KR102011033B1 - Composition for enhancing cognitive function comprising tea extraction which has modified amount of ingredients - Google Patents

Abstract

The present specification is to provide a composition derived from natural products with excellent cognitive improvement effect and a method for preparing the same, extracts and compositions according to an aspect of the present invention is safe from natural products and thereby prevent, improve and treat cognitive decline This can improve the quality of life of the aging population without fear of side effects and promote the development of related industries.

Description

COMPOSITION FOR ENHANCING COGNITIVE FUNCTION COMPRISING TEA EXTRACTION WHICH HAS MODIFIED AMOUNT OF INGREDIENTS}

The present specification relates to a composition for improving cognitive function including green tea extract having a changed component content.

Cognitive function is the ability to manipulate knowledge and information efficiently and encompasses memory, spatial perception, judgment, executive function, and language ability. As medical technology advances and economic growth enters an aging society, both physical and psychological damages associated with neurodegenerative diseases that impair cognitive and cognitive abilities appear.

Deterioration of cognitive function often means the decline of function and ability mainly related to memory, etc., but in recent years, it is not limited to memory, but complex attention, perception ability, execution function, learning and memory, language according to the process of brain change In terms of ability and area, such as ability, perceptual-motor function, and social cognition, it can be shown in various aspects.

Dementia, a representative disease related to cognitive decline, is a pathological phenomenon to be distinguished from normal aging, and according to the cause of Alzheimer's dementia, vascular dementia, other alcoholism, trauma, and Parkinson's disease. It is distinguished by dementia, which comes as an aftereffect.

Alzheimer's dementia has been reported as a chronic mental degenerative disorder that indicates high cerebral dysfunction such as loss of memory, unclear consciousness, disorientation of space-time perception, thinking, computing, judgment, and common sense. Alzheimer's dementia has been reported to occur in a relatively young group, and is the most common form of dementia in the elderly population, with the incidence doubled with every five years of age in the 65-85 range. have. The pathogenesis of Alzheimer's dementia is not well known, but the decrease in acetylcholine function in the central nervous system is most common. Therefore, acetylcholine precursors or drugs that inhibit the degradation of acetylcholine are used to treat the brain. Treatments that increase acetylcholine levels have been used. Therefore, acetylcholinesterase inhibitors alone or in combination with existing cholinesterase inhibitors are used as therapeutic agents. Representative drugs include tacrine, donepezil, and rivastigmine. Galantamine. All of these have slowed down the progression of acetylcholinesterase inhibitors and disease, but have little effect on direct treatment, and have limited therapeutic range in the early stages of disease. Therefore, efforts have been made to develop drugs to treat the root cause of Alzheimer's dementia.

Vascular dementia is mostly caused by damage to brain cells due to a lack of blood supply to various parts of the brain due to cerebrovascular atherosclerosis. Vascular dementia and Alzheimer's dementia have different causes, but the same is true in that damage occurs in memory and learning ability.

As the elderly population increases, the necessity for the treatment and prevention of aging, degenerative neurological diseases and brain diseases is increasing. Accordingly, studies on the prevention, treatment, alleviation and improvement of such aging and diseases have been steadily continued. The substance of the problem is that the effect is unclear or cause side effects, it is necessary to develop a natural-based treatment to solve these problems.

1) Republic of Korea Patent Publication No. 10-2014-0077382 (2014.06.24)

The present specification is to provide a composition derived from a natural product and non-toxic and excellent cognitive function improving efficacy.

In one aspect, the present invention for solving the above problems, 5 to 25% by weight of (-)-gallocatechin gallate ((-)-gallocatechin gallate, GCG) and 7 to 15% by weight based on the total weight of the composition It provides a composition for improving cognitive decline, including a green tea extract containing (-)-epigallocatechin gallate ((-)-epigallocatechin gallate, EGCG) as an active ingredient.

In another aspect, the present invention also provides 5 to 25% by weight of (-)-gallocatechin gallate (GCG) and 7 to 15% by weight of (-), based on the total weight of the composition. It provides a composition for preventing or treating neurodegenerative diseases, including green tea extract comprising epigallocatechin gallate ((-)-epigallocatechin gallate, EGCG) as an active ingredient.

In another aspect, the present invention (1) adding ethanol to green tea and extracting for 30 minutes to 4 hours at 50 to 70 ℃; (2) filtering and depressurizing to remove ethanol; And (3) adding water, stirring at 70 to 100 ° C. for 3 to 6 hours, and then concentrating under reduced pressure.

The extracts and compositions according to one aspect of the present invention are safe from natural products and thereby prevent, improve and treat cognitive decline, thereby improving the quality of life of an aging population without fear of side effects and promoting the development of related industries. can do.

Figure 1 shows a chromatogram for the test-type green tea extract of Example 1 (Sample 1).
Figure 2 shows a chromatogram for the hot-treated green tea extract (Sample 2) according to an aspect of the present invention.
Figure 3 shows the neuronal cell cytotoxicity confirmation results for Samples 1 and 2.
Figure 4 shows the acetylcholinesterase activity assay results for Samples 1 and 2.
Figure 5 shows the results of the DNMT1 inhibitory assay for Sample 1 and 2, 'RG108' is N -Phthaloyl-l-tryptophan which is a DNMT inhibitor (hibitor).
Figure 6 shows the lipid peroxidation (MDA) assay results for Sample 2.
Figure 7 shows the Y labyrinth experiment method and results for the mouse administered to Sample 2.
Figure 8 shows the test results of the amyloid beta aggregation inhibitory ability of Sample 3.
Figure 9 shows the test results of the amyloid beta aggregation inhibitory ability of Sample 3.
Figure 10 shows the results of the cell death protection confirmation experiment through the inhibition of amyloid beta aggregation of Sample 3.
Figure 11 shows the results of the long-term memory reinforcement phenomenon (LTP) induction test of Sample 3.

The term “green tea extract” in this specification, regardless of the extraction method, the solvent, the extracted component or the form of the extract, is extracted from the evergreen tree ( Camellia sinensis ), an evergreen tree belonging to the fruit or Bacillus subtilis spp.) inoculated and extracted from fermented tea leaves and the like, and the extract is fractionated with a specific solvent. The tea includes one or more selected from the group consisting of tea tree leaves, flowers, stems, fruits, roots, stems, and roots of the root, and may preferably be a leaf. In addition, the extract may be in the form of a powder. The extraction or fractionation may be performed using water, an organic solvent, or a mixed solvent thereof. The organic solvent may be alcohol, isopropanol, acetone, hexane, ethyl acetate, carbon dioxide, or a mixture of two or more thereof, but is not limited thereto, and extracted by room temperature or warming under the condition that the active ingredient of green tea is not destroyed or minimized. Or fractionated. The alcohol may be a C ₁ ~ C ₅ lower alcohol. The number or methods of extraction or fractionation are not particularly limited, and for example, cold extraction, ultrasonic extraction, reflux cooling extraction, hot water extraction, and the like may be used. Preferably, the extraction or fractionation of the active ingredient by cooling or warming is performed. After filtration, the filtrate was concentrated under reduced pressure to obtain the green tea extract of the present invention.

As used herein, "epic catechin" refers to epigallocatechin (EGC), (-) epicatechin (EC), (-)-epigallocatechin gallate (EGCG), and epicatechin 3-O-gallate (ECG) It will include.

In one aspect, the present invention, based on the total weight of the composition or extract, 5 to 25% by weight of (-)-gallocatechin gallate ((-)-gallocatechin gallate (GCG) and 7 to 15% by weight of (-) -Epigallocatechin gallate ((-)-epigallocatechin gallate, EGCG) may be related to a composition for improving cognitive function lowering comprising the green tea extract as an active ingredient.

In another aspect, the present invention also provides 5 to 25% by weight of (-)-gallocatechin gallate (GCG) and 7 to 15% by weight of (-), based on the total weight of the composition. It provides a composition for preventing or treating neurodegenerative diseases, including green tea extract comprising epigallocatechin gallate ((-)-epigallocatechin gallate, EGCG) as an active ingredient.

In one aspect the GCG is at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11% by weight based on the total weight of the composition or the extract , At least 12 wt%, at least 12.52 wt%, at least 13 wt%, at least 14 wt%, at least 16 wt%, at least 18 wt%, at least 20 wt%, at least 22 wt%, or at least 24 wt%. In another aspect the GCG is 25% or less, 23% or less, 21% or less, 19% or less, 17% or less, 15% or less, 14% or less by weight based on the total weight of the composition or the extract , Up to 13 wt%, up to 12.55 wt%, up to 12 wt%, up to 11 wt%, up to 10 wt%, up to 9 wt%, up to 8 wt%, up to 7 wt%, or up to 6 wt%.

In one aspect, the EGCG is at least 7% by weight, at least 8% by weight, at least 8.48% by weight, at least 8.5% by weight, at least 9% by weight, at least 10% by weight, 12% by weight, based on the total weight of the composition or the extract. Or at least 14% by weight. In another aspect, the EGCG is 15% by weight, 13% by weight, 11% by weight, 10% by weight, 9% by weight, 8.5% by weight or less, 8.48% by weight based on the total weight of the composition or the extract. Or less, 8.3 wt% or less, 8 wt% or less, or 7.5 wt% or less.

As another embodiment, the total content of GCG and EGCG in the extract may be up to 40% by weight based on the total weight of the composition or extract. In one aspect, the catechin content is 40 wt% or less, 35 wt% or less, 30 wt% or less, 25 wt% or less, 20 wt% or less, 18 wt% or less, 16 wt% or less based on the total weight of the composition or extract. , Up to 15 wt%, up to 14 wt%, up to 12 wt%, up to 10 wt%, up to 8 wt%, up to 6 wt% or up to 4 wt%. In another aspect the total content of the GCG and EGCG is at least 3% by weight, at least 6% by weight, at least 8% by weight, at least 10% by weight, at least 12% by weight, at least 13% by weight, based on the total weight of the composition or extract Or at least 14 wt%, at least 16 wt%, at least 18 wt%, at least 20 wt%, at least 25 wt%, at least 30 wt%, or at least 35 wt%.

As another embodiment, the epi catechin content in the extract may be 20% by weight or less based on the total weight of the composition or extract. In one aspect, the epi catechin content is 20 wt% or less, 18 wt% or less, 16 wt% or less, 15 wt% or less, 14 wt% or less, 12 wt% or less, 10 wt% based on the total weight of the composition or extract. Up to 8 wt%, up to 6 wt% or up to 4 wt%. In another aspect the catechin content is at least 3%, at least 6%, at least 8%, at least 10%, at least 12%, at least 13%, at least 14% by weight, based on the total weight of the composition or extract. , 16 wt% or more, or 18 wt% or more.

As another embodiment, the extract may be an extract extracted one or more times by any one or more of water and C1 to C4 alcohol. In one aspect the alcohol may be ethanol. In another aspect the alcohol may be at least 20%, at least 30%, at least 40%, at least 50%, at least 60% or at least 70% ethanol. In another aspect the alcohol can be 70% or less, 60% or less, 50% or less, 40% or less or 30% or less ethanol.

As another embodiment, the content of the extract in the composition may be 1% to 100% by weight on a dry weight basis. In one aspect, the content of the extract in the composition on a dry weight basis of at least 1% by weight, at least 10% by weight, at least 20% by weight, 30% by weight, 40% by weight, 50% by weight, 60% by weight or more , At least 70% by weight, at least 80% by weight, or at least 90% by weight. In another aspect, the content of the extract in the composition is 100% by weight, 90% by weight, 80% by weight, 70% by weight, 60% by weight, 50% by weight, 40% by weight on a dry weight basis It may be 30 wt% or less or 20 wt% or less.

As another embodiment, the dosage of the active ingredient may be 5 mg / kg / day to 1000 mg / kg / day on a dry weight basis. In one aspect, the dosage is at least 5 mg / kg / day, at least 100 mg / kg / day, at least 200 mg / kg / day, at least 300 mg / kg / day, at least 400 mg / kg / day, at least 500 mg / kg / day, At least 600 mg / kg / day, at least 700 mg / kg / day, at least 800 mg / kg / day, or at least 900 mg / kg / day. In another aspect, the dosage is 1000 mg / kg / day or less, 900 mg / kg / day or less, 800 mg / kg / day or less, 700 mg / kg / day or less, 600 mg / kg / day or less, 500 mg / kg / day or less, Up to 400 mg / kg / day, up to 300 mg / kg / day, up to 200 mg / kg / day, up to 100 mg / kg / day, up to 50 mg / kg / day or up to 10 mg / kg / day.

In one embodiment, the cognitive decline may be due to any one selected from the group consisting of neurotransmitter degradation, neurotransmitter production reduction, and neurotransmitter receptor reduction.

In another embodiment, the neurotransmitter may be acetylcholine.

Dementia is classified into Alzheimer's disease, vascular dementia, and other diseases according to the cause of the dementia. Dementia patients generally have clinically obvious impairments in intellectual ability, emotion, and behavioral changes. They then show signs of severe cortical dysfunction, such as loss of orientation, memory damage, and aphasia. The pathological mechanism suggested by the study of Alzheimer's disease is that the abnormal protein called β-amyloid accumulates in the brain outside the neuron, and then the tau protein aggregates inside the neuron to repair synaptic damage. It is known to cause neuronal dysfunction and brain cell death ultimately. In dementia, neurochemically, cholinergic disorders of the hippocampus and temporal lobe are particularly prominent, and disorders in these areas are directly or indirectly related to memory damage. Biochemical deterioration and neuronal loss of the cholinergic nervous system of the basal forebrain pathway have been consistently reported by dementia researchers. Indeed, choline uptake and acetylcholine synthesis were reduced in the hippocampus and cerebral cortex in patients with dementia. On the contrary, the expression of acetylcholine esterase (AChE), an enzyme that degrades acetylcholine, was high. It is reported. In addition, the activity of choline acetyltransferase (ChAT), an enzyme that synthesizes acetylcholine, drops dramatically, which is greatest in the hippocampus and nicotinic and / or muscarinic acetylcholine receptors in the brain: It was confirmed that the number of nAchR and / or mAchR) was reduced. The present invention is to develop a cognitive ability improving composition as an indicator of acetylcholine metabolism in one aspect, the composition according to one aspect of the present invention significantly improves acetylcholine metabolism, can greatly improve the cognitive decline, Furthermore, it can be of great help in improving the quality of life, improving symptoms, and treating patients with cognitive impairment, such as dementia, amnesia, amnesia.

In another embodiment, the cognitive decline may be due to DNA methylation.

In another embodiment, the DNA methylation may be based on DNA methyltransferase 1 (DNMT1). DNMT1 inhibits gene expression by causing DNA methylation, which causes problems such as BDNF expression, leading to a decrease in cognitive ability. In one aspect, the present invention inhibits DNA methyltransferase 1 (DNMT1) expression, thereby inhibiting DNA methylation, thereby helping to improve cognitive ability.

In one embodiment, the cognitive decline may be due to brain tissue lipid peroxidation.

In another embodiment, the cognitive decline may be due to malondialdehyde, a product of peroxide.

Lipid peroxidation refers to the peroxidation of lipids, which is actually the fatty acid portion of the lipid, which has a perhydroxy structure, an internal peroxidation structure, a perhydroxy radical structure, and in some cases their Decomposition products may be included in this area. Perhydroxyl structures are generally unstable and frequently generate radicals. It is believed that causing the chain of these radical reactions is the cause of tissue disorders in living organisms. In other words, lipofucin, which appears in the nerves, liver, and muscle cells of old animals, is considered to be an insoluble heterogeneous polymer that swept proteins around lipid peroxide, and peroxidation of membrane lipid changes properties such as permeability of membrane. As can be thought of as a factor of vascular lesions there are harmful aspects to the living body. In addition, cholesterol peroxide gas is also known. The chemical structure will be called alcohol peroxide. Clinically known to be related to aging and the like, lipid peroxide is known as the cause of brain aging in particular, this lipid peroxide leads to brain aging and cognitive decline. Therefore, it is possible to improve cognitive function by reducing lipid peroxide, and the present invention can improve brain aging and decrease in cognitive function by lowering brain tissue lipid peroxide products in one aspect.

In another embodiment, the cognitive decline may be one or more selected from the group consisting of lethargy, memory loss, forgetfulness, cognitive decline, learning disability, attention loss, depression, hearing loss, painlessness, involuntary symptoms, and discrimination decline have.

In another embodiment, the cognitive decline may be due to a neurodegenerative disease.

In one embodiment, the neurodegenerative diseases include Alzheimer's disease, dementia, Parkinson's disease, hunting tongue disease, autosomal-dominant cerebellar ataxia, narcolepsy, alcoholism, drug addiction and hereditary sensory and autonomic neuropathy. and autonomic neuropathy).

In one embodiment, the composition may be a food or pharmaceutical composition.

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 appropriately selected and blended by the person skilled in the art according to the formulation or purpose of use, which are conventionally used in the relevant fields, and synergistic effect when applied simultaneously with other raw materials. Can happen. In addition, the food may be a health functional food.

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 one 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. .

Food composition according to one aspect of the present invention, for example, a variety of food products such as chewing gum, caramel products, candy, ice cream, confectionery, beverage products such as soft drinks, mineral water, alcoholic beverages, vitamins and minerals It may be a nutraceutical product.

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 one 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 60 parts by weight per 100 parts by weight of the composition according to one aspect of the invention.

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 composition according to an aspect of the present invention may include a pharmaceutically acceptable salt, which salt is formed of (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.

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. Effective ingredient dosage determination based on these factors is within the level of one of ordinary skill in the art.

In another aspect, (1) adding ethanol to green tea and extracting for 30 minutes to 4 hours at 50 ℃ to 70 ℃; (2) filtering and depressurizing to remove ethanol; And (3) adding water and stirring for 3 to 8 hours at 70 ° C. to 100 ° C., followed by concentration under reduced pressure.

In one aspect, the ethanol may be 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, or 70% or more ethanol. In another aspect the ethanol may be 70% or less, 60% or less, 50% or less, 40% or less or 30% or less ethanol.

In one aspect, the temperature of step (1) may be 50 ° C. or higher, 55 ° C. or higher, 60 ° C. or higher, 65 ° C. or higher, or 68 ° C. or higher. In another aspect, the temperature of step (1) may be 70 ° C. or less, 65 ° C. or less, 60 ° C. or less, 55 ° C. or less, or 52 ° C. or less.

In one aspect, the time of step (1) is at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 60 minutes, at least 70 minutes, at least 80 minutes, at least 90 minutes, at least 100 minutes, at least 120 minutes, at least 140 minutes. , At least 160 minutes, at least 180 minutes, at least 200 minutes, or at least 220 minutes. In another aspect, the time of step (1) is 240 minutes or less, 220 minutes or less, 200 minutes or less, 180 minutes or less, 160 minutes or less, 140 minutes or less, 120 minutes or less, 100 minutes or less, 90 minutes or less, 80 minutes or less , Up to 70 minutes, up to 60 minutes, up to 50 minutes, or up to 40 minutes.

In one aspect, the temperature of step (3) may be at least 70 ℃, at least 75 ℃, at least 80 ℃, at least 90 ℃, at least 95 ℃ or 98 ℃. In another aspect, the temperature of step (3) may be 100 ° C. or less, 95 ° C. or less, 90 ° C. or less, 85 ° C. or less, 80 ° C. or less, or 75 ° C. or less.

In one aspect, the stirring time of step (3) may be 30 minutes or more, 1 hour or more, 2 hours or more, 3 hours or more, 4 hours or more, 5 hours or more, 6 hours or more, or 7 hours or more. In another aspect, the stirring time of step (3) may be 8 hours or less, 7 hours or less, 6 hours or less, 5 hours or less, 4 hours or less, 3 hours or less, 2 hours or less, 1 hour or less.

As another embodiment, the weight ratio of the product of step (2) and the water added in step (3) may be 1: 7 to 1:12. In one aspect the weight ratio is 1: 5 or more, 1: 6 or more, 1: 7 or more, 1: 8 or more, 1: 9 or more, 1:10 or more, 1:12 or more, 1:14 or more, 1:16 or more Or 1:18 or more. In another aspect, the weight ratio is 1:20 or less, 1:18 or less, 1:16 or less, 1:14 or less, 1:12 or less, 1:11 or less, 1:10 or less, 1: 9 or less, or 1: 8 or less. , 1: 7 or less, or 1: 6 or less.

As another embodiment, the yield after the step (3) may be 5 to 30% by weight based on the weight of the green tea in the step (1). In one aspect, the yield is at least 5% by weight, at least 6% by weight, at least 8% by weight, at least 10% by weight, at least 12% by weight, at least 14% by weight, at least 16% by weight, at least 18% by weight, at least 20% by weight. , At least 22 weight percent, at least 24 weight percent, at least 26 weight percent or at least 28 weight percent. In another aspect, the yield is 30 wt% or less, 28 wt% or less, 26 wt% or less, 24 wt% or less, 22 wt% or less, 20 wt% or less, 18 wt% or less, 16 wt% or less, 14 wt% or less , 12 wt% or less, 10 wt% or less, 8 wt% or less, or 6 wt% or less.

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 help the understanding of the present specification, and the scope and scope of the present specification are not limited by the following examples.

Example 1 Preparation of Tested Green Tea Extract and High-Temperature Green Tea Extract

1000 ml of 50% ethanol was added to 100 g of green tea (Camellia sinensis, Jeju Osulloc Farm) and stirred under reflux at 60 ° C for 1 hour. The sample obtained by lowering the temperature of the sample to room temperature and filtration was distilled under reduced pressure to obtain 23 g of a dark green powder of a visible green tea extract (GT-LE-35CAT, sample 1) (yield 23%).

Meanwhile, 10 g of the sample 1 was dissolved in 90 ml of water, and then stirred at 80 ° C. for 30 minutes to 8 hours. After lowering the temperature to room temperature, the insoluble material was filtered and concentrated under reduced pressure to obtain a high-temperature green tea extract 10g. At this time, while measuring the change in the content of GCG, etc. for each time zone using the apparatus shown in Table 1 for the hot-treated green tea extract obtained by the stirring time zone (content of each component in the time zone extract is as Table 4), The time zone in which the most amount of GCG was present was confirmed, and 10 g (GT-LE-10GCG, High temperature processed green tea extract, HTP-GTE) of the obtained high-temperature green tea extract was stopped as the sample time.

In addition, while measuring the change in the content of the GCG, such as time zones using the apparatus shown in Table 5 for the hot-treated green tea extract obtained by the stirring time zone (content of each component in the time zone extract is shown in Table 7), GCG When the content of 5 ~ 8% of the stirring was stopped and the obtained high temperature green tea extract 10g was used as sample 3.

The conditions and the results of analyzing the compositions of the three kinds of extracts obtained are shown in Tables 1 to 3 and 5 to 6, respectively. In addition, chromatograms for the two kinds of extracts are shown in Figure 1 (Sample 1) and Figure 2 (Sample 2). That is, in the case of Sample 2, it was confirmed that the composition is different from the existing green tea extract.

Composition analysis condition column Sun fire C18 5um, 4.6 X 250mm Detector UV 280nm Device Waters 2998 PDA Detector, Waters 1525 Pump,
Waters 2707 Autosampler Dilution Water in Gradient A-0.1% Trifluoro acetic acid (TFA)
B-acetonitrile Gradient profile 0 min A (95): B (5) 1min A (95): B (5)
20min A (71): B (29)
22min A (71): B (29) Flow rate 1ml / min Injection volumn 20 μl

Sample 1 EGC Caffeine EC EGCG ECG Epicatechin Total 9.16 3.21 3.63 20.93 2.62 36.34

Sample 2 EGC Caffeine EC EGCG ECG GCG CG Epicatechin Total GCG + EGCG 2.16 3.28 0.75 8.48 1.90 12.52 2.38 13.28 21

Stirring time EGCG GCG ECG CG 1 hours 11.79 7.6 1.16 1.16 3 hours 9.67 11.08 2.44 1.46 5 hours 8.48 12.52 1.9 2.38 7 hours 6.71 9.44 1.85 1.56

Composition analysis condition column Thermofisher C18 5um, 4.6 X 250mm Detector UV 280nm Dilution Water in Gradient A-0.1% Trifluoro acetic acid (TFA)
B-acetonitrile Gradient profile 0 min A (90): B (10) 30min A (85): B (15)
42min A (80): B (20)
44min A (5): B (95)
49min A (90): B (10) Flow rate 1ml / min Injection volumn 20 μl

Sample 3 EGC Caffeine EC EGCG ECG GCG CG Epicatechin Total GCG + EGCG 4.56 4.58 2.27 10.39 2.79 7.59 0.87 20.01 17.98

Stirring time EGCG GCG ECG CG 1 hours 16.28 3.09 3.82 0.84 3 hours 13.74 6.08 3.08 0.85 5 hours 11.23 7.21 2.83 0.86 6 hours 10.39 7.59 2.79 0.87

(In Table 2 to Table 4, Table 6 and Table 7, EGC is epigallocatechin, EC is (-) epicatechin, ECG is epicatechin 3-O-gallate)

(The units of Tables 2 to 4, Table 6 and Table 7 are all the weight percent of the corresponding substance to the total weight of the sample)

Experimental Example 1 Neuronal Toxicity Confirmation Experiment

The PC12 cell line (neurocytoma) obtained from the Korea Cell Line Bank was seeded by 1 × 10 ⁵ per well in a 96 well plate (FALCON), and cultured in a 37 ° C., 5% CO ₂ incubator for 24 hours, and then the sample. 1 and Sample 2 were treated with 3, 10, 20 and 30 μg / ml, respectively, and further incubated for 24 hours.

Then, the medium was removed and cell viability was confirmed using cell counting kit-8 (Dojindo). 10 μl of the cell counting kit-8 (Dojindo) solution was added to 100 μl of RPMI1640 (Lonza), and the number of living cells was quantified by checking the absorbance at 450 nm after treatment. Cell number or cell viability was calculated by the following formula.

Cell survival rate (%) = (absorbance of sample treated group-absorbance of reaction reagent only) / (absorbance of untreated group-absorbance of reaction reagent only) X 100

The results are the same as in Figure 3, Sample 2 was confirmed that the neuronal toxicity is lower than Sample 1.

Experimental Example 2 Acetylcholinesterase Activity Assay

Acetylcholine (acetylcholine, ACh) is an organic molecule that acts as a neurotransmitter, a substance involved in plasticity, arousal, and compensation systems, and decreases in patients with Alzheimer's disease. As acetylcholinesterase activity is enhanced, acetylcholine decreases. Therefore, inhibition of acetylcholinesterase activity may improve cognitive function.

Acetylcholinesterase activity assay kit (Anaspec) was used to determine the inhibitory effect of acetylcholinesterase (acetylcholinesterase) of each sample.

5, 10 μg / ml of Sample 1 and Sample 2 were reacted with a reagent containing Acetylcholinesterase for 1 hour, and the fluorescence intensity was measured at Ex / Em = 490 nm / 520 nm using a multiplate reader (tecan). Acetylcholinesterase activity was measured in comparison with the fluorescence intensity of the sample untreated group. The formula for comparing the activity was as follows.

Relative activity = (sample treated group fluorescence intensity / untreated group fluorescence intensity) X 100

The result was as shown in FIG. It was confirmed that Sample 2 had a higher inhibitory effect of acetylcholinesterase activity than Sample 1 at the same concentration (2 μg / ml). In addition, in order to inhibit the acetylcholinesterase activity of 2 μg / ml level of Sample 1, Sample 2 is required to 1.39 μg / ml, Sample 2 can be seen that the inhibitory effect of acetylcholinesterase activity in a much smaller amount than Sample 1 .

Experimental Example 3 DNMT1 (DNA (cytosine-5) -methyltransferase 1) Inhibition Assay

DNMT1 enzyme expression was measured using EpiQuik DNMT Activity / Inhibition Assay Ultra Kit (Epigentek).

Specifically, DNMT1 enzyme and the samples 1 and 2 were mixed 10μM, 50μM and then reacted for 90 minutes at 37 ℃. After washing with the wash buffer provided in the kit, the capture antibody that recognizes the methylated DNA was added and incubated for 60 minutes. After washing with the wash buffer again and incubated for 30 minutes with an antibody that recognizes the capture antibody, washed once more with the wash buffer and give an enhancer solution and further incubated for 30 minutes. After washing again, the develop solution was treated for 10 minutes, the color change was observed, and after 10 minutes, the reaction was terminated by adding the solution. Absorbance was measured at 450 nm using a multiplate reader (tecan), and the percentage of DNMT1 enzyme absorbance inhibition was calculated relative to the sample (enzyme alone).

As a result, as shown in Figure 5, it was confirmed that at the same concentration (10, 50 μg / ml) Sample 2 has a much higher inhibitory effect of DNMT1 expression than Sample 1. DNA methyltransferase 1 (DNMT 1) inhibits gene expression by inducing methylation in DNA.BDNF (brain-derived neurotrophic factor) causes methylation and acetylation in the scopolamine induced memory impairment model. It is known that expression is suppressed. Inhibition of DNMT 1 may increase the expression of BDNF in the scopolamine induced memory impairment model, thereby improving cognitive function. Therefore, the sample 2 will be superior to the cognitive function improving effect than the sample 1.

Experimental Example 4 Lipid Peroxidation (MDA) Assay

To determine the antioxidant effect of each sample, the amount of lipid peroxidation (malondialdehyde, MDA) was measured using a lipid peroxidation (MDA) assay kit (SigmaAldrich).

Specifically, oral administration of Sample 2 (100 mg / kg) or physiological saline to mice for 4 weeks and after administration of 3 mg / kg of scopolamine (scopolamine, SigmaAldrich) or physiological saline for the last 6 days, the brain on the last day Extracted and homogenized in malondialdehyde (MDA) lysis buffer (SigmaAldrich) and only the supernatant was used. 200 μl of the supernatant was reacted with 600 μl of TBA solution (SigamaAldrich) at 95 ° C. for 1 hour and then cooled in an ice bath for 10 minutes. Absorbance was measured at 532 nm using a multiplate reader (tecan). The amount of MDA was quantified by comparing the absorbance of the reference sample treated with malondialdehyde (MDA) directly.

The results were as shown in FIG. 6, and it was confirmed that malondialdehyde (MDA) concentration increased by Scopolamine (Sco) administration was lowered through the intake of Sample 2 (100 mg / kg).

Experimental Example 5 Y-maze test

The Y-maze test was performed to confirm the cognitive ability, short-term memory, and spatial working memory protection of Sample 2.

Specifically, rats were administered orally with sample 2 (100 mg / kg) or saline solution for 4 weeks, and after scopolamine (3 mg / kg, sigmaaldrich) or saline solution for the last 6 days, the rat after the last dose. Is located on one arm of a Y-shaped maze (precision B & P) consisting of three arms (120 degrees each) for free navigation. At this time, the movement of the rat was recorded by using a camera and the spontaneous alternation was confirmed by checking the number of times the other arm enters the arm.

Spontaneous alternation (%) = (number of other arms / total number of arms-2 (last choice)) X 100

The results are shown in Figure 7, it was found that the short-term memory damaged by Scopolamine administration is recovered through the intake of Sample 2 (100 mg / kg), the effect is that Aza-2 (5-Aza-2 ' -deoxycytidine) was found to be a similar level. Scopolamine inhibits neuronal cell activity by acetylcholine by acting on a muscaric receptor acting by the neurotransmitter acetylcholine (ACh). Scopolamine plays a role in impairing memory by increasing the expression of DNMT1, a type of DNA methyltransferase, which reduces the expression level of BDNF, a nerve growth factor. Therefore, the intake of Sample 2 prevents and improves the damage of short-term memory capacity by scopolamine.

Experimental Example 6 Amyloid Beta Aggregation Inhibition Assay

Amyloid beta is a common protein found in Alzheimer's patients, and it aggregates abnormally outside the nerve cells to form clumps. The aggregated amyloid beta is toxic in neurons, causing synaptic damage, neuronal cell death, inflammation, mitochondrial damage and oxidative stress, causing Alzheimer's disease. In order to confirm the ability to inhibit the amyloid beta aggregation of Sample 3, it was measured using an amyloid beta aggregation confirmation kit (Anaspec) using Thioflavin T.

Specifically, put in a 96-well plate (Falcon) so that the final concentration of Thioflavin T solution is 20μ, and then put the sample so that the final concentration is 100 μg / ml each, the final volume after adding amyloid beta (1-42) After adjusting to μl and incubating at 37 ° C., fluorescence intensity was measured at Ex / Em = 440 nm / 484 nm using a Multiplate reader (tecan) every 5 minutes. At this time, the experimental group treated with morin, which is one of the polyphenols known to have the aggregation inhibitory effect of amyloid beta, proceeded together as a positive control group. Relative fluorescence intensity was calculated by the following equation.

Relative fluorescence unit = Fluorescence intensity of sample and amyloid beta group-Fluorescence intensity of sample alone group

As shown in FIGS. 8 and 9, amyloid beta aggregation formed over time was more effectively inhibited in Sample 3 than in Sample 1, and the inhibition of aggregation of Sample 3 was similar to that of the positive control group after 1 and a half hours of reaction. It was confirmed that appears.

Experimental Example 7 Experiment confirming cell death protection by inhibiting amyloid beta aggregation

The SH-SY5Y cell line (neurocytoma) obtained from the Korea Cell Line Bank was seeded by 1 × 10 ⁴ per well in a 96 well plate (FALCON), and cultured in a 37 ° C., 5% CO ₂ incubator for 24 hours. Amyloid beta (1-42, abcam) was added to DMEM / F12 (Gibco) to 40 μM and the sample was added to 10 μg / ml, and then thermostater (Eppendorf) at 37 ° C and 500 rpm. Reaction was induced for 24 hours to induce aggregation. After 24 hours after seeding, the culture medium was removed, and the culture medium which induced aggregation of amyloid beta prepared was further cultured for 24 hours.

Thereafter, the medium was removed, and 20 μl of CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega) was added to 100 μl of DMEM / F12 (Gibco) to confirm the absorbance at 490 nm. Cell number or cell viability (%) was calculated by the following formula.

Cell survival rate (%) = (absorbance of sample treated group-absorbance of reaction reagent only) / (absorbance of untreated group-absorbance of reaction reagent only) X 100 The results are shown in FIG. 10 and sample 3 is amyloid beta compared to sample 1. Inhibition of the aggregation was confirmed to protect the cell death caused by the aggregated amyloid beta.

Experimental Example 8 Experiment for Measuring Ex vivo Long-term Potentiation (LTP) Change

Long-term memory reinforcement phenomenon (LTP) is an increase in the transmission efficiency at the synapses of neurons connected to, it is considered as a mechanism of memory formation and storage. In particular, the effect of promoting LTP induction in the hippocampus is thought to be able to enhance memory formation and storage capacity. Thus, the LTP change measurement experiment according to the sample treatment of the present invention was performed. Specifically, the brain of 16-month-old male C57BL / 6 mice was extracted and sucrose artificial cerebrospinal fluid (CSF) solution (195.5 sucrose, 2.5 KCl, 1 NaH ₂ PO ₄ , 32.5 NaHCO ₃ , 11 Glucose, 2 Na pyruvate, 1 Na L-ascorbate, 5 MgS0 ₄ , 0.5 CaCl ₂ ) was prepared. Brain slices containing the schaffer collateral pathway of the hippocampus (the hippocampal neural circuit from CA3 to CA1) were prepared and prepared at 35 ° C. artificial CFS (128.5 NaCl ₂ , 2.5). KCl, 1 NaH ₂ PO ₄ , 21.7 NaHCO ₃ , 11 Glucose, 2 Na pyruvate, 1 Na L-ascorbate, 5 MgSO ₄ , 1 CaCl ₂ ) and reacted for 30 minutes to restore tissue function. Ion currents were recorded using a traditional field potential recording (EPSP) method using a patch clamp amplifier (molecular devices, USA). The measuring electrode was manufactured by borosilicate glass capillary (sutter instrument, USA). When the solution is filled into the electrode, the resistance is 3㏁-4㏁, and the plate containing hippocampal sections is placed on an upright microscope and the extracellular solution (124 NaCl, 2.5 KCl, 1 NaH ₂ PO ₄ , 26.2 NaHCO ₃ , 11 Glucose, 2 Na pyruvate, 1 Na L-ascorbate, 3 MgSO ₄ , 1.5 CaCl ₂ ) were perfused at 1-2 ml / min by gravity. Induction of CA1 post-synapse LTP by applying high frequency stimulation (HFS) electrical stimulation (4 times at 100 Hz, 500 ms, 20 s intervals) to the presynaptic axon fiber from CA3 when the sample was not processed and when the sample was processed. Excitatory postsynaptic potentials (EPS) were measured in the CA1 region. The potential after increased excitatory junction was calculated by the following equation.

Increased excitability after excitation (%) = (potential after electrostimulation) / (potential before electrostimulation) X 100

The results are shown in FIG. 11, and it was confirmed that Sample 3 induces LTP more effectively by increasing the potential increase width after the excitatory connection compared to Sample 1.

Formulation Example 1 Soft Capsule

Sample 2 according to Example 1 was prepared at 150 mg, and a soft capsule filling solution was prepared by mixing lactose 440 mg, corn starch 430 mg, and magnesium stearate 2 mg. In addition, a soft capsule sheet was prepared at a ratio of 66 parts by weight of gelatin, 24 parts by weight of glycerine, and 10 parts by weight of sorbitol solution and filled with the filler to prepare a soft capsule.

Formulation Example 2 Tablet

Sample 2 according to Example 1 was prepared in 150 mg, 15 mg of vitamin E, 15 mg of vitamin C, 250 mg of galactooligosaccharide, 60 mg of lactose and 140 mg of maltose were mixed and granulated using a fluidized bed dryer, followed by sugar ester. (sugar ester) 8 mg was added. The composition was compressed into tablets in a conventional manner.

[Formulation Example 3] Drinks

80 mg of Sample 2 according to Example 1 was prepared, and vitamin E 9 mg, vitamin C 9 mg, glucose 10 g, citric acid 0.6 g, and liquid oligosaccharide 25 g were mixed and filled with 400 ml of purified water. After filling the bottle sterilized for 4 to 5 seconds at 30 ℃ to prepare a drink.

Formulation Example 4 Granules

150 mg of Sample 2 according to Example 1 was prepared, and vitamin E 9 mg, vitamin C 9 mg, 250 mg of anhydrous glucose, and 550 mg of starch were mixed. Was prepared.

Formulation Example 5 Healthy Food

150 mg of Sample 2 according to Example 1 was prepared, and a vitamin mixture (70 μg of vitamin A acetate, 1.0 mg of vitamin E, 0.13 mg of vitamin B1, 0.15 mg of vitamin B2, 0.5 mg of vitamin B6, 0.2 μg of vitamin B12, vitamin C) 10 mg, 10 mg biotin, 1.7 mg nicotinic acid amide, 50 µg folic acid and a mineral mixture (1.75 mg ferrous sulfate, 0.82 mg zinc oxide, 25.3 mg magnesium carbonate, 15 mg potassium phosphate, 55 mg dibasic calcium phosphate) , 90 mg of potassium citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride) were used to prepare a health food.

Formulation Example 6 Healthy Drinks

50 mg of Sample 2 prepared according to Example 1 was prepared, and 900 mL of healthy drink was prepared by adding 1000 mg of citric acid, 100 g of oligosaccharide, 2 g of plum concentrate, 1 g of taurine, and the remaining amount of purified water.

As described above in detail specific parts of the present specification, it is apparent to those skilled in the art that these specific techniques are merely preferred embodiments, and thus the scope of the present specification is not limited thereto. Therefore, the substantial scope of the present specification will be defined by the appended claims and equivalents thereof.

Claims (18)

5 to 25% by weight of (-)-gallocatechin gallate (GCG) and 7 to 15% by weight of (-)-epigallocatechin gallate (( -)-epigallocatechin gallate (EGCG) containing green tea extract as an active ingredient,
The total content of GCG and EGCG in the extract is 35% by weight or less based on the total weight of the composition,
The weight ratio of the GCG: EGCG is 5 to 13: 7 to 13, the composition for improving cognitive decline. The composition of claim 1, wherein the extract is an extract extracted one or more times by any one or more of water and C1 to C4 alcohols. The composition of claim 1, wherein the content of the extract in the composition is 1% to 100% by weight on a dry weight basis. The composition of claim 1, wherein the dosage of the active ingredient is 5 mg / kg / day to 1000 mg / kg / day on a dry weight basis. The composition of claim 1, wherein the cognitive decline is based on any one selected from the group consisting of neurotransmitter degradation, neurotransmitter production reduction, and neurotransmitter receptor reduction. The composition of claim 5, wherein the neurotransmitter is acetylcholine. The composition of claim 1, wherein the cognitive decline is by DNA methylation. 8. The composition of claim 7, wherein the DNA methylation is based on DNA methyltransferase 1 (DNMT1). The composition of claim 1, wherein the cognitive decline is due to brain tissue lipid peroxidation. 10. The composition of claim 9, wherein the cognitive decline is due to malondialdehyde, a product of peroxide. The method of claim 1, wherein the cognitive decline is at least one selected from the group consisting of lethargy, memory loss, forgetfulness, cognitive decline, learning disability, attention loss, depression, hearing loss, painlessness, involuntary illness, and discrimination decline , Composition. The composition of claim 1, wherein the cognitive decline is due to a neurodegenerative disease. 13. The neurodegenerative disease according to claim 12, wherein the neurodegenerative disease is Alzheimer's disease, dementia, Parkinson's disease, hunting tongue disease, autosomal-dominant cerebellar ataxia, narcolepsy, alcoholism, drug addiction and hereditary sensation and autonomic neuropathy. at least one selected from the group consisting of sensory and autonomic neuropathy. The composition of claim 1, wherein the composition is a food composition. The composition of claim 1, wherein the composition is a pharmaceutical composition. (1) adding ethanol to green tea and extracting at 50 ° C. to 70 ° C. for 30 minutes to 4 hours;
(2) filtering and depressurizing to remove ethanol; And
(3) adding a water, stirring at 70 ° C. to 100 ° C. for 3 to 8 hours, and then concentrating under reduced pressure. The method according to claim 16, wherein the weight ratio of the resultant of step (2) and the water added in the step (3) is 1: 5 to 1:20. The method of claim 16, wherein the yield after step (3) is 5 to 30% by weight based on the weight of the green tea in step (1).

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