaque formation, degrading beta-amyloid plaques or aggregated beta-amyloid, enhancing BDNF expression and decreasing DNMT1 expression. It may be through one or more selected from the group.

According to one embodiment of the present invention, the composition may be a composition for protecting nerve cells.

In another embodiment, the neuronal cell protection may be to protect the neuron from the effects of aggregation or plaque of beta amyloid, reduced BDNF expression, or enhanced DNMT1 expression. Since aggregated beta amyloid is known to damage and kill nerve cells, according to one aspect of the present invention, it is possible to protect nerve cells when inhibiting aggregation or plaque formation of beta amyloid. In addition, DNMT1 inhibits gene expression by causing DNA methylation, which causes problems in BDNF expression and the like, leading to a decrease in cognitive ability. In one aspect, the present invention inhibits DNA methyltransferase 1 (DNMT1) activity and inhibits DNA methylation, thereby helping to improve cognitive ability and neurodegenerative disease through neuronal protection.

According to another aspect of the invention, the composition may be a pharmaceutical composition or a food composition. In one aspect, the composition may be a pharmaceutical composition for preventing or treating neurodegenerative diseases. In another aspect, the neurodegenerative disease may be due to one or more selected from the group consisting of beta amyloid (β-amyloid) aggregation, decreased BDNF expression, and increased DNMT1 expression. In another aspect, the neurodegenerative disease includes dementia, Alzheimer's disease, forgetfulness and the like.

The pharmaceutical composition according to an aspect of the present invention may be administered orally, parenteral, rectal, topical, transdermal, intravenous, intramuscular, intraperitoneal, subcutaneous. Formulations for oral administration may be tablets, pills, soft and hard capsules, granules, powders, granules, solutions, emulsions or pellets, but are not limited thereto. It is not. Formulations for parenteral administration may be, but are not limited to, solutions, suspensions, emulsions, gels, injections, drops, suppositories, patches or sprays. The formulations can be readily prepared according to conventional methods in the art and include surfactants, excipients, hydrating agents, emulsifiers, suspending agents, salts or buffers for controlling osmotic pressure, colorants, spices, stabilizers, preservatives, preservatives or Other commercially available auxiliaries may further be included.

The dosage or dosage of the pharmaceutical composition according to one aspect of the present invention will vary depending on the age, sex, weight, pathology and severity of the subject to be administered, the route of administration or the judgment of the prescriber. Application amount determination based on these factors is within the level of skill in the art.

The formulation of the food composition is not particularly limited, but may be, for example, formulated into tablets, granules, pills, powders, liquids such as drinks, caramels, gels, bars, tea bags, and the like. In addition to the active ingredient, the food composition of each formulation may be suitably selected by a person skilled in the art according to the formulation or purpose of use in addition to the active ingredient, and may be synergistic when applied simultaneously with other raw materials.

The composition may be administered by various methods such as simple ingestion, drinking, injection administration, spray administration or squeeze administration.

In the food composition according to an aspect of the present invention, the dosage determination of the active ingredient is within the level of those skilled in the art and may vary depending on various factors such as age, health condition, complications, etc. of the subject to be administered.

The food composition according to an aspect of the present invention may be processed foods of any type, such as, for example, various foods such as chewing gum, caramel products, candy, ice cream, confectionery, and beverage products such as soft drinks, mineral water, and alcoholic beverages. It may be a health functional food, including vitamins and minerals.

In addition to the above, the food composition according to an aspect of the present invention is a flavor, such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and enhancers (such as cheese, chocolate), pectic acid and its Salts, 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. In addition, the food compositions according to an aspect of the present invention may include a pulp for producing natural fruit juice and fruit juice beverage and vegetable beverage. These components can be used independently or in combination. The proportion of such additives is not so critical but is typically included in the range of 0 to about 50 parts by weight per 100 parts by weight of the composition according to one aspect of the invention.

Hereinafter, the configuration and effects of the present specification will be described in more detail with reference to Examples, Experimental Examples, and Formulation Examples. However, these examples are provided only for the purpose of illustration to aid the understanding of the present specification, the scope and scope of the present specification is not limited by the following examples.

Example 1 Preparation of Postfermented Tea Sample

The water content was adjusted to 40 wt% by adding water to the green tea made from green tea ( Camellia sinensis var. Yabukita ) leaves. Bacillus subtillis ( Bacillus subtillis ) 5 × 10 ⁶ cfu / g was inoculated, fermented for 3 days at 50 ℃ and then fermented at 80 ℃ 4 days.

The aged tea sample was ground for 15 seconds and filtered through a stainless steel sieve of mesh size 1 mm. Thereafter, the pulverized 50mg was added to 1.5ml Eppendorf tube, 1ml of deionized water was added, stirred at a constant speed for 30 minutes in a 60 ° C constant temperature water bath, and centrifuged for 15 minutes at 25 ° C 13,000rpm. Only insoluble portions of the dried fermented green tea extract were separated.

Example 2 Fraction Obtained and Compound Separation

150 g of the post-fermented tea sample was fractionated with acetone to remove catechin derivatives and caffeine and to obtain a soluble substance in which other compounds were concentrated. For 40 g of the acetone solubles, a fraction of 5: 1 (v / v) of chloroform: methanol was first obtained by using silica gel column chromatography as a solvent.

8.9 g of caffeine-free chloroform: methanol 5: 1 (v / v) fractions were fractionated using high-performance countercurrent chromatography (HPCCC, Dynamic Extractions Ltd, UK). The solvent used was n-hexane-TBME (Methyl tert-butyl ether) -BuOH-MeCN-Water (0.25: 3: 1: 1: 5, v / v), and the flow rate was 25 ml / min. A total of 10 subfractions were divided using the above conditions, and each fraction was subdivided again into a small volume of Dynamic Extractions Ltd, UK (HPCCC), High-performance liquid chromatography (HPLC), and Sephadex LH-20 column (GE Healthcare Bio). -Sciences, Sweden) and the like were used to isolate the components contained in each fraction.

As a result, camphorol 3-O- [2-O ''-(E) -p-coumaroyl] [beta-D-glucopyranosyl- (1 → 3), a previously unknown compound from the fractions -O-alpha-L-ramnopyranosyl- (1 → 6) -O-beta-D-glucopyranoside] (Kaempferol3-O- [2-O ''-(E) -p-coumaroyl] [beta -D-glucopyranosyl- (1 → 3) -O-alpha-L-rhamnopyranosyl- (1 → 6) -O-beta-D-glucopyranoside] was isolated and ¹ H, ¹³ C-NMR (nuclear magnetic) Resonace spectroscopy, UV (ultraviolet spectroscopy), and ESI-MS (Electro Spray Ionization Mass Specroscopy) were used to identify the structure of each compound. ^{In the case of 1} H and ¹³ C nuclear magnetic resonance (NMR), methanol-d3 was used as a solvent, and the device used Bruker Advance DPX-500 (BRUKER, USA). MS spectra of each compound were analyzed using 6200 Series Accurate-Mass Time-of-Flight (TOF) LC / MS (Agilent, US).

As a result of the analysis, each of the compounds is a novel compound which is not known previously, and is a camphorol 3-O- [2-O ''-(E) -p-coumaroyl] having a molecular weight of 902.2481 of C ₄₂ H ₄₆ O ₂₂ . Beta-D-glucopyranosyl- (1 → 3) -O-alpha-L-lamnopyranosyl- (1 → 6) -O-beta-D-glucopyranoside].

Camperol 3-O- [2-O ''-(E) -p-coumaroyl] [beta-D-glucopyranosyl- (1 → 3) -O-alpha-L-ramnopyranosyl- (1 → 6) -O-beta-D-glucopyranoside] is shown below.

Position		13 C-NMR		1 H-NMR
2		161.26
3		135.07
4		179.31
5		161.5
6		99.87		6.17 (H6, brs)
7		165.74
8		94.8		6.35 (H8, brs)
9		158.58
10		105.84
One'		122.72
2 ', 6'		132.29		7.99 (H2 '/ H6', d, J = 8.3 Hz)
3 ', 5'		116.27		6.87 (H3 '/ H5', d, J = 8.3 Hz)
4'		158.69
p-coumaric acid
One'''		127.3
2 '' ', 6' ''		131.2		7.45 (H2'`` / H6 ''', d, J = 8.1 Hz)
3 '' ', 5' ''		116.82		6.80 (H3'`` / H5 ''', d, J = 8.1 Hz)
4'''		161.26
7 '' '		115.31		6.35 (H7'``, d, J = 15.7 Hz)
8'''		146.88		7.67 (H8'``, d, J = 15.7 Hz)
C = O		168.79
Glc1
One''		101.55		5.46 (H1``, d, J = 7.8 Hz)
2''		74.14		5.34 (H2``, t, J = 9 Hz)
3 ''		73.25		3.76 (H3``, d, J = 10.4 Hz)
4''		70.47		3.85 (H4``, m)
5 ''		75.51		3.73 (H5``, m)
6 ''		67.54		3.76 (H6``, brd, J = 10.4 Hz)
				3.54 (H6``, m)
Rha
One''''		101.85		4.60 (H1 '' '', brs)
2''''		71.34		3.95 (2 '' '', m)
3 ''		83.09		3.61 (H3 '''', dd, J = 9, 3 Hz)
4''''		72.6		3.46 (H4 '' '', m)
5 ''		69.49		3.54 (H5 '' '', m)
6 '' ''		18.08		1.19 (H6 '''', d, J = 6 Hz)
Glc2
One'''''		105.74		4.40 (H1 ''''', d, J = 7.5 Hz
2'''''		75.4		3.25 (H2 '' '' ', m)
3 '' '' '		77.6		3.36 (H3 '' '' ', m)
4'''''		70.84		3.36 (H4 '' '' ', m)
5 '' '' '		77.6		3.25 (H5 '' '' ', m)
6 '' '' '		62.05		3.71 (H6 '' '' ', m)

Camperol 3-O- [2-O ''-(E) -p-coumaroyl] [beta-D-glucopyranosyl- (1 → 3) -O-alpha-L-ramnopyranosyl- (1 6) -O-beta-D-glucopyranoside] is shown in Figure 1, ¹ H-NMR spectrum and ¹³ C-NMR spectrum is shown in Figure 2 and 3, respectively, and HSQC (Heteronuclear Single Quantum Coherence) spectrum is as shown in Figure 4, Heteronuclear Multiple-Bond Coherence (HMBC) spectrum was as shown in FIG.

Experimental Example 1 Beta Amyloid Coagulation Efficacy Experiment

Camphorol 3-O- [2-O ''-(E) -p-coumaroyl] [beta-D-glucopyranosyl- (1 → 3) -O-alpha-L-ramnopyranosyl- ( 1 → 6) -O-beta-D-glucopyranoside] was confirmed by the fluorescence assay (ThioflavinT assay).

Specifically, beta-amyloid (Aβ1-42, AnaSpec Inc, USA) was obtained and used at a concentration of 0.1 mg / ml, and stored at -80 ° C before use. Morin (20 μM), Phenol Red (20 μM), camphorol 3-O- [2-O ''-(E) -p-coumaroyl] [beta-D-glucopyranosyl- in DMSO (1 → 3) -O-alpha-L-lamnopyranosyl- (1 → 6) -O-beta-D-glucopyranoside] (1 mg / ml) was each diluted to adjust to this concentration.

In order to specify the degree of inhibition of Aβ1-42 aggregation, 50 μl of 0.01 M sodium phosphate buffer solution was diluted to 10 μM of each compound prepared at the above concentration, and then 40 μl of 0.1 mg / ml of Aβ1-42 was added. 10 [mu] l of mM Thioflavin T was added and fluorescence was measured by a fluorescence spectrometer (RF-5300PC, SHIMADZU CORPORATION, Japan) for 5 minutes at 37 ° C for 5 minutes.

The results are shown in Table 2 below and FIG.

	Increased RFU	Increased RFU (% of Pos.Cont.)
Pos.Cont.	14595	100.0
New Substances33	11235	77.0
Morin	11471	78.6
Phenol red	13655	93.6

In the above table, RFU is relative fluorescence unit and “Increased RFU” is the amount of aggregated beta amyloid, and “Increased RFU (% of Pos. Cont.)” Is the percentage value of the amount of aggregated beta amyloid compared to the positive control. Indicates. “New Material 33” refers to camphorol 3-O- [2-O ''-(E) -p-coumaroyl] [beta-D-glucopyranosyl- (1 → 3) -O-alpha-L- Rhamnopyranosyl- (1 → 6) -O-beta-D-glucopyranoside].

In other words, when the aggregation of the positive control (positive control (labeled as "Pos.Cont.", Aggregated only beta amyloid without compound treatment) at 100%) Camphorol 3-O- [2-O ''-(E ) -p-coumaroyl] [beta-D-glucopyranosyl- (1 → 3) -O-alpha-L-ramnopyranosyl- (1 → 6) -O-beta-D-glucopyranoside] Was 23.0% more effective than the positive control. These results indicate that beta-amyloid aggregation and plaque formation inhibitory effects are better than the previously known inhibitors, Morin (21.4%) and Phenol red (6.4%). Thus, the two compounds have the same efficacy, and thus can be used for the prevention, treatment, and improvement of cognitive decline associated with beta amyloid aggregation. In addition, it is possible to prevent, prevent, and inhibit nerve cell damage or death by beta amyloid aggregation, etc., thereby protecting the nerve cells.

Experimental Example 2 Cumulative Skin Experiment

Camphorol 3-O- [2-O ''-(E) -p-coumaroyl] [beta-D-glucopyranosyl- (1 → 3) -O-alpha-L-ramnopyranosyl- ( 1 → 6) -O-beta-D-glucopyranoside] was subjected to HRIPT (Human repeated insult patch tests) to determine the cumulative irritation of the skin and to calculate the concentration range that can be used on the skin.

Specifically, 15 healthy adult subjects were randomly selected, and the test composition (the skin composition including an emulsifier, a stabilizer, purified water, etc.) containing 0.5 wt%, 1 wt%, and 3 wt% of the compound was randomly selected. 20 microliters per drop (IQ chamber, Epitest Ltd, Finland) was dripped, and after 24 hours after patching on the upper right part of the upper part of a subject, it replaced with the new patch. In this way, the skin reactions were examined before and after each patch while performing nine patches three times a week for a total of three weeks. The skin reactions were confirmed up to 48 hours after the final patch removal, and the average response was obtained.

The results are shown in Table 3 below.

Test substance and content	Number of subjects with ±, +, or ++ reactivity									Average reactivity
	1 time	Episode 2	3rd time	4 times	5 times	6th	7th	8th	9th
Control	0	0	0	0	0	0	0	0	0	0
	0	0	0	0	0	0	0	0	0
	0	0	0	0	0	0	0	0	0
New Substance 33 0.5wt%	0	0	0	0	0	0	0	0	0	0
	0	0	0	0	0	0	0	0	0
	0	0	0	0	0	0	0	0	0
New Substance 33 1% by weight	0	0	0	0	0	0	0	0	0	0
	0	0	0	0	0	0	0	0	0
	0	0	0	0	0	0	0	0	0
New Substance 33 3% by weight	0	0	0	0	0	0	0	0	0	0
	0	0	0	0	0	0	0	0	0
	0	0	0	0	0	0	0	0	0
Reactivity-: negative (no response) ±: suspicious or insignificant erythema, etc. +: weak reaction (without vesicles), erythema, papules ++: severity response (with vesicles), erythema, papules, vesicles ++ +: Strong response, algebraic response average responsiveness formula average responsiveness = [{(sum of the subjects multiplied by the response index and the response index) / (total subjects x highest score (4 points))} X 100] / test Number of times (9) In the above formula, if the reaction degree is-, the reaction index is 0, if the reaction degree is ±, the reaction index is 1, if the reaction degree is +, the reaction index is 2, and if the reaction degree is ++, the reaction index is 4. Determined as safe composition when average reactivity is less than 3

The skin response was determined according to the criteria of the International Contact Dermatitis Research Group (ICDRG). “New Material 33” in the table refers to camphorol 3-O- [2-O ''-(E) -p-coumaroyl] [beta-D-glucopyranosyl- (1 → 3) -O-alpha. -L-lamnopyranosyl- (1 → 6) -O-beta-D-glucopyranoside]. That is, the substance showed all (-) reactivity in the content range (no subject showed ±, +, ++, or +++ reactivity), through which the substance has no cumulative irritation of the skin, It was found to be safe to use.

Experimental Example 3 Expression of BDNF (Brain-derived Neurotrophic Factor) and DNMT1 (DNA (cytosine-5) -methyltransferase 1) in Cells

It was confirmed whether the novel substance 33 exerts efficacy in cells.

Specifically, SH-SY5Y (neuroblastoma, Korea Cell Line Bank) cell line seeded 2X10 ⁶ per well in a 6-well plate (FALCON), and incubated in 37 ° C, 5% CO ₂ incubator for 24 hours , GCG 10 μM, EGCG 10 μM, existing green tea extract (GTE) 10 μg / ml, the 'new material 33' 10 μg / ml, 5-Aza-2'deoxycytidine (5-Aza, Sigma as a positive control) -aldrich) and further incubated for 24 hours by treatment with 1 μM. Then, the medium was removed and RNA was extracted using an RNA extraction kit (RNeasy mini kit, Quiagen). After quantifying the extracted mRNA using an ultraviolet detector (TECAN) 1 μg of mRNA was synthesized using complementary DNA using a kit (SuperScript VILO cDNA Synthesis Kit, Thermofisher scientific). About 1 μg of complementary DNA was taken and subjected to real-time quantitative chain polymerization using Taqman probe (Life technology) and Quantitect Probe PCR Kit (Quiagen). Through this, the expression levels of BDNF and DNMT1 were confirmed. At this time, GAPDH, a housekeeping gene, was used as a reference mRNA.

Expression levels of BDNF and DNMT1 are shown in Tables 5 and 6, respectively.

Relative Expression of BDNF

division	%
Control group (untreated group)	100
New Substance 33 (10 μg / ml)	128
5-Aza-2'deoxycytidine 1 μM	149

Relative Expression of DNMT1

division	%
Control group (untreated group)	100
New Substance 33 (10 μg / ml)	78
5-Aza-2'deoxycytidine 1 μM	65

Since novel substance 33 lowers DNMT1 expression and increases BDNF expression, the compound can prevent, prevent, and inhibit neuronal damage or death, thereby protecting neurons and preventing and improving neurodegenerative diseases. You can do it.

Hereinafter, one example to describe the formulation of the composition according to an aspect of the present invention, the scope of the present invention is not limited thereto.

Formulation Example 1 Soft Capsule

Camperol 3-O- [2-O ''-(E) -p-coumaroyl] [beta-D-glucopyranosyl- (1 → 3) -O-alpha-L-ramnopyranosyl- (1 → 6) -O-beta-D-glucopyranoside] 20 mg, L-carnitine 80-140 mg, soybean oil 180 mg, palm oil 2 mg, vegetable hardened oil 8 mg, lead 4 mg and lecithin 6 mg are mixed and 1 capsule according to a conventional method. It was filled in to prepare a soft capsule.

Formulation Example 2 Tablet

Camperol 3-O- [2-O ''-(E) -p-coumaroyl] [beta-D-glucopyranosyl- (1 → 3) -O-alpha-L-ramnopyranosyl- (1 → 6) -O-beta-D-glucopyranoside] 30mg, galactooligosaccharide 200mg, lactose 60mg and malt sugar 140mg are mixed and granulated using a fluidized bed drier, and then 6mg of sugar ester is added to a tableting machine. Tablets were prepared by compression.

Formulation Example 3 Granules

Camperol 3-O- [2-O ''-(E) -p-coumaroyl] [beta-D-glucopyranosyl- (1 → 3) -O-alpha-L-ramnopyranosyl- (1 → 6) -O-beta-D-glucopyranoside] 50 mg, 250 mg of anhydrous crystalline glucose and 550 mg of starch were mixed, molded into granules using a fluidized bed granulator, and filled into sachets to prepare granules.

[Formulation Example 4] Drinks

Camperol 3-O- [2-O ''-(E) -p-coumaroyl] [beta-D-glucopyranosyl- (1 → 3) -O-alpha-L-ramnopyranosyl- (1 → 6) -O-beta-D-glucopyranoside] 20 mg, 10 g of glucose, 0.6 g of citric acid, and 25 g of liquid oligosaccharides are mixed and 300 ml of purified water is added to each bottle for 200 ml. After filling the bottle sterilized for 4-5 seconds at 130 ℃ to prepare a drink.

Formulation Example 5 Injection

Camperol 3-O- [2-O ''-(E) -p-coumaroyl] [beta-D-glucopyranosyl- (1 → 3) -O-alpha-L-ramnopyranosyl- (1 → 6) -O-beta-D-glucopyranoside] An injection was prepared in a conventional manner using 50 mg, sterile distilled water titrant for injection, and a pH adjuster titrant for injection.

 Formulation Example 6 Health Food

To prepare a health food in a conventional manner according to the composition shown in Table 6.

ingredient	content
Camperol 3-O- [2-O ''-(E) -p-coumaroyl] [beta-D-glucopyranosyl- (1 → 3) -O-alpha-L-ramnopyranosyl- (1 → 6) -O-beta-D-glucopyranoside]	0.5mg
Vitamin mixtures
Vitamin A Acetate	70 μg
Vitamin E	1.0 mg
Vitamin B1	0.13 mg
Vitamin B2	0.15 mg
Vitamin B6	0.5 mg
Vitamin B12	0.2 μg
Vitamin c	10 mg
Biotin	10 μg
Nicotinic acid amide	1.7 mg
Folic acid	50 μg
Calcium Pantothenate	0.5 mg
Mineral mixture
Ferrous sulfate	1.75 mg
Zinc oxide	0.82 mg
Magnesium carbonate	25.3 mg
Potassium phosphate monobasic	15 mg
Dicalcium Phosphate	55 mg
Potassium citrate	90 mg
Calcium carbonate	100 mg
Magnesium chloride	24.8 mg

Although the composition ratio of the vitamin and inorganic mixture is a composition that is relatively suitable for health foods, for example, the composition ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method, and then the conventional method. According to the health food composition can be used.

Formulation Example 7 Health Drink

ingredient	content
Camperol 3-O- [2-O ''-(E) -p-coumaroyl] [beta-D-glucopyranosyl- (1 → 3) -O-alpha-L-ramnopyranosyl- (1 → 6) -O-beta-D-glucopyranoside]	10mg
Citric acid	1000 mg
oligosaccharide	100 g
Plum concentrate	2 g
Taurine	1 g
Purified water	Remaining amount
Total volume	900 ml

After adding the remaining amount of purified water to a total volume of 900ml as shown in Table 7 and mixing the above components according to a conventional healthy beverage manufacturing method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution was filtered Obtained in a sterilized 2 liter container, sterilized sealed, and then refrigerated to prepare a healthy beverage.

As described above in detail the specific embodiments of the present disclosure, it is apparent to those skilled in the art that these specific techniques are only preferred embodiments, which are not intended to limit the scope of the present disclosure. . Therefore, the substantial scope of the present specification will be defined by the appended claims and equivalents thereof.

Claims (19)

1. A composition for improving cognitive deterioration comprising a compound of Formula 1, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, a solvate thereof, or a post-fermented tea extract comprising the same as an active ingredient

   [Formula 1]

   

   In Formula 1, R ₁ is C ₁₅ H ₉ O ₆ , R ₂ is C ₆ H ₁₁ O ₅ , R ₃ is C ₉ H ₇ O ₂ , The composition for improving cognitive decline.
2. The composition of claim 1, wherein R ₁ is a compound of Formula 2

   [Formula 2]

   

   .
3. The composition of claim 1, wherein R ₂ is a compound of Formula 3

   [Formula 3]

   

   .
4. The composition of claim 1, wherein R ₃ is a compound of Formula 4

   [Formula 4]

   

   .
5. The compound of claim 1, wherein the compound is

   Camperol 3-O- [2-O ''-(E) -p-coumaroyl] [beta-D-glucopyranosyl- (1 → 3) -O-alpha-L-ramnopyranosyl- (1 → 6) -O-beta-D-glucopyranoside] (Kaempferol3-O- [2-O ''-(E) -p-coumaroyl] [beta-D-glucopyranosyl- (1 → 3) -O- alpha-L-rhamnopyranosyl- (1 → 6) -O-beta-D-glucopyranoside]).
6. The composition of claim 1, wherein the extraction is extraction with one or more solvents selected from hot water, C ₁ to C ₆ lower alcohols, and mixed solvents thereof.
7. The composition of claim 6, wherein the lower alcohol is ethanol.
8. The composition of claim 1, wherein the extract is a fraction fractionated into ketones after extraction.
9. The composition of claim 8, wherein the ketone is acetone.
10. The composition of claim 1, wherein the content of the compound of formula 1, isomer thereof, pharmaceutically acceptable salt thereof, hydrate thereof, or solvate thereof in the composition is from 0.00001% to 10% by weight relative to the total weight of the composition.
11. The composition of claim 1, wherein the content of the post-fermented tea extract in the composition is 0.1 wt% to 90 wt% based on the total weight of the composition.
12. The method of claim 1, wherein the extract is a compound of Formula 1, isomers thereof, pharmaceutically acceptable salts thereof, hydrates thereof, or solvates thereof is 0.0001 to 20% by weight based on the total weight of the extract Phosphorus composition.
13. The dosage of the compound of Formula 1, an isomer thereof, a pharmaceutically acceptable salt thereof, a hydrate thereof, or a solvate thereof by administering the composition is 0.001 mg / kg / day to 100 mg / kg. Composition, per day.
14. The method according to any one of claims 1 to 13, wherein the cognitive decline is agglomeration of beta amyloid, decreased brain-derived neurotrophic factor (BDNF) expression, and DNMT1 (DNA (cytosine-5) -methyltransferase 1), the composition is due to at least one selected from the group consisting of increased expression.
15. The composition of claim 1, wherein the cognitive decline comprises one or more selected from the group consisting of memory decline, cognitive decline, discriminant decline, depression, and forgetfulness.
16. The method according to any one of claims 1 to 13, wherein the improvement is through at least one selected from the group consisting of inhibition of aggregation of beta-amyloid, degradation of aggregated beta-amyloid, enhancement of BDNF expression and reduction of DNMT1 expression. Phosphorus composition.
17. The composition of any one of claims 1 to 13, wherein the composition is a neuronal cell protective composition.
18. 18. The method of claim 17, wherein the neuronal cell protection comprises the aggregation of beta amyloid (β-amyloid), decreased brain-derived neurotrophic factor (BDNF) expression, and DNMT1 (DNA (cytosine-5) -methyltransferase 1) to protect neurons from the effects of one or more selected from the group consisting of increased expression.
19. The composition of claim 1, wherein the composition is a food or pharmaceutical composition.

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