WO2020096299A1 - Green tea extract having modified constituent content and composition comprising same - Google Patents

Green tea extract having modified constituent content and composition comprising same Download PDF

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Publication number
WO2020096299A1
WO2020096299A1 PCT/KR2019/014810 KR2019014810W WO2020096299A1 WO 2020096299 A1 WO2020096299 A1 WO 2020096299A1 KR 2019014810 W KR2019014810 W KR 2019014810W WO 2020096299 A1 WO2020096299 A1 WO 2020096299A1
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Prior art keywords
composition
green tea
weight
extract
tea extract
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PCT/KR2019/014810
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French (fr)
Korean (ko)
Inventor
김아영
김형수
박원석
조시영
홍용덕
권구상
Original Assignee
(주)아모레퍼시픽
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Priority claimed from KR1020190037810A external-priority patent/KR20200051452A/en
Application filed by (주)아모레퍼시픽 filed Critical (주)아모레퍼시픽
Priority to EP19881736.3A priority Critical patent/EP3878460A4/en
Priority to CN201980073102.7A priority patent/CN112996526B/en
Publication of WO2020096299A1 publication Critical patent/WO2020096299A1/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
    • A23L5/20—Removal of unwanted matter, e.g. deodorisation or detoxification
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/82—Theaceae (Tea family), e.g. camellia

Definitions

  • the present invention is a green tea extract with a changed component content, comprising a green tea extract with reduced hepatotoxicity, and an active ingredient thereof, for improving blood circulation, for suppressing fat accumulation in hepatocytes, for inhibiting ginger ballis bacteria, or for increasing cerebral blood flow. It is about.
  • Fatty liver is the main cause of chronic drinking, overeating and diabetes due to overeating, and other toxic drugs, antibiotics taken incorrectly during pregnancy, and fever-infected infections in children. As a result, fatty liver may occur even if aspirin is abused.
  • fatty livers are easily recovered by regeneration of hepatocytes when physical requirements such as immune status are good, but otherwise, as fat accumulates in the hepatocytes, important components of cells, including the nucleus, are pushed to one side, and the function of hepatocytes decreases, accumulating in the cells Due to the fat, the expanded hepatocytes press the microvascular and lymph glands between the hepatocytes, causing disorders in the circulation of blood and lymphatic fluid in the liver, and the liver cells are unable to receive oxygen and nutrients properly, resulting in deterioration of liver function.
  • the blood circulation disease maintains homeostasis by balancing the activation and suppression reactions of the hemostatic mechanism in normal blood vessels, and excessive hemostatic action or formation of blood clots interferes with the flow of blood flow, thereby causing thrombus.
  • Normal vascular endothelial cells act to inhibit the adhesion and activation of platelets by protecting the blood vessel walls by releasing inhibitors such as nitrogen monoxide (NO) and prostacyclin (PGI2), but when endothelial cells are damaged, platelets Coagulation is promoted.
  • NO nitrogen monoxide
  • PKI2 prostacyclin
  • the thrombosis is a disease that occurs when blood clots block blood vessels.
  • Periodontitis has a complex onset and variable symptoms.
  • One of the main causes of periodontitis is due to microorganisms in the oral cavity. They primarily produce insoluble glucan using sugars and the like, and they attach to the tooth surface like various cells to form tartar. Dental cavities and periodontitis are caused by these calculus. Therefore, the most effective way to prevent periodontitis can be achieved by using substances that inhibit the activity of these microorganisms and improve the blood flow in the gums where inflammation has occurred.
  • Microorganisms distributed in the oral cavity cause periodontal disease, periodontal disease, etc., and some of these microorganisms are known to have the possibility of disease development as opportunistic pathogens.
  • Several species are known as pathogenic microorganisms that cause periodontitis in the oral cavity, but among them, Prevotella intermedia and Porphyromonas gingivalis are known to be the main pathogens that cause periodontitis. have.
  • the brain performs its functions by receiving blood ejected from the heart and obtaining nutrients such as oxygen and glucose.
  • the brain has little ability to accumulate oxygen and glucose in the tissues. Therefore, when the blood vessels supplied to the brain become obstructed by the narrowing or closing of blood vessels by arteriosclerosis or blood clots, oxygen deficiency and glucose
  • the lack of causes brain disease, including damage to nerve cells.
  • Diseases that are directly or indirectly associated with disorders of cerebral blood flow include anemia, stroke, vascular dementia, Parkinson's syndrome, and Huntington's disease.
  • green tea has a weight-loss and cholesterol-lowering effect, and is widely used as a supplement to improve obesity or metabolic diseases.
  • ingesting a large amount of green tea extract may damage the liver, and safety issues for green tea extract have emerged. Therefore, there is a need to discover the composition of green tea extract that reduces liver damage in green tea.
  • the present invention is a hepatotoxicity caused by green tea, specifically, green tea extract in which hepatic cell damage is alleviated, and high safety, including it as an active ingredient, improves blood circulation, inhibits fat accumulation in hepatocytes, inhibits ginger ballis bacteria, or increases brain blood flow It is an object to provide this excellent composition.
  • the present invention in one aspect, based on the total weight of the extract, comprising 8% by weight or less of epigallocatechin (EGC), provides a green tea extract with reduced hepatotoxicity.
  • ECG epigallocatechin
  • based on the total weight of the extract comprising a green tea extract with less than 8% by weight epigallocatechin (EGC), hepatotoxicity is reduced, as an active ingredient, provides a composition for improving blood circulation .
  • ELC epigallocatechin
  • the present invention based on the total weight of the extract, containing less than 8% by weight of epigallocatechin (epigallocatechin, EGC), containing the green tea extract with reduced liver toxicity as an active ingredient, fat accumulation in liver cells Provided is a composition for inhibition.
  • epigallocatechin epigallocatechin, EGC
  • the present invention based on the total weight of the extract, containing less than 8% by weight of epigallocatechin (epigallocatechin, EGC), containing the green tea extract with reduced hepatotoxicity as an active ingredient, inhibiting ginger germs
  • EGC epigallocatechin
  • the present invention based on the total weight of the extract, containing less than 8% by weight of epigallocatechin (epigallocatechin, EGC), comprising a green tea extract with reduced hepatotoxicity as an active ingredient, for increasing cerebral blood flow
  • epigallocatechin epigallocatechin, EGC
  • EGC epigallocatechin
  • Green tea extract according to an aspect of the present invention is a low-concentration level that causes hepatotoxicity compared to the existing green tea extract, by effectively changing the components of the green tea extract through high-temperature treatment, it is possible to effectively alleviate hepatocyte damage caused by the existing green tea extract and a large amount High safety even when ingested.
  • composition according to another aspect of the present invention including the green tea extract as an active ingredient, has high safety and excellent blood circulation improvement efficacy, thus exhibiting excellent thrombosis disease prevention or treatment efficacy.
  • composition according to another aspect of the present invention including the green tea extract as an active ingredient, has high safety and has excellent efficacy in inhibiting fat accumulation in liver cells, thereby exhibiting excellent fatty liver prevention or treatment efficacy.
  • composition according to another aspect of the present invention includes the green tea extract as an active ingredient, and exhibits excellent periodontitis prevention or treatment efficacy because it has high safety and excellent inhibitory effect against Porphyromonas gingivalis .
  • composition according to another aspect of the present invention includes the green tea extract as an active ingredient, and exhibits excellent cognitive function improvement efficacy with excellent safety and increased brain blood flow efficiency.
  • Example 1 shows a chromatogram for the general green tea extract of Example 1 (Sample 1).
  • Figure 2 shows a chromatogram for the high-temperature treated green tea extract (sample 2) according to an aspect of the present invention.
  • Figure 3 shows the results of the hepatotoxicity test of the high-temperature treated green tea extract according to Experimental Example 1.
  • Figure 4 shows the results of the experiment confirming the effect on the hepatocyte damage of the high-temperature treated green tea extract according to Experimental Example 2.
  • FIG. 5 shows the results of experiments confirming the effects of three catechins EGCG, GCG and EGC on hepatocyte damage according to Experimental Example 4.
  • Figure 6 shows the results of the experiment to evaluate the blood circulation improvement efficacy of the high-temperature treated green tea extract according to Experimental Example 5.
  • Figure 7 shows the results of the evaluation of the effect of inhibiting fat accumulation in liver cells of the high-temperature treated green tea extract according to Experimental Example 6.
  • Figure 8 shows the results of the experiment of evaluating the efficacy of inhibiting fat accumulation in liver cells of GCG and EGCG according to Experimental Example 7.
  • Figure 9 shows the results of the evaluation of the effect of increasing the blood flow of cerebral blood flow of the high-temperature treated green tea extract according to Experimental Example 9.
  • Figure 10 shows the results of the evaluation of the efficacy of increasing the blood flow of cerebral blood flow of the high-temperature treated green tea extract according to Experimental Example 9.
  • green tea extract is extracted from tea ( Camellia sinensis ), which is an evergreen tree belonging to the family, regardless of the extraction method, extraction solvent, or extracted component or extract type, or Bacillus subtilis spp.), and extracts from fermented tea leaves, etc., and the fractions obtained by fractionation with a specific solvent.
  • the tea includes at least one selected from the group consisting of tea tree leaves, flowers, stems, fruits, roots, stems, and cores of the roots, and may be preferably leaves.
  • the form of the extract may be preferably a powder form.
  • the extraction or fractionation can 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 mixed solvent of two or more of them, but is not limited thereto, and the active ingredient of green tea is not destroyed or extracted by heating at room temperature or in a minimized condition Or fractionation.
  • the alcohol may be a lower alcohol of C 1 ⁇ C 5 .
  • the number or method of extraction or fractionation is not particularly limited, and for example, methods such as cold immersion extraction, ultrasonic extraction, reflux cooling extraction, and hot water extraction can be used. After filtration, the filtrate is concentrated under reduced pressure to obtain a green tea extract of the present invention.
  • epigallocatechin refers to epigallocatechin (EGC), (-) epicatechin ((-) epicatechin, EC), (-)-epigalocatechin gallate ((-)-epigallocatechin gallate, EGCG), and epicatechin 3-O-gallate (epicatechin-3-O-gallate, ECG).
  • epi-catechin epimer refers to gallocatechin (GC), catechin (C), (-)-gallocatechin gallate (GCG) and catechin gallate (catechin). gallate, CG).
  • the present invention may include an epigallocatechin (EGC) of 8% by weight or less, and may relate to green tea extract with reduced hepatotoxicity.
  • ECC epigallocatechin
  • the present invention to prepare a composition for alleviating liver toxicity, based on the total weight of the extract, comprising less than 8% by weight epigallocatechin (EGC), the use of green tea extract with reduced hepatotoxicity It may be about
  • the present invention may relate to the use of green tea extract with reduced hepatotoxicity, including epigallocatechin (EGC) of 8% by weight or less, based on the total weight of the extract, for alleviating liver toxicity.
  • ECG epigallocatechin
  • the present invention may be related to a composition for improving blood circulation, including the green tea extract as an active ingredient.
  • the composition may be to increase nitrogen monoxide (NO) in vascular endothelial cells, and may exhibit blood circulation improvement effects through increased nitrogen monoxide in vascular endothelial cells.
  • NO nitrogen monoxide
  • the present invention based on the total weight of the extract, comprising the step of administering an effective amount of green tea extract with reduced hepatotoxicity, containing less than 8% by weight epigallocatechin (EGC) It may be related to a method for improving blood circulation, including.
  • the present invention relates to the use of green tea extract with reduced hepatotoxicity, comprising up to 8% by weight of epigallocatechin (EGC), based on the total weight of the extract, for preparing a composition for improving blood circulation.
  • ECG epigallocatechin
  • the present invention may be related to the use of green tea extract with reduced hepatotoxicity, including up to 8% by weight of epigallocatechin (EGC), based on the total weight of the extract, for improving blood circulation.
  • ECG epigallocatechin
  • the present invention may include a composition for inhibiting fat accumulation in liver cells, comprising the green tea extract as an active ingredient.
  • the present invention based on the total weight of the extract, comprising the step of administering an effective amount of green tea extract with reduced hepatotoxicity, containing less than 8% by weight epigallocatechin (EGC) Including, it may be related to a method for inhibiting fat accumulation in liver cells.
  • ECG epigallocatechin
  • the present invention in another aspect, for producing a composition for inhibiting fat accumulation in liver cells, based on the total weight of the extract, containing less than 8% by weight epigallocatechin (EGC), green tea extract with reduced hepatotoxicity It may be related to the use of.
  • ECG epigallocatechin
  • the present invention relates to the use of green tea extract with reduced hepatotoxicity, comprising up to 8% by weight of epigallocatechin (EGC), based on the total weight of the extract, for inhibiting fat accumulation in liver cells.
  • ECG epigallocatechin
  • the present invention may include a green tea extract as an active ingredient, it may be related to a composition for inhibiting ginger ballis.
  • the present invention based on the total weight of the extract, comprising the step of administering an effective amount of green tea extract with reduced hepatotoxicity, containing less than 8% by weight epigallocatechin (EGC) Including, it may be related to a method for inhibiting Ginger Valeris.
  • ECG epigallocatechin
  • the present invention to prepare a composition for inhibiting Ginger Bacillus, based on the total weight of the extract, comprising less than 8% by weight epigallocatechin (EGC), the use of green tea extract with reduced hepatotoxicity It may be about
  • the present invention may be related to the use of green tea extract with reduced hepatotoxicity, including 8% by weight or less of epigallocatechin (EGC), based on the total weight of the extract for inhibiting ginger germ bacteria. have.
  • ECG epigallocatechin
  • the ginger ballis bacteria may be Porphyromonas gingivalis bacteria.
  • the present invention may be related to a composition for increasing cerebral blood flow, comprising the green tea extract as an active ingredient.
  • the composition may be to increase the amount of oxygen in the cerebral blood flow, specifically , May increase blood flow in the frontal lobe portion of the brain.
  • the present invention based on the total weight of the extract, comprising the step of administering an effective amount of green tea extract with reduced hepatotoxicity, containing less than 8% by weight epigallocatechin (EGC) Including, it may be related to a method for increasing cerebral blood flow.
  • ECG epigallocatechin
  • the present invention to prepare a composition for increasing cerebral blood flow, based on the total weight of the extract, comprising less than 8% by weight epigallocatechin (EGC), the use of green tea extract with reduced hepatotoxicity It may be about
  • the present invention may be related to the use of green tea extract with reduced hepatotoxicity, including up to 8% by weight of epigallocatechin (EGC), based on the total weight of the extract, for increasing cerebral blood flow.
  • ECG epigallocatechin
  • administration of the method may be performed according to the administration method and administration dose described herein.
  • the EGC is based on the total weight of the extract 8 wt% or less, 7 wt% or less, 6 wt% or less, 5 wt% or less, 4.7 wt% or less, 4.56 wt% or less, 4.3 wt% or less, 4 It may be less than or equal to 0.3% by weight, less than or equal to 3.53%, less than or equal to 3.5%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.8%, less than or equal to 0.5%, less than or equal to 0.3% or 0.1% by weight, In another aspect, the EGC is 0.0001% by weight or more, 0.001% by weight or more, 0.01% by weight or more, 1% by weight or more, 2% by weight or more, 3% by weight or more, 3.5% by weight or more, 3.53 based on the total weight of the extract It may be at least 4 wt%, at least 4 wt%, at least 4.3 wt%, at least 4.56
  • the extract is based on the total weight of the extract 4 to 15% by weight of (-)-gallocatechin gallate ((-)-gallocatechin gallate, GCG) and 4 to 15% by weight of (-)-epigal Locatechin gallate ((-)-epigallocatechin gallate, EGCG).
  • the GCG is 4 wt% or more, 5 wt% or more, 5.3 wt% or more, 5.59 wt% or more, 5.7 wt% or more, 6 wt% or more, 7 wt% or more, 8 wt% or more, based on the total weight of the extract, It may be 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, or 14% or more.
  • the GCG is 15% by weight, 14% by weight, 13% by weight, 12% by weight, 11% by weight, 10% by weight, 9% by weight, 8% by weight based on the total weight of the extract %, 7% or less, 6% or less, 5.7% or less, 5.59% or less, 5.3% or less, or 5% or less.
  • the EGCG is based on the total weight of the extract 4 wt% or more, 5 wt% or more, 5.2 wt% or more, 5.27 wt% or more, 5.5 wt% or more, 6 wt% or more, 7 wt% or more, 8 It may be at least 10% by weight, at least 9% by weight, at least 10% by weight, at least 11% by weight, at least 12% by weight, at least 13% by weight, or at least 14% by weight.
  • the EGCG is 15% by weight or less, 14% by weight or less, 13% by weight or less, 12% by weight or less, 11% by weight or less, 10% by weight or less, 9% by weight or less, 8 based on the total weight of the extract It may be less than or equal to 7% by weight, less than or equal to 7%, less than or equal to 5.5%, less than or equal to 5.27%, less than or equal to 5.2%, or less than or equal to 5%.
  • the total content of GCG and EGCG in the extract may be 30% by weight or less based on the total weight of the extract.
  • the total content of the GCG and EGCG is 30 wt% or less, 25 wt% or less, 20 wt% or less, 18 wt% or less, 16 wt% or less, 15 wt% or less, 14 wt% based on the total weight of the extract
  • it may be 12% by weight or less, 11% by weight or less, 10.86% by weight or less, 10% by weight or less, or 9% by weight or less.
  • the total content of the GCG and EGCG is 8% by weight, 10% by weight, 10.86% by weight, 11% by weight, 12% by weight, 13% by weight, 14% by weight based on the total weight of the extract % Or more, 16% or more, 18% or more, 20% or more, or 25% or more.
  • the epicatechin content in the extract may be 20% by weight or less based on the total weight of the extract.
  • the epicatechin 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, 11.1 wt% or less based on the total weight of the extract, or It may be 10% by weight or less.
  • the epicatechin content is at least 9% by weight, at least 10% by weight, at least 11.1% by weight, at least 12% by weight, at least 13% by weight, at least 14% by weight, at least 16% by weight based on the total weight of the extract, or It may be 18% by weight or more.
  • the total content of 8 catechins in the extract i.e., (-)-epigallocatechin gallate (EGCG), epigallocatechin (EGC) in the extract (-) Epicatechin ((-) epicatechin (EC)), epicatechin 3-O-gallate (ECG), gallocatechin gallate (GCG), gallocatechin (GC),
  • the total content of catechin (C) and catechin gallate (CG) may be 19 to 30% by weight based on the total weight of the extract.
  • the total content of the eight catechins is 19% by weight or more, 21% by weight or more, 23% by weight or more, 24% by weight or more, 24.5% by weight or more, 25% by weight or more, 26% by weight based on the total weight of the extract % Or more, 27% or more, 28% or more, or 29% or more.
  • the total content of the eight catechins is 30% by weight or less, 29% by weight or less, 28% by weight or less, 27% by weight or less, 26% by weight or less, 25% by weight or less, 24.5% by weight based on the total weight of the extract % Or less, 24% or less, 23% or less, or 21% or less.
  • the extract may be an extract extracted one or more times by any one or more of water and C1 to C4 alcohol.
  • the alcohol may be ethanol.
  • the alcohol may be 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, or 70% or more ethanol.
  • the alcohol may be 70% or less, 60% or less, 50% or less, 40% or less, or 30% or less ethanol.
  • the extract may be contained in a dietary supplement or pharmaceutical composition.
  • the content of the green tea extract in the dietary supplement or pharmaceutical composition may be 1% to 100% by weight relative to the total weight of the composition.
  • the content of the extract in the composition is 1 wt% or more, 10 wt% or more, 20 wt% or more, 30 wt% or more, 40 wt% or more, 50 wt% or more, 60 wt% or more, 70 wt% Or more, 80% or more, or 90% or more.
  • the content of the extract in the composition is 100% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less Or less or 20% by weight.
  • the dosage of the green tea extract may be 5 mg / kg / day to 1000 mg / kg / day.
  • the dosage is 5 mg / kg / day or more, 100 mg / kg / day or more, 200 mg / kg / day or more, 300 mg / kg / day or more, 400 mg / kg / day or more, 500 mg / kg / day or more, It may be 600 mg / kg / day or more, 700 mg / kg / day or more, 800 mg / kg / day or more, or 900 mg / kg / day or more.
  • 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, It may be 400 mg / kg / day or less, 300 mg / kg / day or less, 200 mg / kg / day or less, 100 mg / kg / day or less, 50 mg / kg / day or less, or 10 mg / kg / day or less.
  • the formulation of the food composition is not particularly limited, for example, tablets, granules, pills, powders, liquids such as drinks, caramels, gels, bars, tea bags, etc. may be formulated.
  • the food composition of each formulation can be formulated by appropriately selecting the ingredients commonly used in the field other than the active ingredient according to the formulation or purpose of use without difficulty, and synergistic effect when applied simultaneously with other ingredients. Can happen.
  • the food may be a health functional food.
  • composition may be administered by various methods such as simple ingestion, drinking, injection, spray administration, or squeeze administration.
  • the dosage 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, and complications of the subject to be administered. .
  • Food composition for example, chewing gum, caramel products, candy, ice cream, confectionery, various foods, soft drinks, mineral water, alcoholic beverages, beverage products, vitamins and minerals, etc. It may be a functional food product.
  • the food composition according to one aspect of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, colorants and enhancers (cheese, chocolate, etc.), pectic acid and the like. Salts, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonic acid used in carbonated beverages, and the like.
  • food compositions according to one aspect of the present invention may include natural fruit juice and fruit juice for the production of fruit juice drinks and vegetable drinks. These ingredients can be used independently or in combination. The proportion of these additives is not so critical, but is generally 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 present invention.
  • the food composition according to an aspect of the present invention may be that the liver toxicity of green tea extract is alleviated. More specifically, the food composition may be that the liver toxicity by EGC in green tea extract is alleviated.
  • compositions according to an aspect of the present invention may be administered orally, parenteral, rectal, topical, transdermal, intravenous, intramuscular, intraperitoneal, subcutaneous, and the like.
  • Formulations for oral administration may be tablets, pills, soft and hard capsules, granules, powders, granules, liquids, emulsions or pellets, but are not limited thereto. It is not.
  • Formulations for parenteral administration may be solutions, suspensions, emulsions, gels, injections, drops, suppositories, patches or sprays, but are not limited thereto.
  • formulations can be readily prepared according to conventional methods in the art, surfactants, excipients, hydrating agents, emulsifying accelerators, suspending agents, salts or buffers for controlling osmotic pressure, colorants, spices, stabilizers, preservatives, preservatives or Other commercial adjuvants may be further included.
  • the pharmaceutical composition according to an aspect of the present invention may include a pharmaceutically acceptable salt, wherein the salt is formed of an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc .; 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-Tolu
  • the applied amount or dosage of the pharmaceutical composition according to an aspect of the present invention will vary depending on the age, gender, weight, pathology and severity of the subject to be administered, the route of administration or the judgment of the prescriber.
  • the determination of the active ingredient dosage based on these factors is within the level of those skilled in the art.
  • composition analysis conditions of Samples 1 and 2 composition analysis conditions of Samples 1 and 2
  • Table 2 composition analysis results of Sample 1
  • Table 3 composition analysis results of Sample 2
  • chromatograms for the two types of extracts are shown in Fig. 1 (sample 1) and 2 (sample 2). That is, it was confirmed that the composition of Sample 2 is different from the existing green tea extract.
  • Sample 2 contains significantly lower EGC (3.53% by weight), EGCG (5.27% by weight), and total catechin (24.41% by weight) than Sample 1, whereas Sample 1 has four components that are not found. It was confirmed that the epicatechin epimer of was additionally produced.
  • GC gallocatechin
  • EGC epigallocatechin
  • C catechin
  • EC (-) epicatechin
  • GCG gallocatechin gallate
  • EGCG epigallocatechin gallate
  • CG Catechin gallate
  • ECG epicatechin 3-O-gallate
  • Example 2 the hepatocyte cell line (HepG2 cell line) obtained from the Korean Cell Line Bank was placed in a 96 well plate (well plate, FALCON) 1X10 5 per well. After seeding each time, and cultured in a 37 ° C, 5% CO 2 incubator for 24 hours, the samples 1 and 2 were treated with 5, 10, 25, 50, 100 ⁇ g / ml, respectively, and further cultured for 24 hours. After removing the medium, cell viability was confirmed using a CellTiter 96 AQueous One Solution Cell Proliferation Assay Kit (MTS, Promega Co. Madison, WI, USA).
  • MTS CellTiter 96 AQueous One Solution Cell Proliferation Assay Kit
  • Cell viability (%) (Absorbance of the sample treated group-Absorbance of only the reaction reagent) / (Absorbance of the untreated group-Absorbance of the reaction reagent only) X 100
  • the sample 2 according to the present invention has low hepatocellular toxicity compared to the sample 1, which is a normal green tea extract.
  • Example 2 In order to confirm the effect of the high-temperature-treated green tea extract (Sample 2) obtained in Example 1 on liver cell damage, an analysis program (MILLIPLEX Analyst, Millipore) was used to analyze the degree of hepatocellular enzyme outflow due to hepatocyte damage. Analysis. Experimental conditions are the same as in Experimental Example 1, and after treatment of Sample 1 and Sample 2, the level of MDH 1 (malate dehydrogenase 1), GST ⁇ ( ⁇ -glutathione S-transferase), and SDH (sorbitol dehydrogenase) in the cell culture solution was 24 hours. It was measured.
  • MDH 1 malate dehydrogenase 1
  • GST ⁇ ⁇ -glutathione S-transferase
  • SDH sorbitol dehydrogenase
  • hepatocyte cell line obtained from the Korea Cell Line Bank
  • hepatocyte cell line obtained from the Korea Cell Line Bank
  • EGC has the highest effect on hepatocyte damage compared to EGCG and GCG.
  • Endothelial nitric oxide synthase is present in human vascular endothelial cells, and its activity is increased to produce nitrogen monoxide (NO) to expand blood vessels and promote blood circulation.
  • NO nitrogen monoxide
  • vascular endothelial cells were seeded at 2X10 4 per well in a 96-well plate (clear-plate, black, well plate, FALCON) with a transparent bottom and a black wall, and seeded for 24 hours at 37 ° C and 5% CO 2 After incubation in an incubator, cells were starvated for 4 hours with phenol red free medium from which fetal bovine serum (FBS) was removed, and then the medium was removed and washed twice with PBS (phosphate buffer saline).
  • FBS fetal bovine serum
  • DAF-2DA was dissolved in HBSS (Hank's Balanced Salt Solution) with 5 ⁇ M, treated with 100 ⁇ l of cells, and then incubated in a 5% CO 2 incubator for 20 minutes by blocking the light with foil. Fluorescence was measured before sample treatment (excitation: 485 nM, emission: 535 nM), and sample 1 and sample 2 were prepared at 6, 20, and 60 ⁇ g / ml, respectively, and treated with 100 ⁇ l in each well, followed by microplate reader ( Fluorescence is measured with Tecan Salzburg, Austria). Meanwhile, instead of samples 1 and 2, DMSO (dimethylsulfoxide) was treated with 0.1% (v / v) as a control. The relative NO production was calculated by the following equation.
  • Relative NO production (absorbance of sample treatment group-absorbance of reaction reagent only) / (absorbance of control group (DMSO treatment group)-absorbance of reaction reagent only)
  • the high-temperature-treated green tea extract according to the present invention has high hepatotoxicity and low safety compared to the conventional green tea extract, but also contains a large amount of NO in a smaller amount in vascular endothelial cells. It can be seen that it exhibits excellent blood circulation improvement efficacy.
  • the hepatocyte cell line (HepG2 cell line) is seeded in a 12-well plate (well plate, FALCON) for 1 ⁇ 10 6 per well, 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, 30 ⁇ g / ml and bovine serum albumin (BSA) and conjugated stearic acid 100 ⁇ M, respectively, and cultured for 48 hours. The medium was removed, washed with PBS, and fixed with 4% formaldehyde.
  • BSA bovine serum albumin
  • Amount of oil red O (%) (absorbance of sample treatment group-control absorbance) X 100
  • the high-temperature-treated green tea extract according to the present invention has low liver toxicity compared to the conventional green tea extract, has high safety, and has excellent remarkable ability to inhibit fat accumulation in liver cells, and has excellent fatty liver prevention efficacy. It can be seen that indicates.
  • the results are as shown in FIG. 8, and it was confirmed that GCG has a significantly superior inhibitory effect on fat accumulation in liver cells compared to EGCG at the same concentration. Accordingly, it was found that the high-temperature-treated green tea extract according to the present invention has a much lower EGCG content than the normal green tea extract and contains GCG components not found in the general green tea extract, thereby showing excellent ability to inhibit fat accumulation in liver cells.
  • samples were prepared by mixing the ingredients and composition ratios shown in Table 5 below. Each sample was prepared in stock 100 times the corresponding content and used for the experiment, and the content of each component in Sample 2 is the same as the content of 8 kinds of catechins in Table 3.
  • the high-temperature-treated green tea extract according to the present invention has low liver toxicity compared to the conventional green tea extract, has high safety, and has excellent efficacy in inhibiting Gingervalis bacteria, and has excellent periodontitis prevention efficacy. It can be seen that indicates.
  • NIRS Near infrared spectroscopy
  • NIRSIT functional near infrared spectroscopy
  • the high-temperature-treated green tea extract according to the present invention has low hepatotoxicity compared to the conventional green tea extract, and has high safety while increasing the blood flow in the frontal lobe of the brain, thereby improving the cognitive function. It shows that it shows.

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Abstract

The present invention discloses a green tea extract having modified constituent content, which exhibits reduced hepatotoxicity, and a composition comprising same as an active ingredient for enhancing blood circulation, inhibiting lipid accumulation in hepatocytes, inhibiting gingivalis bacteria, or increasing cerebral blood flow. Specifically, a green tea extract according to an aspect of the present invention has constituents that are modified by treatment at a high temperature to thus have lower concentration of that which causes hepatotoxicity than conventional green tea extracts, thereby allowing hepatcyte damage to be reduced. The composition comprising such a green tea extract according to the present invention is of high safety even when being ingested in large amounts and exhibits excellent efficacy in enhancing blood circulation, inhibiting lipid accumulation in hepatocytes, inhibiting gingivalis bacteria, or increasing cerebral blood flow.

Description

성분 함량이 변화된 녹차 추출물, 및 이를 포함하는 조성물Green tea extract with changed component content, and composition comprising same
관련 출원에 대한 상호 참조Cross reference to related applications
본 출원은 2018년 11월 5일자로 출원된 대한민국 특허출원 제10-2018-0134620호, 2019년 4월 1일자로 출원된 대한민국 특허출원 제10-2019-0037810호, 및 2019년 10월 17일자로 출원된 대한민국 특허출원 제10-2019-0129034호에 대한 우선권을 주장하며, 그 출원 내용 전체가 본 출원에 참조로서 통합된다.This application is filed on November 5, 2018, Korean Patent Application No. 10-2018-0134620, filed April 1, 2019, Korean Patent Application No. 10-2019-0037810, and October 17, 2019 Priority is claimed on Korean Patent Application No. 10-2019-0129034 filed as, and the entire contents of the application are incorporated herein by reference.
기술분야Technology field
본 발명은 성분 함량이 변화된 녹차 추출물로서, 간독성이 완화된 녹차 추출물, 및 이를 유효성분으로 포함하는, 혈행개선용, 간세포 내 지방 축적 억제용, 진저발리스균 저해용, 또는 뇌혈류량 증가용 조성물에 관한 것이다.The present invention is a green tea extract with a changed component content, comprising a green tea extract with reduced hepatotoxicity, and an active ingredient thereof, for improving blood circulation, for suppressing fat accumulation in hepatocytes, for inhibiting ginger ballis bacteria, or for increasing cerebral blood flow. It is about.
지방간은 만성적인 음주, 과식에 의한 비만증과 당뇨병이 주요 원인으로 꼽히며, 그 밖에 독성이 강한 약제의 복용, 임신 중에 항생제를 잘못 복용하였을 경우, 어린아이에게서의 발열성 감염질환이 있을 경우에 해열목적으로 아스피린을 함부로 남용하였을 경우에도 지방간이 생길 수 있다. 이러한 지방간은 면역상태 등의 신체적 요건이 좋으면 간세포의 재생으로 쉽게 회복되지만 그렇지 못할 경우에는 간세포에서 지방이 축적되면서 핵을 포함한 세포의 중요한 구성성분이 한쪽으로 밀려 간세포의 기능이 저하되며, 세포 내에 축적된 지방으로 인하여 팽창된 간세포들이 간세포 사이에 있는 미세혈관과 임파선을 압박하여 간 내의 혈액과 임파액의 순환에 장애가 생기게 되고, 간세포는 산소와 영양공급을 적절히 받을 수 없어 간 기능이 저하된다.Fatty liver is the main cause of chronic drinking, overeating and diabetes due to overeating, and other toxic drugs, antibiotics taken incorrectly during pregnancy, and fever-infected infections in children. As a result, fatty liver may occur even if aspirin is abused. These fatty livers are easily recovered by regeneration of hepatocytes when physical requirements such as immune status are good, but otherwise, as fat accumulates in the hepatocytes, important components of cells, including the nucleus, are pushed to one side, and the function of hepatocytes decreases, accumulating in the cells Due to the fat, the expanded hepatocytes press the microvascular and lymph glands between the hepatocytes, causing disorders in the circulation of blood and lymphatic fluid in the liver, and the liver cells are unable to receive oxygen and nutrients properly, resulting in deterioration of liver function.
혈액순환 질환은 정상 혈관에서 지혈기전의 활성화 반응과 억제 반응이 균형을 이루면서 항상성을 유지하는데 과도한 지혈 작용 또는 혈괴의 생성은 혈류의 흐름을 방해하여 그로 인해 혈전이 유발된다. 정상의 혈관 내피세포에서는 일산화질소(NO)와 프로스타시클린(prostacyclin, PGI2)과 같은 억제 인자를 방출하여 혈관벽을 보호함으로써 혈소판의 부착과 활성화를 억제하는 작용을 하지만, 내피세포가 손상되면 혈소판 응고가 촉진된다. 이렇게 혈전이 혈관을 막아 일어나는 병을 통틀어 혈전증이라 한다.The blood circulation disease maintains homeostasis by balancing the activation and suppression reactions of the hemostatic mechanism in normal blood vessels, and excessive hemostatic action or formation of blood clots interferes with the flow of blood flow, thereby causing thrombus. Normal vascular endothelial cells act to inhibit the adhesion and activation of platelets by protecting the blood vessel walls by releasing inhibitors such as nitrogen monoxide (NO) and prostacyclin (PGI2), but when endothelial cells are damaged, platelets Coagulation is promoted. The thrombosis is a disease that occurs when blood clots block blood vessels.
치주염은 발병요인도 복잡하고 증상도 다양하다. 이중 치주염의 주요 발생원인의 하나가 구강 내 미생물에 의한 것으로 이들은 일차적으로 당류등을 이용하여 불용성 글루칸을 생성하고 이들이 각종 균체와 같이 치아표면에 부착하여 치석을 형성하게 된다. 이러한 치석에 의해 충치 및 치주염이 발생하게 된다. 따라서, 치주염을 예방하기 위한 가장 효과적인 방법은 이러한 미생물들의 활성을 억제하고, 염증이 발생한 잇몸의 혈류를 좋게하는 물질을 사용함으로써 이루어질 수 있다. 구강 내 분포하는 미생물들은 구치, 치주질환 등을 유발하며, 이들 미생물들의 일부는 기회성 병원체로서 질병 발병의 가능성을 가지고 있는 것으로 알려져 있다. 구강 내 상재하면서 치주염을 발생시키는 병원성 미생물로 여러 종들이 알려져 있으나, 그 중 프레보텔라 인터메디아( Prevotella intermedia), 포피로모나스 진저발리스( Porphyromonas gingivalis) 등이 치주염을 발생시키는 주 병원체인 것으로 알려져 있다.Periodontitis has a complex onset and variable symptoms. One of the main causes of periodontitis is due to microorganisms in the oral cavity. They primarily produce insoluble glucan using sugars and the like, and they attach to the tooth surface like various cells to form tartar. Dental cavities and periodontitis are caused by these calculus. Therefore, the most effective way to prevent periodontitis can be achieved by using substances that inhibit the activity of these microorganisms and improve the blood flow in the gums where inflammation has occurred. Microorganisms distributed in the oral cavity cause periodontal disease, periodontal disease, etc., and some of these microorganisms are known to have the possibility of disease development as opportunistic pathogens. Several species are known as pathogenic microorganisms that cause periodontitis in the oral cavity, but among them, Prevotella intermedia and Porphyromonas gingivalis are known to be the main pathogens that cause periodontitis. have.
뇌는 심장으로부터 박출되는 혈액을 공급받아 산소나 포도당 등의 영양물질을 얻음으로써 제기능을 수행하게 된다. 그러나 뇌는 다른 신체조직과는 달리 조직내에 산소와 포도당을 비축할 능력이 거의 없기 때문에, 뇌혈관이 동맥경화나 혈전 등에 의하여 좁아지거나 폐쇠됨으로써 뇌로 공급되는 혈류에 장애가 발생하게 되면, 산소 결핍과 포도당의 부족으로 신경 세포의 손상을 포함한 뇌질환이 발생하게 된다. 이러한 뇌혈류의 장애와 직간접적으로 관련되어 발병하는 질환은 빈혈, 뇌졸중, 혈관성 치매, 파킨슨 증후군, 헌팅턴병 등을 들 수 있다.The brain performs its functions by receiving blood ejected from the heart and obtaining nutrients such as oxygen and glucose. However, unlike other body tissues, the brain has little ability to accumulate oxygen and glucose in the tissues. Therefore, when the blood vessels supplied to the brain become obstructed by the narrowing or closing of blood vessels by arteriosclerosis or blood clots, oxygen deficiency and glucose The lack of causes brain disease, including damage to nerve cells. Diseases that are directly or indirectly associated with disorders of cerebral blood flow include anemia, stroke, vascular dementia, Parkinson's syndrome, and Huntington's disease.
한편, 녹차는 체중감량 및 콜레스테롤 저하 효능을 가지고 있어 비만 혹은 대사 질환을 개선시키기 위한 보조 식품 등으로 널리 쓰인다. 하지만 최근 다량의 녹차 추출물을 섭취하게 되면 간에 손상을 줄 수 있다는 연구 결과들이 보고되고 있어 녹차 추출물에 대한 안전성 이슈가 대두되고 있다. 따라서 녹차의 간 손상을 줄인 녹차 추출물의 조성 발굴이 필요한 실정이다.On the other hand, green tea has a weight-loss and cholesterol-lowering effect, and is widely used as a supplement to improve obesity or metabolic diseases. However, recently, research results have been reported that ingesting a large amount of green tea extract may damage the liver, and safety issues for green tea extract have emerged. Therefore, there is a need to discover the composition of green tea extract that reduces liver damage in green tea.
본 발명은 녹차에 의한 간독성, 구체적으로 간세포 손상이 완화된 녹차 추출물, 및 이를 유효성분으로 포함하여 안전성이 높으면서, 혈행개선 효능, 간세포 내 지방 축적 억제 효능, 진저발리스균 저해 효능 또는 뇌혈류량 증가 효능이 뛰어난 조성물을 제공하는 것을 목적으로 한다.The present invention is a hepatotoxicity caused by green tea, specifically, green tea extract in which hepatic cell damage is alleviated, and high safety, including it as an active ingredient, improves blood circulation, inhibits fat accumulation in hepatocytes, inhibits ginger ballis bacteria, or increases brain blood flow It is an object to provide this excellent composition.
상기한 목적을 달성하기 위하여, 본 발명은 일 측면에서, 추출물 총 중량을 기준으로, 8 중량% 이하의 에피갈로카테킨(EGC)을 포함하는, 간독성이 완화된 녹차 추출물을 제공한다.In order to achieve the above object, the present invention, in one aspect, based on the total weight of the extract, comprising 8% by weight or less of epigallocatechin (EGC), provides a green tea extract with reduced hepatotoxicity.
본 발명은 다른 측면에서, 추출물 총 중량을 기준으로, 8 중량% 이하의 에피갈로카테킨(EGC)을 포함하는, 간독성이 완화된 녹차 추출물을 유효성분으로 포함하는, 혈행개선용 조성물을 제공한다.In another aspect, in another aspect, based on the total weight of the extract, comprising a green tea extract with less than 8% by weight epigallocatechin (EGC), hepatotoxicity is reduced, as an active ingredient, provides a composition for improving blood circulation .
본 발명은 또 다른 측면에서, 추출물 총 중량을 기준으로, 8 중량% 이하의 에피갈로카테킨(epigallocatechin, EGC)을 포함하는, 간독성이 완화된 녹차 추출물을 유효성분으로 포함하는, 간세포 내 지방 축적 억제용 조성물을 제공한다.In another aspect, the present invention, based on the total weight of the extract, containing less than 8% by weight of epigallocatechin (epigallocatechin, EGC), containing the green tea extract with reduced liver toxicity as an active ingredient, fat accumulation in liver cells Provided is a composition for inhibition.
본 발명은 또 다른 측면에서, 추출물 총 중량을 기준으로, 8 중량% 이하의 에피갈로카테킨(epigallocatechin, EGC)을 포함하는, 간독성이 완화된 녹차 추출물을 유효성분으로 포함하는, 진저발리스균 저해용 조성물을 제공한다.In another aspect, the present invention, based on the total weight of the extract, containing less than 8% by weight of epigallocatechin (epigallocatechin, EGC), containing the green tea extract with reduced hepatotoxicity as an active ingredient, inhibiting ginger germs Provided is a composition.
본 발명은 또 다른 측면에서, 추출물 총 중량을 기준으로, 8 중량% 이하의 에피갈로카테킨(epigallocatechin, EGC)을 포함하는, 간독성이 완화된 녹차 추출물을 유효성분으로 포함하는, 뇌혈류량 증가용 조성물을 제공한다.In another aspect, the present invention, based on the total weight of the extract, containing less than 8% by weight of epigallocatechin (epigallocatechin, EGC), comprising a green tea extract with reduced hepatotoxicity as an active ingredient, for increasing cerebral blood flow Provided is a composition.
본 발명의 일 측면에 따른 녹차 추출물은 고온처리를 통해 녹차 추출물의 성분 변화를 시킴으로써, 기존의 녹차 추출물에 비하여 간독성을 일으키는 농도가 낮아, 기존의 녹차 추출물에 의한 간세포 손상을 효과적으로 완화시킬 수 있고 다량 섭취 시에도 안전성이 높다.Green tea extract according to an aspect of the present invention is a low-concentration level that causes hepatotoxicity compared to the existing green tea extract, by effectively changing the components of the green tea extract through high-temperature treatment, it is possible to effectively alleviate hepatocyte damage caused by the existing green tea extract and a large amount High safety even when ingested.
본 발명의 다른 측면에 따른 조성물은, 상기 녹차 추출물을 유효성분으로 포함하여 안전성이 높으면서도 혈행 개선 효능이 우수하여 뛰어난 혈전 질환 예방 또는 치료 효능을 나타낸다.The composition according to another aspect of the present invention, including the green tea extract as an active ingredient, has high safety and excellent blood circulation improvement efficacy, thus exhibiting excellent thrombosis disease prevention or treatment efficacy.
본 발명의 또 다른 측면에 따른 조성물은, 상기 녹차 추출물을 유효성분으로 포함하여 안전성이 높으면서도 간 세포 내 지방 축적 억제 효능이 우수하여 뛰어난 지방간 예방 또는 치료 효능을 나타낸다.The composition according to another aspect of the present invention, including the green tea extract as an active ingredient, has high safety and has excellent efficacy in inhibiting fat accumulation in liver cells, thereby exhibiting excellent fatty liver prevention or treatment efficacy.
본 발명의 또 다른 측면에 따른 조성물은, 상기 녹차 추출물을 유효성분으로 포함하여 안전성이 높으면서도 포피로모나스 진저발리스( Porphyromonas gingivalis)균 저해 효능이 우수하여 뛰어난 치주염 예방 또는 치료 효능을 나타낸다.The composition according to another aspect of the present invention includes the green tea extract as an active ingredient, and exhibits excellent periodontitis prevention or treatment efficacy because it has high safety and excellent inhibitory effect against Porphyromonas gingivalis .
본 발명의 또 다른 측면에 따른 조성물은, 상기 녹차 추출물을 유효성분으로 포함하여 안전성이 높으면서도 뇌혈류량 증가 효능이 우수하여 뛰어난 인지기능 개선 효능을 나타낸다.The composition according to another aspect of the present invention includes the green tea extract as an active ingredient, and exhibits excellent cognitive function improvement efficacy with excellent safety and increased brain blood flow efficiency.
도 1은 실시예 1의 일반 녹차 추출물(시료 1)에 대한 크로마토그램을 나타낸 것이다.1 shows a chromatogram for the general green tea extract of Example 1 (Sample 1).
도 2는 본 발명의 일 측면에 따른 고온처리 녹차 추출물(시료 2)에 대한 크로마토그램을 나타낸 것이다.Figure 2 shows a chromatogram for the high-temperature treated green tea extract (sample 2) according to an aspect of the present invention.
도 3은 실험예 1에 따른 고온처리 녹차 추출물의 간세포 독성 확인 실험 결과를 나타낸 것이다.Figure 3 shows the results of the hepatotoxicity test of the high-temperature treated green tea extract according to Experimental Example 1.
도 4는 실험예 2에 따른 고온처리 녹차 추출물의 간세포 손상에 대한 영향 확인 실험 결과를 나타낸 것이다.Figure 4 shows the results of the experiment confirming the effect on the hepatocyte damage of the high-temperature treated green tea extract according to Experimental Example 2.
도 5는 실험예 4에 따른 3종의 카테킨 EGCG, GCG 및 EGC의 간세포 손상에 대한 영향 확인 실험 결과를 나타낸 것이다.FIG. 5 shows the results of experiments confirming the effects of three catechins EGCG, GCG and EGC on hepatocyte damage according to Experimental Example 4.
도 6은 실험예 5에 따른 고온처리 녹차 추출물의 혈행개선 효능 평가 실험 결과를 나타낸 것이다.Figure 6 shows the results of the experiment to evaluate the blood circulation improvement efficacy of the high-temperature treated green tea extract according to Experimental Example 5.
도 7은 실험예 6에 따른 고온처리 녹차 추출물의 간 세포 내 지방 축적 억제 효능 평가 실험 결과를 나타낸 것이다.Figure 7 shows the results of the evaluation of the effect of inhibiting fat accumulation in liver cells of the high-temperature treated green tea extract according to Experimental Example 6.
도 8은 실험예 7에 따른 GCG 및 EGCG의 간 세포 내 지방 축적 억제 효능 평가 실험 결과를 나타낸 것이다.Figure 8 shows the results of the experiment of evaluating the efficacy of inhibiting fat accumulation in liver cells of GCG and EGCG according to Experimental Example 7.
도 9는 실험예 9에 따른 고온처리 녹차 추출물의 뇌혈류량 증가 효능 평가 실험 결과를 나타낸 것이다.Figure 9 shows the results of the evaluation of the effect of increasing the blood flow of cerebral blood flow of the high-temperature treated green tea extract according to Experimental Example 9.
도 10은 실험예 9에 따른 고온처리 녹차 추출물의 뇌혈류량 증가 효능 평가 실험 결과를 나타낸 것이다.Figure 10 shows the results of the evaluation of the efficacy of increasing the blood flow of cerebral blood flow of the high-temperature treated green tea extract according to Experimental Example 9.
본 명세서에서 "녹차 추출물"은 추출방법, 추출용매, 추출된 성분 또는 추출물의 형태를 불문하고, 차과에 속하는 상록수인 차(카멜리아 시넨시스; Camellia sinensis)로부터 추출한 것 또는 바실러스 서브틸리스( Bacillus subtilis spp.)를 접종하고 발효시킨 차잎 등으로부터 추출한 것을 포함하고, 상기 추출한 것을 특정 용매로 분획한 분획물을 포함한다. 상기 차는 차나무 잎, 꽃, 줄기, 열매, 뿌리, 줄기, 및 뿌리의 심재로 구성된 군에서 선택되는 하나 이상이 포함되며, 바람직하게는 잎일 수 있다. 또한, 상기 추출물의 형태는 바람직하게는 분말형태일 수 있다. 상기 추출 또는 분획은 물, 유기 용매, 또는 이들의 혼합용매를 사용하여 할 수 있다. 유기용매는 알코올, 이소프로판올, 아세톤, 헥산, 에틸아세테이트, 이산화탄소, 또는 이들 중 둘 이상의 혼합용매를 사용할 수 있으나 이에 한정되지는 않으며, 녹차의 유효 성분이 파괴되지 않거나 최소화된 조건에서 실온 또는 가온하여 추출 또는 분획할 수 있다. 상기 알코올은 C 1~C 5의 저급 알코올일 수 있다. 추출 또는 분획의 횟수나 방법은 특별히 제한되지 않으며, 예를 들어 냉침 추출, 초음파 추출, 환류 냉각 추출, 열수 추출 등의 방법을 사용할 수 있고, 바람직하게는 냉침 또는 가온하여 유효성분을 추출 또는 분획하고 여과한 다음, 그 여과액을 감압농축하여 본 발명의 녹차 추출물을 얻을 수 있다.In the present specification, "green tea extract" is extracted from tea ( Camellia sinensis ), which is an evergreen tree belonging to the family, regardless of the extraction method, extraction solvent, or extracted component or extract type, or Bacillus subtilis spp.), and extracts from fermented tea leaves, etc., and the fractions obtained by fractionation with a specific solvent. The tea includes at least one selected from the group consisting of tea tree leaves, flowers, stems, fruits, roots, stems, and cores of the roots, and may be preferably leaves. In addition, the form of the extract may be preferably a powder form. The extraction or fractionation can 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 mixed solvent of two or more of them, but is not limited thereto, and the active ingredient of green tea is not destroyed or extracted by heating at room temperature or in a minimized condition Or fractionation. The alcohol may be a lower alcohol of C 1 ~ C 5 . The number or method of extraction or fractionation is not particularly limited, and for example, methods such as cold immersion extraction, ultrasonic extraction, reflux cooling extraction, and hot water extraction can be used. After filtration, the filtrate is concentrated under reduced pressure to obtain a green tea extract of the present invention.
본 명세서에서 "에피 카테킨"이란, 에피갈로카테킨(epigallocatechin, EGC), (-)에피카테킨((-)epicatechin, EC), (-)-에피갈로카테킨 갈레이트((-)-epigallocatechin gallate, EGCG), 및 에피카테킨 3-O-갈레이트(epicatechin-3-O-gallate, ECG)를 포함하는 것이다.As used herein, "epi-catechin" refers to epigallocatechin (EGC), (-) epicatechin ((-) epicatechin, EC), (-)-epigalocatechin gallate ((-)-epigallocatechin gallate, EGCG), and epicatechin 3-O-gallate (epicatechin-3-O-gallate, ECG).
본 명세서에서 "에피 카테킨 에피머"란, 갈로카테킨(gallocatechin, GC), 카테킨(catechin, C), (-)-갈로카테킨 갈레이트((-)-gallocatechin gallate, GCG) 및 카테킨 갈레이트(catechin gallate, CG)를 포함하는 것이다.As used herein, the term "epi-catechin epimer" refers to gallocatechin (GC), catechin (C), (-)-gallocatechin gallate (GCG) and catechin gallate (catechin). gallate, CG).
본 발명은 일 측면에서, 추출물 총 중량을 기준으로, 8 중량% 이하의 에피갈로카테킨(EGC)을 포함하는, 간독성이 완화된 녹차 추출물에 관한 것일 수 있다.In one aspect, the present invention, based on the total weight of the extract, may include an epigallocatechin (EGC) of 8% by weight or less, and may relate to green tea extract with reduced hepatotoxicity.
본 발명은 다른 측면에서, 추출물 총 중량을 기준으로, 8 중량% 이하의 에피갈로카테킨(EGC)을 포함하는, 간독성이 완화된 녹차 추출물의 유효량을 이를 필요로 하는 대상에게 투여하는 단계를 포함하는, 간 독성 완화 방법에 관한 것일 수 있다.In another aspect, in another aspect, based on the total weight of the extract, comprising the step of administering an effective amount of green tea extract with reduced hepatotoxicity, containing less than 8% by weight epigallocatechin (EGC) It may be related to a method for alleviating liver toxicity.
본 발명은 또 다른 측면에서, 간 독성 완화용 조성물을 제조하기 위한, 추출물 총 중량을 기준으로, 8 중량% 이하의 에피갈로카테킨(EGC)을 포함하는, 간독성이 완화된 녹차 추출물의 용도에 관한 것일 수 있다.In another aspect, the present invention, to prepare a composition for alleviating liver toxicity, based on the total weight of the extract, comprising less than 8% by weight epigallocatechin (EGC), the use of green tea extract with reduced hepatotoxicity It may be about
본 발명은 또 다른 측면에서, 간 독성 완화를 위한, 추출물 총 중량을 기준으로, 8 중량% 이하의 에피갈로카테킨(EGC)을 포함하는, 간독성이 완화된 녹차 추출물의 용도에 관한 것일 수 있다.In another aspect, the present invention may relate to the use of green tea extract with reduced hepatotoxicity, including epigallocatechin (EGC) of 8% by weight or less, based on the total weight of the extract, for alleviating liver toxicity. .
본 발명은 또 다른 측면에서, 상기 녹차 추출물을 유효성분으로 포함하는, 혈행개선용 조성물에 관한 것일 수 있다.In another aspect, the present invention may be related to a composition for improving blood circulation, including the green tea extract as an active ingredient.
또 다른 측면에서, 상기 조성물은 혈관내피세포에서 일산화질소(NO)를 증가시키는 것일 수 있으며, 혈관내피세포 내 일산화질소 증가를 통한 혈행개선 효능을 나타낼 수 있다.In another aspect, the composition may be to increase nitrogen monoxide (NO) in vascular endothelial cells, and may exhibit blood circulation improvement effects through increased nitrogen monoxide in vascular endothelial cells.
본 발명은 또 다른 측면에서, 추출물 총 중량을 기준으로, 8 중량% 이하의 에피갈로카테킨(EGC)을 포함하는, 간독성이 완화된 녹차 추출물의 유효량을 이를 필요로 하는 대상에게 투여하는 단계를 포함하는, 혈행 개선 방법에 관한 것일 수 있다.In another aspect, the present invention, based on the total weight of the extract, comprising the step of administering an effective amount of green tea extract with reduced hepatotoxicity, containing less than 8% by weight epigallocatechin (EGC) It may be related to a method for improving blood circulation, including.
본 발명은 또 다른 측면에서, 혈행 개선용 조성물을 제조하기 위한, 추출물 총 중량을 기준으로, 8 중량% 이하의 에피갈로카테킨(EGC)을 포함하는, 간독성이 완화된 녹차 추출물의 용도에 관한 것일 수 있다.In another aspect, the present invention relates to the use of green tea extract with reduced hepatotoxicity, comprising up to 8% by weight of epigallocatechin (EGC), based on the total weight of the extract, for preparing a composition for improving blood circulation. May be
본 발명은 또 다른 측면에서, 혈행 개선을 위한, 추출물 총 중량을 기준으로, 8 중량% 이하의 에피갈로카테킨(EGC)을 포함하는, 간독성이 완화된 녹차 추출물의 용도에 관한 것일 수 있다.In another aspect, the present invention may be related to the use of green tea extract with reduced hepatotoxicity, including up to 8% by weight of epigallocatechin (EGC), based on the total weight of the extract, for improving blood circulation.
본 발명은 또 다른 측면에서, 상기 녹차 추출물을 유효성분으로 포함하는, 간 세포 내 지방 축적 억제용 조성물에 관한 것일 수 있다.In another aspect, the present invention may include a composition for inhibiting fat accumulation in liver cells, comprising the green tea extract as an active ingredient.
본 발명은 또 다른 측면에서, 추출물 총 중량을 기준으로, 8 중량% 이하의 에피갈로카테킨(EGC)을 포함하는, 간독성이 완화된 녹차 추출물의 유효량을 이를 필요로 하는 대상에게 투여하는 단계를 포함하는, 간 세포 내 지방 축적 억제 방법에 관한 것일 수 있다.In another aspect, the present invention, based on the total weight of the extract, comprising the step of administering an effective amount of green tea extract with reduced hepatotoxicity, containing less than 8% by weight epigallocatechin (EGC) Including, it may be related to a method for inhibiting fat accumulation in liver cells.
본 발명은 또 다른 측면에서, 간 세포 내 지방 축적 억제용 조성물을 제조하기 위한, 추출물 총 중량을 기준으로, 8 중량% 이하의 에피갈로카테킨(EGC)을 포함하는, 간독성이 완화된 녹차 추출물의 용도에 관한 것일 수 있다.The present invention, in another aspect, for producing a composition for inhibiting fat accumulation in liver cells, based on the total weight of the extract, containing less than 8% by weight epigallocatechin (EGC), green tea extract with reduced hepatotoxicity It may be related to the use of.
본 발명은 또 다른 측면에서, 간 세포 내 지방 축적 억제를 위한, 추출물 총 중량을 기준으로, 8 중량% 이하의 에피갈로카테킨(EGC)을 포함하는, 간독성이 완화된 녹차 추출물의 용도에 관한 것일 수 있다.In another aspect, the present invention relates to the use of green tea extract with reduced hepatotoxicity, comprising up to 8% by weight of epigallocatechin (EGC), based on the total weight of the extract, for inhibiting fat accumulation in liver cells. May be
본 발명은 또 다른 측면에서, 상기 녹차 추출물을 유효성분으로 포함하는, 진저발리스균 저해용 조성물에 관한 것일 수 있다.In another aspect, the present invention may include a green tea extract as an active ingredient, it may be related to a composition for inhibiting ginger ballis.
본 발명은 또 다른 측면에서, 추출물 총 중량을 기준으로, 8 중량% 이하의 에피갈로카테킨(EGC)을 포함하는, 간독성이 완화된 녹차 추출물의 유효량을 이를 필요로 하는 대상에게 투여하는 단계를 포함하는, 진저발리스균 저해 방법에 관한 것일 수 있다.In another aspect, the present invention, based on the total weight of the extract, comprising the step of administering an effective amount of green tea extract with reduced hepatotoxicity, containing less than 8% by weight epigallocatechin (EGC) Including, it may be related to a method for inhibiting Ginger Valeris.
본 발명은 또 다른 측면에서, 진저발리스균 저해용 조성물을 제조하기 위한, 추출물 총 중량을 기준으로, 8 중량% 이하의 에피갈로카테킨(EGC)을 포함하는, 간독성이 완화된 녹차 추출물의 용도에 관한 것일 수 있다.In another aspect, the present invention, to prepare a composition for inhibiting Ginger Bacillus, based on the total weight of the extract, comprising less than 8% by weight epigallocatechin (EGC), the use of green tea extract with reduced hepatotoxicity It may be about
본 발명은 또 다른 측면에서, 진저발리스균 저해를 위한, 추출물 총 중량을 기준으로, 8 중량% 이하의 에피갈로카테킨(EGC)을 포함하는, 간독성이 완화된 녹차 추출물의 용도에 관한 것일 수 있다.In another aspect, the present invention may be related to the use of green tea extract with reduced hepatotoxicity, including 8% by weight or less of epigallocatechin (EGC), based on the total weight of the extract for inhibiting ginger germ bacteria. have.
또 다른 측면에서, 상기 진저발리스균은 포피로모나스 진저발리스( Porphyromonas gingivalis)균일 수 있다.In another aspect, the ginger ballis bacteria may be Porphyromonas gingivalis bacteria.
본 발명은 또 다른 측면에서, 상기 녹차 추출물을 유효성분으로 포함하는, 뇌혈류량 증가용 조성물에 관한 것일 수 있다.또 다른 측면에서, 상기 조성물은 뇌혈류 내 산소량을 증가시키는 것일 수 있고, 구체적으로는, 뇌의 전두엽 부분의 혈류량을 증가시키는 것일 수 있다.In another aspect, the present invention may be related to a composition for increasing cerebral blood flow, comprising the green tea extract as an active ingredient. In another aspect, the composition may be to increase the amount of oxygen in the cerebral blood flow, specifically , May increase blood flow in the frontal lobe portion of the brain.
본 발명은 또 다른 측면에서, 추출물 총 중량을 기준으로, 8 중량% 이하의 에피갈로카테킨(EGC)을 포함하는, 간독성이 완화된 녹차 추출물의 유효량을 이를 필요로 하는 대상에게 투여하는 단계를 포함하는, 뇌혈류량 증가 방법에 관한 것일 수 있다.In another aspect, the present invention, based on the total weight of the extract, comprising the step of administering an effective amount of green tea extract with reduced hepatotoxicity, containing less than 8% by weight epigallocatechin (EGC) Including, it may be related to a method for increasing cerebral blood flow.
본 발명은 또 다른 측면에서, 뇌혈류량 증가용 조성물을 제조하기 위한, 추출물 총 중량을 기준으로, 8 중량% 이하의 에피갈로카테킨(EGC)을 포함하는, 간독성이 완화된 녹차 추출물의 용도에 관한 것일 수 있다.In another aspect, the present invention, to prepare a composition for increasing cerebral blood flow, based on the total weight of the extract, comprising less than 8% by weight epigallocatechin (EGC), the use of green tea extract with reduced hepatotoxicity It may be about
본 발명은 또 다른 측면에서, 뇌혈류량 증가를 위한, 추출물 총 중량을 기준으로, 8 중량% 이하의 에피갈로카테킨(EGC)을 포함하는, 간독성이 완화된 녹차 추출물의 용도에 관한 것일 수 있다.In another aspect, the present invention may be related to the use of green tea extract with reduced hepatotoxicity, including up to 8% by weight of epigallocatechin (EGC), based on the total weight of the extract, for increasing cerebral blood flow. .
본 발명의 일 관점에서, 상기 방법의 투여는 본 명세서에 기재된 투여 방법 및 투여 용량에 따라 수행될 수 있다.In one aspect of the present invention, administration of the method may be performed according to the administration method and administration dose described herein.
일 측면에서, 상기 EGC는 상기 추출물 총 중량을 기준으로 8 중량% 이하, 7 중량% 이하, 6 중량% 이하, 5 중량% 이하, 4.7 중량% 이하, 4.56 중량% 이하, 4.3 중량% 이하, 4 중량% 이하, 3.53 중량% 이하, 3.5 중량% 이하, 3 중량% 이하, 2 중량% 이하, 1 중량% 이하, 0.8 중량% 이하, 0.5 중량% 이하, 0.3 중량% 이하 또는 0.1 중량% 이하일 수 있으며, 다른 측면에서 상기 EGC는 상기 추출물 총 중량을 기준으로 0.0001 중량% 이상, 0.001 중량% 이상, 0.01 중량% 이상, 1 중량% 이상, 2 중량% 이상, 3 중량% 이상, 3.5 중량% 이상, 3.53 중량% 이상, 4 중량% 이상, 4.3 중량% 이상, 4.56 중량% 이상, 4.7 중량% 이상, 5 중량% 이상, 6 중량% 이상 또는 7 중량% 이상일 수 있다. 상기 EGC가 상기 함량 범위로 포함되는 경우에 우수한 간독성 완화 효능을 나타낼 수 있다.In one aspect, the EGC is based on the total weight of the extract 8 wt% or less, 7 wt% or less, 6 wt% or less, 5 wt% or less, 4.7 wt% or less, 4.56 wt% or less, 4.3 wt% or less, 4 It may be less than or equal to 0.3% by weight, less than or equal to 3.53%, less than or equal to 3.5%, less than or equal to 2%, less than or equal to 1%, less than or equal to 0.8%, less than or equal to 0.5%, less than or equal to 0.3% or 0.1% by weight, In another aspect, the EGC is 0.0001% by weight or more, 0.001% by weight or more, 0.01% by weight or more, 1% by weight or more, 2% by weight or more, 3% by weight or more, 3.5% by weight or more, 3.53 based on the total weight of the extract It may be at least 4 wt%, at least 4 wt%, at least 4.3 wt%, at least 4.56 wt%, at least 4.7 wt%, at least 5 wt%, at least 6 wt%, or at least 7 wt%. When the EGC is included in the content range, it may exhibit excellent hepatotoxicity-relieving efficacy.
일 측면에서, 상기 추출물은 추출물 총 중량을 기준으로 4 내지 15 중량%의 (-)-갈로카테킨 갈레이트((-)-gallocatechin gallate, GCG) 및 4 내지 15 중량%의 (-)-에피갈로카테킨 갈레이트((-)-epigallocatechin gallate, EGCG)를 포함할 수 있다.In one aspect, the extract is based on the total weight of the extract 4 to 15% by weight of (-)-gallocatechin gallate ((-)-gallocatechin gallate, GCG) and 4 to 15% by weight of (-)-epigal Locatechin gallate ((-)-epigallocatechin gallate, EGCG).
상기 GCG는 상기 추출물 총 중량을 기준으로 4 중량% 이상, 5 중량% 이상, 5.3 중량% 이상, 5.59 중량% 이상, 5.7 중량% 이상, 6 중량% 이상, 7 중량% 이상, 8 중량% 이상, 9 중량% 이상, 10 중량% 이상, 11 중량% 이상, 12 중량% 이상, 13 중량% 이상 또는 14 중량% 이상일 수 있다. 다른 측면에서 상기 GCG는 상기 추출물 총 중량을 기준으로 15 중량% 이하, 14 중량% 이하, 13 중량% 이하, 12 중량% 이하, 11 중량% 이하, 10 중량% 이하, 9 중량% 이하, 8 중량% 이하, 7 중량% 이하, 6 중량% 이하, 5.7 중량% 이하, 5.59 중량% 이하, 5.3 중량% 이하 또는 5 중량% 이하일 수 있다.The GCG is 4 wt% or more, 5 wt% or more, 5.3 wt% or more, 5.59 wt% or more, 5.7 wt% or more, 6 wt% or more, 7 wt% or more, 8 wt% or more, based on the total weight of the extract, It may be 9% or more, 10% or more, 11% or more, 12% or more, 13% or more, or 14% or more. In another aspect, the GCG is 15% by weight, 14% by weight, 13% by weight, 12% by weight, 11% by weight, 10% by weight, 9% by weight, 8% by weight based on the total weight of the extract %, 7% or less, 6% or less, 5.7% or less, 5.59% or less, 5.3% or less, or 5% or less.
일 측면에서, 상기 EGCG는 상기 추출물 총 중량을 기준으로 4 중량% 이상, 5 중량% 이상, 5.2 중량% 이상, 5.27 중량% 이상, 5.5 중량% 이상, 6 중량% 이상, 7중량% 이상, 8중량% 이상, 9 중량% 이상, 10 중량% 이상, 11 중량% 이상, 12 중량% 이상, 13 중량% 이상 또는 14 중량% 이상일 수 있다. 다른 측면에서, 상기 EGCG는 상기 추출물 총 중량을 기준으로 15 중량% 이하, 14 중량% 이하, 13 중량% 이하, 12 중량% 이하, 11 중량% 이하, 10 중량% 이하, 9 중량% 이하, 8 중량% 이하, 7 중량% 이하, 6 중량% 이하, 5.5 중량% 이하, 5.27 중량% 이하, 5.2 중량% 이하 또는 5 중량% 이하일 수 있다.In one aspect, the EGCG is based on the total weight of the extract 4 wt% or more, 5 wt% or more, 5.2 wt% or more, 5.27 wt% or more, 5.5 wt% or more, 6 wt% or more, 7 wt% or more, 8 It may be at least 10% by weight, at least 9% by weight, at least 10% by weight, at least 11% by weight, at least 12% by weight, at least 13% by weight, or at least 14% by weight. In another aspect, the EGCG is 15% by weight or less, 14% by weight or less, 13% by weight or less, 12% by weight or less, 11% by weight or less, 10% by weight or less, 9% by weight or less, 8 based on the total weight of the extract It may be less than or equal to 7% by weight, less than or equal to 7%, less than or equal to 5.5%, less than or equal to 5.27%, less than or equal to 5.2%, or less than or equal to 5%.
다른 구현 예로서, 상기 추출물 내의 GCG 및 EGCG의 총 함량은 상기 추출물 총 중량을 기준으로 30 중량% 이하일 수 있다. 일 측면에서 상기 GCG 및 EGCG의 총 함량은 추출물 총 중량을 기준으로 30 중량% 이하, 25 중량% 이하, 20 중량% 이하, 18 중량% 이하, 16 중량% 이하, 15 중량% 이하, 14 중량% 이하, 12 중량% 이하, 11 중량% 이하, 10.86 중량% 이하, 10 중량% 이하 또는 9 중량% 이하일 수 있다. 다른 측면에서 상기 GCG 및 EGCG의 총 함량은 상기 추출물 총 중량을 기준으로 8 중량% 이상, 10 중량% 이상, 10.86 중량% 이상, 11 중량% 이상, 12 중량% 이상, 13 중량% 이상, 14 중량% 이상, 16 중량% 이상, 18 중량% 이상, 20 중량% 이상 또는 25 중량% 이상일 수 있다.As another embodiment, the total content of GCG and EGCG in the extract may be 30% by weight or less based on the total weight of the extract. In one aspect, the total content of the GCG and EGCG is 30 wt% or less, 25 wt% or less, 20 wt% or less, 18 wt% or less, 16 wt% or less, 15 wt% or less, 14 wt% based on the total weight of the extract Hereinafter, it may be 12% by weight or less, 11% by weight or less, 10.86% by weight or less, 10% by weight or less, or 9% by weight or less. In another aspect, the total content of the GCG and EGCG is 8% by weight, 10% by weight, 10.86% by weight, 11% by weight, 12% by weight, 13% by weight, 14% by weight based on the total weight of the extract % Or more, 16% or more, 18% or more, 20% or more, or 25% or more.
다른 구현 예로서, 상기 추출물 내의 에피 카테킨 함량은 상기 추출물 총 중량을 기준으로 20 중량% 이하일 수 있다. 일 측면에서 상기 에피 카테킨 함량은 상기 추출물 총 중량을 기준으로 20 중량% 이하, 18 중량% 이하, 16 중량% 이하, 15 중량% 이하, 14 중량% 이하, 12 중량% 이하, 11.1 중량% 이하 또는 10 중량% 이하일 수 있다. 다른 측면에서 상기 에피 카테킨 함량은 상기 추출물 총 중량을 기준으로 9 중량% 이상, 10 중량% 이상, 11.1 중량% 이상, 12 중량% 이상, 13 중량% 이상, 14 중량% 이상, 16 중량% 이상 또는 18 중량% 이상일 수 있다.As another embodiment, the epicatechin content in the extract may be 20% by weight or less based on the total weight of the extract. In one aspect, the epicatechin 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, 11.1 wt% or less based on the total weight of the extract, or It may be 10% by weight or less. In another aspect, the epicatechin content is at least 9% by weight, at least 10% by weight, at least 11.1% by weight, at least 12% by weight, at least 13% by weight, at least 14% by weight, at least 16% by weight based on the total weight of the extract, or It may be 18% by weight or more.
다른 구현 예로서, 상기 추출물 내의 8종 카테킨의 총 함량, 즉, 상기 추출물 내의 (-)-에피갈로카테킨 갈레이트((-)-epigallocatechin gallate, EGCG), 에피갈로카테킨(epigallocatechin, EGC) (-)에피카테킨((-)epicatechin, EC), 에피카테킨 3-O-갈레이트(epicatechin 3-O-gallate, ECG), 갈로카테킨 갈레이트(gallocatechin gallate, GCG), 갈로카테킨(gallocatechin, GC), 카테킨(catechin, C) 및 카테킨 갈레이트(catechin gallate, CG)의 총 함량은 상기 추출물 총 중량을 기준으로 19 내지 30 중량%일 수 있다. 일 측면에서 상기 8종 카테킨의 총 함량은 상기 추출물 총 중량을 기준으로 19 중량% 이상, 21 중량% 이상, 23 중량% 이상, 24 중량% 이상, 24.5 중량% 이상, 25 중량% 이상, 26 중량% 이상, 27 중량% 이상, 28 중량% 이상 또는 29 중량% 이상일 수 있다. 다른 측면에서 상기 8종 카테킨의 총 함량은 상기 추출물 총 중량을 기준으로 30 중량% 이하, 29 중량% 이하, 28 중량% 이하, 27 중량% 이하, 26 중량% 이하, 25 중량% 이하, 24.5 중량% 이하, 24 중량% 이하, 23 중량% 이하 또는 21 중량% 이하일 수 있다.As another embodiment, the total content of 8 catechins in the extract, i.e., (-)-epigallocatechin gallate (EGCG), epigallocatechin (EGC) in the extract (-) Epicatechin ((-) epicatechin (EC)), epicatechin 3-O-gallate (ECG), gallocatechin gallate (GCG), gallocatechin (GC), The total content of catechin (C) and catechin gallate (CG) may be 19 to 30% by weight based on the total weight of the extract. In one aspect, the total content of the eight catechins is 19% by weight or more, 21% by weight or more, 23% by weight or more, 24% by weight or more, 24.5% by weight or more, 25% by weight or more, 26% by weight based on the total weight of the extract % Or more, 27% or more, 28% or more, or 29% or more. In another aspect, the total content of the eight catechins is 30% by weight or less, 29% by weight or less, 28% by weight or less, 27% by weight or less, 26% by weight or less, 25% by weight or less, 24.5% by weight based on the total weight of the extract % Or less, 24% or less, 23% or less, or 21% or less.
또 다른 구현 예로서, 상기 추출물은 물 및 C1 내지 C4의 알코올 중 어느 하나 이상에 의해 1회 이상 추출한 추출물일 수 있다. 일 측면에서 상기 알코올은 에탄올일 수 있다. 다른 측면에서 상기 알코올은 20% 이상, 30% 이상, 40% 이상, 50% 이상, 60% 이상 또는 70% 이상의 에탄올일 수 있다. 또 다른 측면에서 상기 알코올은 70% 이하, 60% 이하, 50% 이하, 40% 이하 또는 30% 이하의 에탄올일 수 있다. 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 20% or more, 30% or more, 40% or more, 50% or more, 60% or more, or 70% or more ethanol. In another aspect, the alcohol may be 70% or less, 60% or less, 50% or less, 40% or less, or 30% or less ethanol.
일 실시양태로서, 상기 추출물은 건강기능식품 또는 약학 조성물에 함유될 수 있다.As an embodiment, the extract may be contained in a dietary supplement or pharmaceutical composition.
일 구현 예로서, 상기 건강기능식품 또는 약학 조성물 내 상기 녹차 추출물의 함량은 상기 조성물 전체 중량 대비 1 중량% 내지 100 중량% 일 수 있다. 일 측면에서, 상기 조성물 내 상기 추출물의 함량은 1 중량% 이상, 10 중량% 이상, 20 중량% 이상, 30 중량% 이상, 40 중량% 이상, 50 중량% 이상, 60 중량% 이상, 70 중량% 이상, 80 중량% 이상 또는 90 중량% 이상일 수 있다. 다른 측면에서, 상기 조성물 내 상기 추출물의 함량은 100 중량% 이하, 90 중량% 이하, 80 중량% 이하, 70 중량% 이하, 60 중량% 이하, 50 중량% 이하, 40 중량% 이하, 30 중량% 이하 또는 20 중량% 이하일 수 있다.As an embodiment, the content of the green tea extract in the dietary supplement or pharmaceutical composition may be 1% to 100% by weight relative to the total weight of the composition. In one aspect, the content of the extract in the composition is 1 wt% or more, 10 wt% or more, 20 wt% or more, 30 wt% or more, 40 wt% or more, 50 wt% or more, 60 wt% or more, 70 wt% Or more, 80% or more, or 90% or more. In another aspect, the content of the extract in the composition is 100% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less Or less or 20% by weight.
다른 구현 예로서, 상기 녹차 추출물의 투여량은 5mg/kg/일 내지 1000mg/kg/일일 수 있다. 일 측면에서, 상기 투여량은 5mg/kg/일 이상, 100mg/kg/일 이상, 200mg/kg/일 이상, 300mg/kg/일 이상, 400mg/kg/일 이상, 500mg/kg/일 이상, 600mg/kg/일 이상, 700mg/kg/일 이상, 800mg/kg/일 이상, 또는 900mg/kg/일 이상일 수 있다. 다른 측면에서, 상기 투여량은 1000mg/kg/일 이하, 900mg/kg/일 이하, 800mg/kg/일 이하, 700mg/kg/일 이하, 600mg/kg/일 이하, 500mg/kg/일 이하, 400mg/kg/일 이하, 300mg/kg/일 이하, 200mg/kg/일 이하, 100mg/kg/일 이하, 50mg/kg/일 이하 또는 10mg/kg/일 이하일 수 있다.As another embodiment, the dosage of the green tea extract may be 5 mg / kg / day to 1000 mg / kg / day. In one aspect, the dosage is 5 mg / kg / day or more, 100 mg / kg / day or more, 200 mg / kg / day or more, 300 mg / kg / day or more, 400 mg / kg / day or more, 500 mg / kg / day or more, It may be 600 mg / kg / day or more, 700 mg / kg / day or more, 800 mg / kg / day or more, or 900 mg / kg / day or more. 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, It may be 400 mg / kg / day or less, 300 mg / kg / day or less, 200 mg / kg / day or less, 100 mg / kg / day or less, 50 mg / kg / day or less, or 10 mg / kg / day or less.
상기 식품 조성물의 제형은 특별히 한정되지 않으나, 예를 들어, 정제, 과립제, 환제, 분말제, 드링크제와 같은 액제, 캐러멜, 겔, 바, 티백 등으로 제형화될 수 있다. 각 제형의 식품 조성물은 유효 성분 이외에 해당 분야에서 통상적으로 사용되는 성분들을 제형 또는 사용 목적에 따라 당업계의 통상의 기술자가 어려움 없이 적의 선정하여 배합할 수 있으며, 다른 원료와 동시에 적용할 경우 상승 효과가 일어날 수 있다. 또한 상기 식품은 건강기능식품일 수도 있다.The formulation of the food composition is not particularly limited, for example, tablets, granules, pills, powders, liquids such as drinks, caramels, gels, bars, tea bags, etc. may be formulated. The food composition of each formulation can be formulated by appropriately selecting the ingredients commonly used in the field other than the active ingredient according to the formulation or purpose of use without difficulty, and synergistic effect when applied simultaneously with other ingredients. Can happen. Also, the food may be a health functional food.
상기 조성물은 단순 섭취, 음용, 주사 투여, 스프레이 투여 또는 스퀴즈 투여 등 다양한 방법으로 투여될 수 있다.The composition may be administered by various methods such as simple ingestion, drinking, injection, spray administration, or squeeze administration.
본 발명의 일측면에 따른 식품 조성물에 있어서, 상기 유효 성분의 투여량 결정은 당업계의 통상의 기술자의 수준 내에 있으며, 투여하고자 하는 대상의 연령, 건강 상태, 합병증 등 다양한 요인에 따라 달라질 수 있다.In the food composition according to an aspect of the present invention, the dosage 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, and complications of the subject to be administered. .
본 발명의 일측면에 따른 식품 조성물은, 예를 들어, 츄잉껌, 캐러멜 제품, 캔디류, 빙과류, 과자류 등의 각종 식품류, 청량 음료, 미네랄 워터, 알코올 음료 등의 음료 제품, 비타민이나 미네랄 등을 포함한 건강기능식품 제품일 수 있다.Food composition according to one aspect of the present invention, for example, chewing gum, caramel products, candy, ice cream, confectionery, various foods, soft drinks, mineral water, alcoholic beverages, beverage products, vitamins and minerals, etc. It may be a functional food product.
상기 외에, 본 발명의 일 측면에 따른 식품 조성물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 증진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알콜, 탄산 음료에 사용되는 탄산화제 등을 포함할 수 있다. 그 밖에 본 발명의 일측면에 따른 식품 조성물들은 천연 과일 쥬스 및 과일 쥬스 음료 및 야채 음료의 제조를 위한 과육을 포함할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 그렇게 중요하진 않으나 본 발명의 일측면에 따른 조성물 100 중량부 당 0 내지 약 60 중량부의 범위에서 포함되는 것이 일반적이다.In addition to the above, the food composition according to one aspect of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, colorants and enhancers (cheese, chocolate, etc.), pectic acid and the like. Salts, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonic acid used in carbonated beverages, and the like. In addition, food compositions according to one aspect of the present invention may include natural fruit juice and fruit juice for the production of fruit juice drinks and vegetable drinks. These ingredients can be used independently or in combination. The proportion of these additives is not so critical, but is generally 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 present invention.
본 발명의 일 측면에 따른 상기 식품 조성물은 녹차 추출물의 간독성이 완화된 것일 수 있다. 보다 구체적으로, 상기 식품 조성물은 녹차 추출물 중 EGC에 의한 간독성이 완화된 것일 수 있다.The food composition according to an aspect of the present invention may be that the liver toxicity of green tea extract is alleviated. More specifically, the food composition may be that the liver toxicity by EGC in green tea extract is alleviated.
본 발명의 일측면에 따른 상기 약학 조성물은 경구, 비경구, 직장, 국소, 경피, 정맥 내, 근육 내, 복강 내, 피하 등으로 투여될 수 있다. 경구 투여를 위한 제형은 정제(錠劑), 환제(丸劑), 연질 및 경질 캅셀제, 과립제(顆粒劑), 산제, 세립제, 액제, 유탁제(乳濁濟) 또는 펠렛제일 수 있으나, 이에 제한되는 것은 아니다. 비경구 투여를 위한 제형은 용액제, 현탁제, 유액제, 겔, 주사제, 점적제, 좌제(坐劑), 패취 또는 분무제일 수 있으나, 이에 제한되는 것은 아니다. 상기 제형은 당해 분야의 통상적인 방법에 따라 용이하게 제조될 수 있으며, 계면 활성제, 부형제, 수화제, 유화 촉진제, 현탁제, 삼투압 조절을 위한 염 또는 완충제, 착색제, 향신료, 안정화제, 방부제, 보존제 또는 기타 상용하는 보조제를 추가로 포함할 수 있다.The pharmaceutical composition according to an aspect of the present invention may be administered orally, parenteral, rectal, topical, transdermal, intravenous, intramuscular, intraperitoneal, subcutaneous, and the like. Formulations for oral administration may be tablets, pills, soft and hard capsules, granules, powders, granules, liquids, emulsions or pellets, but are not limited thereto. It is not. Formulations for parenteral administration may be solutions, suspensions, emulsions, gels, injections, drops, suppositories, patches or sprays, but are not limited thereto. The formulations can be readily prepared according to conventional methods in the art, surfactants, excipients, hydrating agents, emulsifying accelerators, suspending agents, salts or buffers for controlling osmotic pressure, colorants, spices, stabilizers, preservatives, preservatives or Other commercial adjuvants may be further included.
본 발명의 일 측면에 따른 상기 약학 조성물은 약학적으로 허용가능한 염을 포함할 수도 있는데, 상기 염은 (1) 염산, 브롬화수소산, 황산, 질산, 인산 등과 같은 무기산으로 형성되거나; 또는 아세트산, 프로파이온산, 헥사노산, 시클로펜테인프로피온산, 글라이콜산, 피루브산, 락트산, 말론산, 숙신산, 말산, 말레산, 푸마르산, 타르타르산, 시트르산, 벤조산, 3-(4-히드록시벤조일) 벤조산, 신남산, 만델산, 메테인설폰산, 에테인설폰산, 1,2-에테인-디설폰산, 2-히드록시에테인설폰산, 벤젠설폰산, 4-클로로벤젠설폰산, 2-나프탈렌설폰산, 4-톨루엔설폰산, 캄퍼설폰산, 4-메틸바이시클로 [2,2,2]-oct-2-엔-1-카르복실산, 글루코헵톤산, 3-페닐프로파이온산, 트리메틸아세트산, tert-부틸아세트산, 라우릴 황산, 글루콘산, 글루탐산, 히드록시나프토산, 살리실산, 스테아르산, 뮤콘산과 같은 유기산으로 형성되는 산 부가염(acid addition salt); 또는 (2) 모 화합물에 존재하는 산성 프로톤이 치환될 때 형성되는 염을 포함할 수 있다.The pharmaceutical composition according to an aspect of the present invention may include a pharmaceutically acceptable salt, wherein the salt is formed of an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, etc .; 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 from organic acids such as butylacetic acid, lauryl sulfate, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, and muconic acid; Or (2) a salt formed when the acidic proton present in the parent compound is substituted.
본 발명의 일측면에 따른 상기 약학 조성물의 적용량 또는 투여량은 투여 받을 대상의 연령, 성별, 체중, 병리 상태 및 그 심각도, 투여 경로 또는 처방자의 판단에 따라 달라질 것이다. 이러한 인자에 기초한 유효성분 투여량 결정은 당업계 통상의 기술자의 수준 내에 있다.The applied amount or dosage of the pharmaceutical composition according to an aspect of the present invention will vary depending on the age, gender, weight, pathology and severity of the subject to be administered, the route of administration or the judgment of the prescriber. The determination of the active ingredient dosage based on these factors is within the level of those skilled in the art.
이하, 실시예, 실험예, 및 제형예를 들어 본 명세서의 구성 및 효과를 보다 구체적으로 설명한다. 그러나 이들 예는 본 명세서에 대한 이해를 돕기 위해 예시의 목적으로만 제공된 것일 뿐 본 명세서의 범주 및 범위가 하기 예에 의해 제한되는 것은 아니다.Hereinafter, examples, experimental examples, and formulation examples, the composition and effects of the present specification will be described in more detail. However, these examples are provided for purposes of illustration only to aid understanding of the present specification, and the scope and scope of the present specification are not limited by the following examples.
[실시예 1] 일반 녹차 추출물과 고온처리 녹차 추출물의 제조[Example 1] Preparation of general green tea extract and high temperature-treated green tea extract
100g의 녹차(Camellia sinensis, 제주 오설록 농장)에 50% 에탄올 1000ml 를 가하여 60℃에서 1시간 환류 교반하였다. 상기 시료의 온도를 실온으로 낮추고 여과하여 얻은 용액을 감압증류하여 진한갈색분말의 일반 고시형 녹차 추출물(GT-LE-35CAT, 시료 1) 23g을 수득하였다(수율 23%).To 100 g of green tea (Camellia sinensis, Jeju Osulloc Farm), 1000 ml of 50% ethanol was added and stirred at reflux for 1 hour at 60 ° C. The solution obtained by lowering the temperature of the sample to room temperature and filtering was distilled under reduced pressure to obtain 23 g of a general notice green tea extract (GT-LE-35CAT, sample 1) of a dark brown powder (yield 23%).
한편, 고온처리 녹차 추출물을 제조하기 위하여, 100g의 녹차(Camellia sinensis, 제주 오설록 농장)에 50% 에탄올 1000ml 를 가하여 60℃에서 1시간 환류 교반하고 여과 및 감압증류하여 제1 녹차 추출물을 수득한 후 이를 농축한 다음 스팀 1.5 kgf/cm 2에서 1~7시간 동안 교반시켰다. 이후 온도를 실온으로 낮추고 불용성물질을 여과하고 감압농축하여 고온처리 녹차 추출물 10g을 수득하였다. 이 때, 상기 교반 시간대(1~7시간) 별로 얻은 상기 고온처리 녹차 추출물에 대해 하기 표 1과 같은 장치를 이용하여 8종 카테킨의 함량 변화를 측정하였고, 5시간 째에 열에 의한 EGCG의 GCG로의 전환이 가장 많으면서, 8종 카테킨의 양도 더 이상 줄어들지 않음을 확인하였다. 이때 수득한 고온처리 녹차 추출물 10g(High temperature processed green tea extract, HTP-GTE)을 시료 2로 하였다.Meanwhile, in order to prepare a high-temperature-treated green tea extract, 100 ml of green tea (Camellia sinensis, Jeju Osulloc Farm) was added with 1000 ml of 50% ethanol, stirred under reflux at 60 ° C. for 1 hour, filtered and distilled under reduced pressure to obtain a first green tea extract. It was concentrated and then stirred for 1-7 hours at 1.5 kgf / cm 2 of steam. Thereafter, the temperature was lowered to room temperature, the insoluble material was filtered, and concentrated under reduced pressure to obtain 10 g of a hot-treated green tea extract. At this time, the change in the content of 8 catechins was measured for the high-temperature treated green tea extract obtained for each stirring time period (1 to 7 hours) using the device shown in Table 1 below, and after 5 hours, heat was converted into EGCG to GCG. It was confirmed that the most conversion, the amount of 8 kinds of catechins did not decrease any more. At this time, the obtained high temperature processed green tea extract 10g (High temperature processed green tea extract, HTP-GTE) was used as Sample 2.
수득된 상기 두 종류의 추출물의 조성을 분석한 조건과 결과는 각각 하기 표 1(시료 1 및 2의 조성 분석 조건), 표 2(시료 1의 조성 분석 결과) 및 표 3(시료 2의 조성 분석 결과)와 같다. 또한, 상기 두 종류의 추출물에 대한 크로마토그램은 도 1(시료 1) 및 도 2(시료 2)와 같다. 즉, 시료 2의 경우 기존의 녹차 추출물과는 조성이 다르다는 것을 확인하였다. 구체적으로, 시료 2의 경우 시료 1에 비해 현저하게 낮은 EGC(3.53중량%), EGCG(5.27중량%) 및 총 카테킨(24.41중량%)을 함유하는 반면, 시료 1에서는 발견되지 않은 성분인 4종의 에피카테킨 에피머가 추가로 생성되는 것을 확인할 수 있었다.The conditions and results for analyzing the composition of the obtained two types of extracts are shown in Table 1 (composition analysis conditions of Samples 1 and 2), Table 2 (composition analysis results of Sample 1), and Table 3 (composition analysis results of Sample 2), respectively. ). In addition, the chromatograms for the two types of extracts are shown in Fig. 1 (sample 1) and 2 (sample 2). That is, it was confirmed that the composition of Sample 2 is different from the existing green tea extract. Specifically, Sample 2 contains significantly lower EGC (3.53% by weight), EGCG (5.27% by weight), and total catechin (24.41% by weight) than Sample 1, whereas Sample 1 has four components that are not found. It was confirmed that the epicatechin epimer of was additionally produced.
컬럼column Thermofisher C18 5um, 4.6 X 250mmThermofisher C18 5um, 4.6 X 250mm
디텍터Detector UV 280nmUV 280nm
희석Dilution 그래디언트(Gradient) A-0.1% TFA (Trifluoro acetic acid) 내의 물 B-아세토나이트릴(Acetonitrile)Water in Gradient A-0.1% Trifluoro acetic acid (TFA) B-Acetonitrile
그래디언트 프로파일 (Gradient profile)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)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 )
속도 (Flow rate)Flow rate 1ml/min1ml / min
주입량 (Injection volume)Injection volume 20μl20μl
시료 1 Sample 1 GCGC EGCEGC CaffeineCaffeine CC ECEC GCGGCG EGCGEGCG CGCG ECGECG 에피 카테킨 총량Total amount of epicatechin 에피카테킨에피머총량Epicatechin Epimer Total 8종카테킨총량8 kinds of catechins
00 9.169.16 3.213.21 00 3.633.63 00 20.9320.93 00 2.622.62 36.3436.34 00 36.3436.34
시료 2 Sample 2 GCGC EGCEGC CaffeineCaffeine CC ECEC GCGGCG EGCGEGCG CGCG ECGECG 에피카테킨총량Total amount of epicatechin 에피 카테킨에피머 총량Total amount of epicatechin epimer 8종카테킨총량8 kinds of catechins
4.94.9 3.533.53 3.93.9 1.571.57 0.950.95 5.595.59 5.275.27 1.31.3 1.31.3 11.0511.05 13.3613.36 24.4124.41
(상기 표 2 및 표 3에서, GC는 갈로카테킨, EGC는 에피갈로카테킨, C는 카테킨, EC는 (-)에피카테킨, GCG는 갈로카테킨 갈레이트, EGCG는 에피갈로카테킨 갈레이트, CG는 카테킨 갈레이트, ECG는 에피카테킨 3-O-갈레이트) (In Tables 2 and 3, GC is gallocatechin, EGC is epigallocatechin, C is catechin, EC is (-) epicatechin, GCG is gallocatechin gallate, EGCG is epigallocatechin gallate, CG is Catechin gallate, ECG is epicatechin 3-O-gallate)
(상기 표 2 및 표 3의 단위는 모두 녹차 추출물(시료 1 및 2) 전체 중량 대비 해당 물질의 중량%)(All of the units in Tables 2 and 3 above are green tea extracts (Samples 1 and 2) by weight of the substance relative to the total weight)
[실험예 1] 고온처리 녹차 추출물의 간세포 독성 확인 실험[Experimental Example 1] Hepatotoxicity test of high temperature treated green tea extract
상기 실시예 1에서 수득한 고온처리 녹차 추출물(시료 2)에 의한 간독성을 확인하기 위하여, 한국세포주은행에서 구한 간세포주(HepG2 cell line)를 96웰 플레이트(well plate, FALCON)에 웰 당 1X10 5씩 시딩하고(seeding), 24시간 동안 37℃, 5% CO 2 인큐베이터에서 배양 후, 상기 시료 1과 시료 2를 각각 5, 10, 25, 50, 100 μg/ml로 처리하여 24시간 동안 추가 배양한 뒤 배지를 제거하고 CellTiter 96 AQueous One Solution Cell Proliferation Assay Kit(MTS, Promega Co. Madison, WI, USA)를 이용하여 세포 생존율을 확인하였다. RPMI1640 (Lonza) 100 μl에 CellTiter 96 AQueous One Solution Cell Proliferation Assay Kit(MTS, Promega Co. Madison, WI, USA) 용액 20 μl를 첨가하여 세포에 처리 후 490 nm에서 흡광도를 확인하여 살아있는 세포의 수를 정량화 하였다. 세포 수 또는 세포 생존율(%)은 하기 식에 의해 계산되었다.In order to confirm hepatotoxicity by the high-temperature treated green tea extract obtained in Example 1 (sample 2), the hepatocyte cell line (HepG2 cell line) obtained from the Korean Cell Line Bank was placed in a 96 well plate (well plate, FALCON) 1X10 5 per well. After seeding each time, and cultured in a 37 ° C, 5% CO 2 incubator for 24 hours, the samples 1 and 2 were treated with 5, 10, 25, 50, 100 μg / ml, respectively, and further cultured for 24 hours. After removing the medium, cell viability was confirmed using a CellTiter 96 AQueous One Solution Cell Proliferation Assay Kit (MTS, Promega Co. Madison, WI, USA). After adding 20 μl of CellTiter 96 AQueous One Solution Cell Proliferation Assay Kit (MTS, Promega Co. Madison, WI, USA) solution to 100 μl of RPMI1640 (Lonza), treat the cells, check the absorbance at 490 nm to determine the number of live cells. It was quantified. Cell number or cell viability (%) was calculated by the following formula.
세포 생존율 (%) = (시료 처리군 흡광도 - 반응시약만의 흡광도) / (무처리군 흡광도 - 반응시약만의 흡광도) X 100Cell viability (%) = (Absorbance of the sample treated group-Absorbance of only the reaction reagent) / (Absorbance of the untreated group-Absorbance of the reaction reagent only) X 100
그 결과는 도 3과 같으며, 본원 발명에 따른 시료 2는 일반 녹차 추출물인 시료 1에 비하여 간세포 독성이 낮음을 확인할 수 있었다.The results are as shown in FIG. 3, and it was confirmed that the sample 2 according to the present invention has low hepatocellular toxicity compared to the sample 1, which is a normal green tea extract.
[실험예 2] 고온처리 녹차 추출물의 간세포 손상에 대한 영향 확인 실험[Experimental Example 2] Experiment to confirm the effect of high temperature treated green tea extract on liver cell damage
상기 실시예 1에서 수득한 고온처리 녹차 추출물(시료 2)의 간세포 손상에 대한 영향을 확인하기 위하여, 간세포 손상으로 인하여 세포 외로 간세포 효소가 유출된 정도를 분석 프로그램(MILLIPLEX Analyst, Millipore)을 사용하여 분석하였다. 실험 조건은 실험예 1과 같으며, 시료 1과 시료 2를 처리하고 24시간 후 세포 배양액에서 MDH 1(malate dehydrogenase 1), GSTα(α-glutathione S-transferase), SDH(sorbitol dehydrogenase)의 레벨을 측정하였다.In order to confirm the effect of the high-temperature-treated green tea extract (Sample 2) obtained in Example 1 on liver cell damage, an analysis program (MILLIPLEX Analyst, Millipore) was used to analyze the degree of hepatocellular enzyme outflow due to hepatocyte damage. Analysis. Experimental conditions are the same as in Experimental Example 1, and after treatment of Sample 1 and Sample 2, the level of MDH 1 (malate dehydrogenase 1), GSTα (α-glutathione S-transferase), and SDH (sorbitol dehydrogenase) in the cell culture solution was 24 hours. It was measured.
그 결과는 도 4와 같으며, 고온처리 녹차 추출물(시료 2)을 처리한 경우, 일반 녹차 추출물(시료 1)을 처리한 경우에 비하여 세 효소 모두 세포 배양액에서의 레벨이 낮은 것으로 나타났다.The results are as shown in FIG. 4, and when the high-temperature-treated green tea extract (Sample 2) was treated, the levels of all three enzymes were lower in the cell culture than when the green tea extract (Sample 1) was treated.
즉, 본 발명에 따른 시료 2의 녹차 추출물이 일반 녹차 추출물에 비하여 간세포 손상에 미치는 영향이 낮은 것을 확인할 수 있었다.That is, it was confirmed that the green tea extract of Sample 2 according to the present invention had a lower effect on hepatocyte damage than the green tea extract.
[실험예 3] 카테킨 8종의 간세포 독성 확인 실험[Experimental Example 3] Hepatocellular toxicity confirmation experiment of 8 catechins
일반 녹차 추출물 또는 고온처리 녹차 추출물에 존재하는 카테킨 8종(EGCG, GCG, EGC, GC, ECG, CG, EC, C)에 의한 간독성을 확인하기 위하여, 한국세포주은행에서 구한 간세포주(HepG2 cell line)를 96웰 플레이트(well plate, FALCON)에 웰 당 1X10 5씩 시딩하고(seeding), 24시간 동안 37℃, 5% CO 2 인큐베이터에서 배양 후, 카테킨 8종(EGCG, GCG, EGC, GC, ECG, CG, EC, C)을 각각 10, 50, 100, 200 μM로 처리하여 24시간 동안 추가 배양한 뒤 배지를 제거하고 CellTiter 96 AQueous One Solution Cell Proliferation Assay Kit(MTS, Promega Co. Madison, WI, USA)를 이용하여 세포 생존율을 확인하였다. RPMI1640 (Lonza) 100 μl에 CellTiter 96 AQueous One Solution Cell Proliferation Assay Kit(MTS, Promega Co. Madison, WI, USA) 용액 20 μl를 첨가하여 세포에 처리 후 490 nm에서 흡광도를 확인하여 살아있는 세포의 수를 정량화 하였다. 세포 수 또는 세포 생존율(%)은 상기 실험예 1에서의 식과 동일한 식에 의해 계산되었다. 농도에 따른 생존율을 바탕으로 LD50(lethal dose 50) 값을 구하였으며, 그 결과는 하기 표 4와 같다.In order to confirm hepatotoxicity by 8 kinds of catechins (EGCG, GCG, EGC, GC, ECG, CG, EC, C) present in general green tea extract or high temperature treated green tea extract, hepatocyte cell line (HepG2 cell line) obtained from the Korea Cell Line Bank ) In a 96-well plate (well plate, FALCON) 1X10 5 per well (seeding), and incubated in a 5% CO 2 incubator for 24 hours, 37 ℃, 8 catechins (EGCG, GCG, EGC, GC, ECG, CG, EC, and C) were treated with 10, 50, 100, and 200 μM, respectively, for 24 hours to further incubate, the medium was removed, and the CellTiter 96 AQueous One Solution Cell Proliferation Assay Kit (MTS, Promega Co. Madison, WI) , USA). After adding 20 μl of CellTiter 96 AQueous One Solution Cell Proliferation Assay Kit (MTS, Promega Co. Madison, WI, USA) solution to 100 μl of RPMI1640 (Lonza), treat the cells, check the absorbance at 490 nm to determine the number of live cells. It was quantified. Cell number or cell viability (%) was calculated by the same formula as in Experimental Example 1. Based on the survival rate according to the concentration, a LD50 (lethal dose 50) value was obtained, and the results are shown in Table 4 below.
EGCGEGCG GCGGCG EGCEGC GCGC ECGECG CGCG ECEC CC
LD50 (μM)LD50 (μM) 355.5355.5 212.7212.7 183.9183.9 236.2236.2 209.0209.0 260.8260.8 505.0505.0 569.0569.0
상기 표 4의 결과로부터, 8종의 카테킨 중에서 EGC의 LD50 값이 가장 낮은 것으로 나타났으며, 이에 따라 EGC의 간세포 독성이 가장 높은 것을 확인할 수 있었다.From the results of Table 4, the LD50 value of EGC was the lowest among the 8 catechins, and accordingly, it was confirmed that the hepatocellular toxicity of EGC was the highest.
[실험예 4] 카테킨 3종의 간세포 손상에 대한 영향 확인 실험[Experimental Example 4] Experiment to check the effects of catechins on hepatocyte damage
8종의 카테킨 중 EGCG, GCG, EGC 세 성분의 간세포 손상에 대한 영향을 확인하기 위하여, 간세포 손상으로 인하여 세포 외로 간세포 효소가 유출된 정도를 분석 프로그램(MILLIPLEX Analyst, Millipore)을 사용하여 분석하였다. 실험 조건은 실험예 3과 같으며, EGCG, GCG, EGC를 처리하고 24시간 후 세포 배양액에서 MDH 1(malate dehydrogenase 1), GSTα(α-glutathione S-transferase), SDH(sorbitol dehydrogenase)의 레벨을 측정하였다.In order to confirm the effect of EGCG, GCG, and EGC three components among eight catechins on the damage to hepatocytes, the degree of hepatocellular enzyme outflow due to hepatocyte damage was analyzed using an analysis program (MILLIPLEX Analyst, Millipore). Experimental conditions are the same as in Experimental Example 3, and after treatment with EGCG, GCG, and EGC, levels of MDH 1 (malate dehydrogenase 1), GSTα (α-glutathione S-transferase), and SDH (sorbitol dehydrogenase) were treated in the cell culture solution after 24 hours. It was measured.
그 결과는 도 5와 같으며, EGC를 처리한 경우, 나머지 EGCG, GCG를 처리한 경우에 비하여 세 효소 모두 세포 배양액에서의 레벨이 가장 높은 것으로 나타났다.The results are as shown in FIG. 5, and when the EGC treatment was performed, all three enzymes were found to have the highest level in the cell culture solution compared to the other EGCG and GCG treatments.
즉, EGC가 EGCG 및 GCG에 비하여 간세포 손상에 미치는 영향이 가장 높은 것을 확인할 수 있었다.That is, it was confirmed that EGC has the highest effect on hepatocyte damage compared to EGCG and GCG.
[실험예 5] 고온처리 녹차 추출물의 혈행개선 효능 평가 실험[Experimental Example 5] Experiment for evaluating blood circulation improvement efficacy of high-temperature treated green tea extract
사람의 혈관내피세포에는 eNOS(endothelial nitric oxide synthase)가 존재하며, 그 활성이 증가하는 것으로 일산화질소(NO)를 생성시켜 혈관을 확장시키고, 혈행을 촉진하는 역할을 하게 된다. 이에, 상기 실시예 1의 시료 1 및 시료 2의 녹차 추출물의 혈행개선 효능을 평가하기 위하여 인간 혈관내피세포(human umbilical vein endothelial cell, HUVEC)를 배양한 후 각 시료를 배지에 처리하여 생성되는 NO의 양을 비교하였다.Endothelial nitric oxide synthase (eNOS) is present in human vascular endothelial cells, and its activity is increased to produce nitrogen monoxide (NO) to expand blood vessels and promote blood circulation. Thus, in order to evaluate the blood circulation improvement efficacy of the green tea extracts of Sample 1 and Sample 2 of Example 1, after culturing human umbilical vein endothelial cells (HUVEC), NO is generated by treating each sample in the medium. The amount of was compared.
구체적으로, 혈관내피세포를 바닥은 투명하고 벽은 검은 96웰 플레이트(clear-plate, black, well plate, FALCON)에 웰 당 2X10 4씩 시딩하고(seeding), 24시간 동안 37℃, 5% CO 2 인큐베이터에서 배양 후, 소태아혈청(fetal bovine serum, FBS)를 제거한 phenol red free 배지로 세포를 4시간 동안 starvation 시킨 다음, 배지를 제거하고 PBS(phosphate buffer saline)로 2번 세척하였다. 그 후, DAF-2DA를 HBSS(Hank's Balanced Salt Solution)에 5 μM로 녹여 세포에 100μl 처리한 뒤, 호일로 빛을 차단하여 20분 동안 37℃, 5% CO 2 인큐베이터에서 배양하였다. 시료 처리 전 형광을 먼저 측정하였으며 (excitation: 485 nM, emission: 535 nM), 시료 1과 시료 2를 각각 6, 20, 60 μg/ml로 준비하여 각 웰에 100 μl씩 처리한 뒤 microplate reader(Tecan Salzburg, Austria)로 형광을 측정한다. 한편 상기 시료 1 및 2 대신 DMSO(dimethylsulfoxide)를 0.1%(v/v) 처리한 것을 대조군으로 사용하였다. 상대적인 NO 생성량은 하기의 식에 의해서 계산되었다. Specifically, vascular endothelial cells were seeded at 2X10 4 per well in a 96-well plate (clear-plate, black, well plate, FALCON) with a transparent bottom and a black wall, and seeded for 24 hours at 37 ° C and 5% CO 2 After incubation in an incubator, cells were starvated for 4 hours with phenol red free medium from which fetal bovine serum (FBS) was removed, and then the medium was removed and washed twice with PBS (phosphate buffer saline). Thereafter, DAF-2DA was dissolved in HBSS (Hank's Balanced Salt Solution) with 5 μM, treated with 100 μl of cells, and then incubated in a 5% CO 2 incubator for 20 minutes by blocking the light with foil. Fluorescence was measured before sample treatment (excitation: 485 nM, emission: 535 nM), and sample 1 and sample 2 were prepared at 6, 20, and 60 μg / ml, respectively, and treated with 100 μl in each well, followed by microplate reader ( Fluorescence is measured with Tecan Salzburg, Austria). Meanwhile, instead of samples 1 and 2, DMSO (dimethylsulfoxide) was treated with 0.1% (v / v) as a control. The relative NO production was calculated by the following equation.
상대적 NO 생성량 = (시료 처리군 흡광도 - 반응시약만의 흡광도) / (대조군(DMSO 처리군) 흡광도 - 반응시약만의 흡광도) Relative NO production = (absorbance of sample treatment group-absorbance of reaction reagent only) / (absorbance of control group (DMSO treatment group)-absorbance of reaction reagent only)
그 결과는 도 6과 같으며, 시료 1 및 2 모두 농도 의존적으로 혈관내피세포에서 NO를 증가시키는 것을 확인할 수 있었으나, 본 발명에 따른 시료 2의 고온처리 녹차 추출물이 시료 1의 일반 녹차 추출물에 비하여 동일 농도에서 NO 생성량이 현저히 높은 것을 확인할 수 있었다.The results are as shown in FIG. 6, and it was confirmed that both samples 1 and 2 increased NO in vascular endothelial cells in a concentration-dependent manner, but the high-temperature treated green tea extract of Sample 2 according to the present invention compared to the normal green tea extract of Sample 1 It was confirmed that the NO production amount was remarkably high at the same concentration.
위의 실험예 1 및 2의 결과와 종합하면, 본 발명에 따른 고온처리 녹차 추출물은 기존의 일반 녹차 추출물에 비하여 간독성이 낮아 안전성이 높으면서도 혈관내피세포에서 보다 적은 양으로도 많은 양의 NO를 생성시켜 우수한 혈행개선 효능을 나타내는 것을 알 수 있다.Combined with the results of Experimental Examples 1 and 2 above, the high-temperature-treated green tea extract according to the present invention has high hepatotoxicity and low safety compared to the conventional green tea extract, but also contains a large amount of NO in a smaller amount in vascular endothelial cells. It can be seen that it exhibits excellent blood circulation improvement efficacy.
[실험예 6] 고온처리 녹차 추출물의 간 세포 내 지방 축적 억제 효능 평가 실험[Experimental Example 6] Evaluation of the effect of high-temperature-treated green tea extract on the inhibition of fat accumulation in liver cells
상기 실시예 1의 시료 1 및 시료 2의 녹차 추출물의 간 세포에서 지방산에 의한 지방 축적 억제 효능을 평가하기 위하여 인간 간암 세포주(human hepatoma cell line)인 HepG2를 배양한 후 각 시료를 배지에 처리하여 지방 축적 저해력을 비교하였다.In order to evaluate the effect of inhibiting fat accumulation by fatty acids in liver cells of the green tea extracts of Sample 1 and Sample 2 of Example 1, after culturing HepG2, a human hepatoma cell line, each sample was treated in a medium. Fat accumulation inhibition was compared.
구체적으로, 간세포주(HepG2 cell line)를 12웰 플레이트(well plate, FALCON)에 웰 당 1X10 6씩 시딩하고(seeding), 24시간 동안 37℃, 5% CO 2 인큐베이터에서 배양 후, 상기 시료 1과 시료 2를 각각 3, 10, 30 μg/ml 과 소혈청알부민(bovine serum albumin, BSA)과 컨쥬게이션(conjugation) 된 스테아릭산 (stearic acid) 100 μM을 같이 처리하여 48시간 동안 배양하였다. 배지를 제거하고 PBS로 세척한 뒤 4% 포름알데히드(formaldehyde)로 고정하였다. 100% 프로필렌글리콜(prophyleneglycol)로 5분간 추가 배양 후, Oil-red O 용액 (sigma)을 웰당 500 μL 넣고 2시간동안 배양하여 세포 내 지방을 염색하였다. Oil-red O 용액을 제거한 뒤 85% 프로필렌글리콜로 5분간 배양하고, 60% 이소프로판올(isoprophanol)로 15 분간 배양하여 세포에 축적된 지방에 들어갔던 Oil-red O 용액을 추출하였다. 이소프로판올 200 μL을 96 웰 플레이트에 옮겨 담은 뒤 485 nM에서 흡광도를 측정하여 세포에 축적된 지방량을 정량화 하였다. 대조군 (DMSO 0.1%(v/v)) 대비 상대적인 Oil-red O 양 (%)은 하기 식에 의해 계산되었다. Specifically, the hepatocyte cell line (HepG2 cell line) is seeded in a 12-well plate (well plate, FALCON) for 1 × 10 6 per well, 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, 30 μg / ml and bovine serum albumin (BSA) and conjugated stearic acid 100 μM, respectively, and cultured for 48 hours. The medium was removed, washed with PBS, and fixed with 4% formaldehyde. After further incubation for 5 minutes with 100% propylene glycol (prophyleneglycol), 500 μL of oil-red O solution (sigma) was added per well and cultured for 2 hours to stain intracellular fat. After removing the Oil-red O solution, incubated with 85% propylene glycol for 5 minutes and incubated with 60% isoprophanol for 15 minutes to extract the Oil-red O solution that had entered the fat accumulated in the cells. After transferring 200 μL of isopropanol to a 96-well plate, the absorbance was measured at 485 nM to quantify the amount of fat accumulated in the cells. The relative amount of oil-red O (%) relative to the control (DMSO 0.1% (v / v)) was calculated by the following equation.
Oil red O 양 (%) = (시료처리군 흡광도 - 대조군 흡광도) X 100Amount of oil red O (%) = (absorbance of sample treatment group-control absorbance) X 100
그 결과는 도 7과 같으며, 시료 1의 경우 3μg/ml 처리 시 오히려 지방축적이 증가하고, 10μg/ml 및 30μg/ml 처리 시 농도 의존적으로 지방축적을 저해하는 것을 확인할 수 있었다. 반면, 본 발명에 따른 시료 2의 경우, 3μg/ml 처리 농도부터 농도의존적으로 지방축적을 저해하는 것을 확인할 수 있었으며, 시료 2의 지방축적 저해력이 시료 1에 비하여 현저히 높은 것을 확인할 수 있었다.The results are as shown in FIG. 7, and it was confirmed that in the case of Sample 1, fat accumulation increased when 3 μg / ml was treated, and concentration-dependently inhibited fat accumulation when treated with 10 μg / ml and 30 μg / ml. On the other hand, in the case of Sample 2 according to the present invention, it was confirmed that the concentration-dependent inhibition of fat accumulation from the concentration of 3 μg / ml was observed, and it was confirmed that the inhibition ability of Fat 2 in Sample 2 was significantly higher than that in Sample 1.
위의 실험예 1 및 2의 결과와 종합하면, 본 발명에 따른 고온처리 녹차 추출물은 기존의 일반 녹차 추출물에 비하여 간독성이 낮아 안전성이 높으면서도 간 세포에서 지방축적 억제능이 현저하게 뛰어나 우수한 지방간 예방 효능을 나타내는 것을 알 수 있다.Combined with the results of Experimental Examples 1 and 2 above, the high-temperature-treated green tea extract according to the present invention has low liver toxicity compared to the conventional green tea extract, has high safety, and has excellent remarkable ability to inhibit fat accumulation in liver cells, and has excellent fatty liver prevention efficacy. It can be seen that indicates.
[실험예 7] GCG 및 EGCG의 간 세포 내 지방 축적 억제 효능 평가 실험[Experimental Example 7] GCG and EGCG evaluation of the effect of inhibiting fat accumulation in liver cells
시료 1 및 시료 2 대신 GCG 및 EGCG를 처리한 것을 제외하고는 상기 실험예 6과 동일한 방법으로 실험을 진행하였다.Experiments were conducted in the same manner as in Experimental Example 6, except that GCG and EGCG were treated instead of Sample 1 and Sample 2.
그 결과는 도 8과 같으며, 동일한 농도에서 EGCG에 비하여 GCG의 간 세포 내 지방 축적 억제 효능이 현저히 뛰어남을 확인할 수 있었다. 이에 따라, 본 발명에 따른 고온처리 녹차 추출물은 일반 녹차 추출물에 비하여 EGCG 함량이 훨씬 낮고 일반 녹차 추출물에는 없는 GCG 성분을 함유하여 우수한 간세포 내 지방 축적 억제능을 나타내는 것을 알 수 있었다.The results are as shown in FIG. 8, and it was confirmed that GCG has a significantly superior inhibitory effect on fat accumulation in liver cells compared to EGCG at the same concentration. Accordingly, it was found that the high-temperature-treated green tea extract according to the present invention has a much lower EGCG content than the normal green tea extract and contains GCG components not found in the general green tea extract, thereby showing excellent ability to inhibit fat accumulation in liver cells.
[실험예 8] 고온처리 녹차 추출물의 진저발리스균 저해 효능 평가 실험[Experimental Example 8] Evaluation experiment for inhibiting ginger ballis bacteria of high temperature treated green tea extract
고온처리 녹차 추출물의 진저발리스균 저해 효능을 평가하기 위하여, 하기 표 5와 같은 성분과 조성비율로 시료를 혼합하여 준비하였다. 각각의 시료는 해당 함량의 100배 stock으로 준비하여 실험에 사용하였으며, 시료 2의 각 성분 함량은 상기 표 3에서의 8종 카테킨의 함량과 동일하다.In order to evaluate the efficacy of high-temperature treated green tea extract to inhibit ginger germs, samples were prepared by mixing the ingredients and composition ratios shown in Table 5 below. Each sample was prepared in stock 100 times the corresponding content and used for the experiment, and the content of each component in Sample 2 is the same as the content of 8 kinds of catechins in Table 3.
에피카테킨 혼합 시료Epicatechin mixed sample 에피카테킨 에피머혼합 시료Epicatechin Epimer Mix Sample 고온처리녹차 추출물(시료 2)High temperature treated green tea extract (sample 2)
EGCGEGCG 421.6 μg/ml421.6 μg / ml -- 5.27 중량% (206.5 μg/ml)5.27% by weight (206.5 μg / ml)
GCGGCG -- 421.6 μg/ml421.6 μg / ml 5.59 중량% (219.1 μg/ml)5.59% by weight (219.1 μg / ml)
EGCEGC 337 μg/ml337 μg / ml -- 3.53 중량% (138.3 μg/ml)3.53% by weight (138.3 μg / ml)
GCGC -- 337 μg/ml337 μg / ml 4.9 중량% (192.0 μg/ml)4.9% by weight (192.0 μg / ml)
ECGECG 104 μg/ml104 μg / ml -- 1.3 중량% (51.0 μg/ml)1.3% by weight (51.0 μg / ml)
CGCG -- 104 μg/ml104 μg / ml 1.3 중량% (51.0 μg/ml)1.3% by weight (51.0 μg / ml)
ECEC 102.8 μg/ml102.8 μg / ml -- 0.95 중량% (37.2 μg/ml)0.95% by weight (37.2 μg / ml)
CC -- 102.8 μg/ml102.8 μg / ml 1.57 중량% (61.5 μg/ml)1.57% by weight (61.5 μg / ml)
8종 카테킨 총합8 kinds of catechins 956.6 μg/ml956.6 μg / ml 956.6 μg/ml956.6 μg / ml 24.41 중량% (956.6 μg/ml)24.41% by weight (956.6 μg / ml)
포피로모나스 진저발리스( Porphyromonas gingivalis)를 비타민 K (1 μg/mL)와 헤민(hemin, 5 μg/mL), 5% 탈섬유소 말 혈액(defibrinated horse blood)을 포함한 Brucella agar (Becton Dickison, Le Pont-de-Claix, France)에 접종하여 37℃에서 혐기적으로 40시간 동안 배양하였다. 배양 후, 원심분리에 의해 집균한 다음, 그 침전부를 인산완충액(PBS)으로 3회 세정하였다. 상기 표 5의 시료 각각에 포피로모나스 진저발리스 100μl를 2x10 5 CFU/mL가 되도록 첨가하고 37℃에서 16시간 이상 배양한 후 탁도를 나타내지 않는 최소저해농도를 측정하여 그 결과를 하기 표 6에 나타내었다.Brucella agar (Becton Dickison, Le) with Porphyromonas gingivalis containing vitamin K (1 μg / mL), hemin (hemin, 5 μg / mL) and 5% defibrinated horse blood Pont-de-Claix, France) and incubated anaerobicly at 37 ° C for 40 hours. After incubation, the cells were collected by centrifugation, and then the precipitates were washed three times with phosphate buffer (PBS). To each sample of Table 5, 100 μl of Porphyromonas ginger ballis was added to be 2 × 10 5 CFU / mL, and after incubation at 37 ° C. for 16 hours or more, the minimum inhibitory concentration that did not exhibit turbidity was measured and the results are shown in Table 6 below. Shown.
최소저해농도 (μg/ml)Minimum inhibitory concentration (μg / ml)
에피카테킨혼합 시료Epicatechin Mix Sample 에피카테킨 에피머혼합 시료Epicatechin Epimer Mix Sample 고온처리녹차 추출물(시료 2)High temperature treated green tea extract (sample 2)
포피로모나스 진저발리스Popiromonas Gingerbread >8000> 8000 20002000 10001000
상기 표 6의 결과로부터, 본 발명에 따른 고온처리 녹차 추출물의 경우, 에피카테킨 4종을 포함하는 시료 및 에피카테킨 에피머 4종을 포함하는 시료에 비하여 포피로모나스 진저발리스균 저해 효능이 현저히 우수한 것을 확인할 수 있었다. 특히, 에피카테킨 4종을 포함하는 시료의 경우 포피로모나스 진저발리스균 저해 효능이 크게 떨어지며, 에피카테킨 에피머 4종을 포함하는 시료의 경우 포피로모나스 진저발리스균 저해 효능을 나타내기는 하나, 본 발명에 따른 시료 2에 비해서는 그 효능이 현저히 떨어지는 것을 확인할 수 있었다.From the results of Table 6, in the case of the high-temperature-treated green tea extract according to the present invention, it was confirmed that the effect of inhibiting the activity of Porphyromonas ginger alevalis is significantly superior to the sample containing 4 epicatechins and the sample containing 4 epicatechin epimers. Could. Particularly, in the case of a sample containing 4 epicatechins, the efficacy of inhibiting the activity of Popyromonas ginger germs is greatly reduced. It was confirmed that the efficacy was significantly lower than that of Sample 2 according to the invention.
위의 실험예 1 및 2의 결과와 종합하면, 본 발명에 따른 고온처리 녹차 추출물은 기존의 일반 녹차 추출물에 비하여 간독성이 낮아 안전성이 높으면서도 포피로모나스 진저발리스균 저해 효능이 뛰어나 우수한 치주염 예방 효능을 나타내는 것을 알 수 있다.Combined with the results of Experimental Examples 1 and 2 above, the high-temperature-treated green tea extract according to the present invention has low liver toxicity compared to the conventional green tea extract, has high safety, and has excellent efficacy in inhibiting Gingervalis bacteria, and has excellent periodontitis prevention efficacy. It can be seen that indicates.
[실험예 9] 고온처리 녹차 추출물의 뇌혈류량 증가 효능 평가 실험[Experimental Example 9] Efficacy test for increasing cerebral blood flow of high-temperature treated green tea extract
근적외선 분광법(Near infrared spectroscopy, NIRS)에 따르면 신경활성에 따른 혈액 내 헤모글로빈의 산화 정도를 반영한 신호를 측정함으로써 뇌혈류량을 비침습적으로 확인할 수 있다.According to Near infrared spectroscopy (NIRS), it is possible to non-invasively check cerebral blood flow by measuring signals reflecting the degree of hemoglobin oxidation in the blood according to nerve activity.
구체적으로, 다 채널 근적외선 분광법 기기인 fNIRS (functional near infrared spectroscopy, NIRSIT, OBELAB)을 이용하여 전두엽에서 헤모글로빈 포화정도를 측정함으로써 뇌 혈류량의 변화를 관찰하였다. 먼저 피험자가 도착하면 연구 참여 동의서를 작성한 다음 전반적 실험 진행에 대해 안내하였다. 기본 활성도 측정을 위해 NIRSIT을 착용한 뒤 3분간 휴식상태를 측정하고, 이 후 본 과제를 시작하여 30초간 휴식 상태 측정 후 총 10문제의 곱셈문제를 풀게 하였다. 과제가 끝난 뒤 시료 2의 고온처리 녹차추출물 또는 대조군 약 (450 mg/2 정)을 충분한 물과 함께 섭취하게 하였다. 총 피험자는 23명 (여성 12명, 남성 11명)으로 맹검 및 무작위로 시험군 (총 12명, 여성 6명, 남성 6명, 나이 32.17±1.84세) 또는 대조군 (총 11명, 여성 6명, 남성 5명, 나이 30.55±1.26세)으로 배정되었다. 섭취 후 45분 동안 휴식 및 흡수 기간을 가진 뒤 다시 NIRSIT을 착용한 이후 3분간 휴식상태를 측정하고, 본 과제를 진행하였다. 30초 간 휴식상태 (Baseline) 측정 후 총 30문제의 곱셈문제 (Math arithmetics, MA)를 풀도록 하였다. 이 때 근적외선 분광법을 이용해 측정된 결과는 low pass filter (LPF) 0.1Hz (LPF(DCT 0.1)), high pass filter (HPF) 0.005 Hz (HPF(DCT 0.005)) 또는 HPF 없이 (HPF X) 분석하여 시간대별 총 헤모글로빈의 양 (도 9) 및 전체 과제 동안의 산화헤모글로빈의 양 (도 10)을 확인하였다. Specifically, changes in brain blood flow were observed by measuring the degree of hemoglobin saturation in the frontal lobe using a multi-channel near infrared spectroscopy instrument, functional near infrared spectroscopy (NIRSIT, OBELAB). First, when the subject arrived, a consent form for study participation was drawn up, and then the overall experiment was guided. To measure the basic activity, the NIRSIT was worn and the resting state was measured for 3 minutes, and then the task was started to measure the resting state for 30 seconds and solve the multiplication problem of a total of 10 questions. After the task was over, the high temperature treated green tea extract of Sample 2 or the control drug (450 mg / 2 tablets) was ingested with sufficient water. A total of 23 subjects (12 women, 11 men) were blinded and randomly tested (12 total, 6 female, 6 male, 32.17 ± 1.84 years of age) or a control group (11 total, 6 female) , 5 males, 30.55 ± 1.26 years of age). After taking a rest and absorption period for 45 minutes after ingestion, the rest state was measured for 3 minutes after wearing NIRSIT again, and this task was performed. After measuring the baseline for 30 seconds, a total of 30 multiplication problems (Math arithmetics, MA) were solved. At this time, the results measured using near-infrared spectroscopy are analyzed by low pass filter (LPF) 0.1 Hz (LPF (DCT 0.1)), high pass filter (HPF) 0.005 Hz (HPF (DCT 0.005)) or without HPF (HPF X). The total amount of hemoglobin over time (FIG. 9) and the amount of hemoglobin oxide during the entire task (FIG. 10) were confirmed.
그 결과는 도 9 및 10과 같으며, 본 발명에 따른 시료 2의 고온처리 녹차 추출물을 복용한 경우, 복용 45분 후 진행된 MA 과제 수행 시 대조군에 비하여 뇌 전두엽 부분의 총 헤모글로빈의 양 및 산화된 헤모글로빈의 양이 현저하게 증가하는 것을 확인할 수 있었다.The results are as shown in FIGS. 9 and 10, and when taking the high temperature treated green tea extract of Sample 2 according to the present invention, the amount of hemoglobin and the oxidized amount of the total hemoglobin in the brain frontal region compared to the control group when performing the MA task conducted 45 minutes after taking It was confirmed that the amount of hemoglobin increased significantly.
위의 실험예 1 및 2의 결과와 종합하면, 본 발명에 따른 고온처리 녹차 추출물은 기존의 일반 녹차 추출물에 비하여 간독성이 낮아 안전성이 높으면서도 뇌 전두엽 부분의 혈류량을 증가시켜 우수한 인지기능 개선 효능을 나타내는 것을 알 수 있다.Combined with the results of Experimental Examples 1 and 2 above, the high-temperature-treated green tea extract according to the present invention has low hepatotoxicity compared to the conventional green tea extract, and has high safety while increasing the blood flow in the frontal lobe of the brain, thereby improving the cognitive function. It shows that it shows.

Claims (14)

  1. 추출물 총 중량을 기준으로, 8 중량% 이하의 에피갈로카테킨(epigallocatechin, EGC)을 포함하는, 간독성이 완화된 녹차 추출물.Green tea extract with reduced hepatotoxicity, comprising up to 8% by weight of epigallocatechin (EGC), based on the total weight of the extract.
  2. 제1항에 있어서, 상기 추출물은 추출물 총 중량을 기준으로 4 내지 15 중량%의 (-)-갈로카테킨 갈레이트((-)-gallocatechin gallate, GCG) 및 4 내지 15 중량%의 (-)-에피갈로카테킨 갈레이트((-)-epigallocatechin gallate, EGCG)를 포함하는, 녹차 추출물.The method of claim 1, wherein the extract is 4 to 15% by weight based on the total weight of the extract (-)-gallocatechin gallate ((-)-gallocatechin gallate, GCG) and 4 to 15% by weight of (-)- Green tea extract comprising epigallocatechin gallate ((-)-epigallocatechin gallate (EGCG)).
  3. 제2항에 있어서, 상기 추출물 내의 (-)-에피갈로카테킨 갈레이트((-)-epigallocatechin gallate, EGCG), 에피갈로카테킨(epigallocatechin, EGC) (-)에피카테킨((-)epicatechin, EC), 에피카테킨 3-O-갈레이트(epicatechin 3-O-gallate, ECG), 갈로카테킨 갈레이트(gallocatechin gallate, GCG), 갈로카테킨(gallocatechin, GC), 카테킨(catechin, C) 및 카테킨 갈레이트(catechin gallate, CG) 의 총 함량은 상기 추출물 총 중량을 기준으로 19 내지 30 중량%인, 녹차 추출물.According to claim 2, (-)-Epigallocatechin gallate ((-)-epigallocatechin gallate, EGCG), Epigallocatechin (EGC) (-) Epicatechin ((-) epicatechin, EC in the extract) ), Epicatechin 3-O-gallate (ECG), gallocatechin gallate (GCG), gallocatechin (GC), catechin (C) and catechin gallate ( The total content of catechin gallate, CG) is 19 to 30% by weight based on the total weight of the extract, green tea extract.
  4. 제1항에 있어서, 상기 추출물은 물 및 C1 내지 C4의 알코올 중 어느 하나 이상에 의해 1회 이상 추출한 추출물인, 녹차 추출물.The green tea extract of claim 1, wherein the extract is an extract extracted one or more times with any one or more of water and C1 to C4 alcohols.
  5. 제1항 내지 제4항 중 어느 한 항의 녹차 추출물을 유효성분으로 포함하는, 조성물.A composition comprising the green tea extract of any one of claims 1 to 4 as an active ingredient.
  6. 제5항에 있어서, 상기 녹차 추출물은 조성물 총 중량 대비 1 내지 100 중량%의 양으로 포함되는, 조성물.The composition of claim 5, wherein the green tea extract is included in an amount of 1 to 100% by weight based on the total weight of the composition.
  7. 제5항에 있어서, 상기 조성물은 건강기능식품 또는 약학 조성물인, 조성물.The composition of claim 5, wherein the composition is a health functional food or pharmaceutical composition.
  8. 제5항에 있어서, 상기 조성물은 간독성 완화용인, 조성물.The composition of claim 5, wherein the composition is for relieving liver toxicity.
  9. 제5항에 있어서, 상기 조성물은 혈행개선용인, 조성물.The composition of claim 5, wherein the composition is for improving blood circulation.
  10. 제9항에 있어서, 상기 조성물은 혈관내피세포에서 일산화질소(NO)를 증가시키는, 조성물.The composition of claim 9, wherein the composition increases nitrogen monoxide (NO) in vascular endothelial cells.
  11. 제5항에 있어서, 상기 조성물은 간 세포 내 지방 축적 억제용인, 조성물.The composition of claim 5, wherein the composition is for inhibiting fat accumulation in liver cells.
  12. 제5항에 있어서, 상기 조성물은 진저발리스균 저해용인, 조성물.The composition of claim 5, wherein the composition is for inhibiting Ginger Valeris.
  13. 제12항에 있어서, 상기 진저발리스균은 포피로모나스 진저발리스( Porphyromonas gingivalis)인, 조성물.13. The composition of claim 12, wherein the ginger germ is Porphyromonas gingivalis .
  14. 제5항에 있어서, 상기 조성물은 뇌혈류량 증가용인, 조성물.The composition of claim 5, wherein the composition is for increasing cerebral blood flow.
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