WO2016182161A1 - Composition containing rubus coreanus extract or fraction thereof as active ingredient for increasing low-density lipoprotein receptor by inhibiting pcsk9 gene expression, and use thereof - Google Patents

Composition containing rubus coreanus extract or fraction thereof as active ingredient for increasing low-density lipoprotein receptor by inhibiting pcsk9 gene expression, and use thereof Download PDF

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WO2016182161A1
WO2016182161A1 PCT/KR2015/013365 KR2015013365W WO2016182161A1 WO 2016182161 A1 WO2016182161 A1 WO 2016182161A1 KR 2015013365 W KR2015013365 W KR 2015013365W WO 2016182161 A1 WO2016182161 A1 WO 2016182161A1
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fraction
active ingredient
extract
bokbunja
expression
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PCT/KR2015/013365
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French (fr)
Korean (ko)
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송광훈
김윤희
이혜원
김영화
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한국 한의학 연구원
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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
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
    • 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)
    • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn

Definitions

  • the present invention relates to a composition for increasing the expression of low-density lipoprotein receptor (LDLR) by suppressing the expression of the PCSK9 gene, containing bokbunja extract or fractions thereof as an active ingredient, in particular increased PCSK9 gene expression by statin compounds To a bokbunja extract or fractions thereof.
  • LDLR low-density lipoprotein receptor
  • Hyperlipidemia is an abnormal increase in lipids such as free cholesterol, cholesterol esters, phospholipids, and triglycerides in the blood. It can come in the form of branches. Hyperlipidemia generally does not show specific symptoms by itself, but excess lipids in the blood cling to the walls of blood vessels to narrow blood vessels and cause atherosclerosis through inflammatory reactions. This can lead to coronary heart disease, cerebrovascular disease, obstruction of peripheral blood vessels, and the like. Excessive blood lipids also accumulate in liver tissue, which can lead to fatty liver.
  • Drugs that reduce the concentration of lipids developed to date include bile acid-binding resins, drugs that lower cholesterol, such as HMG-CoA reductase inhibitors, which are important enzymes for cholesterol biosynthesis, fibrin derivatives, and nicotinic acid. Drugs that lower back triglycerides have been developed.
  • statin compounds inhibit the activity of HMG-CoA reductase, an enzyme that regulates the rate of cholesterol synthesis in the cell, thereby reducing the amount of cholesterol in the cell, thereby reducing the amount of cholesterol secreted into the blood, thereby reducing the concentration of cholesterol in the blood.
  • HMG-CoA reductase an enzyme that regulates the rate of cholesterol synthesis in the cell
  • Statin compounds inhibit the activity of HMG-CoA reductase, an enzyme that regulates the rate of cholesterol synthesis in the cell, thereby reducing the amount of cholesterol in the cell, thereby reducing the amount of cholesterol secreted into the blood, thereby reducing the concentration of cholesterol in the blood.
  • HMG-CoA reductase an enzyme that regulates the rate of cholesterol synthesis in the cell
  • HMG-CoA reductase an enzyme that regulates the rate of cholesterol synthesis in the cell
  • reducing the amount of cholesterol in the cell thereby reducing the amount of cholesterol secreted into the blood
  • statin compound is a very effective drug of hypercholesterolemia
  • clinically doubling the dose of the statin compound does not reduce cholesterol in proportion to the dose, but additionally at a reduced level by the initial dose. It's only about 5-6% less. This is because the statin compound increases PCSK9 as described above.
  • PCSK9 prote convertase subtilisin / kexin type 9
  • LDLR low density lipoprotein receptor
  • a protein that allows LDLR to be degraded Horton, J. et al. 2007 Trends in Biochemical Sciences 32: 71-77.
  • Increasing the expression of the PCSK9 gene causes a rapid increase in blood cholesterol levels due to a decrease in LDLR, and an autosomal dominant familial hypercholesterolemia in patients with gain-of-function mutations that increase PCSK9 activity.
  • the inhibition of the expression of the PCSK9 gene can increase the amount of LDLR and decrease the blood cholesterol level has been revealed as a new therapeutic target of hypercholesterolemia that can significantly lower the incidence of coronary artery disease.
  • the method of inducing the decrease of blood cholesterol level through the regulation of the PCSK9 gene is to inhibit the synthesis of the PCSK9 gene using siRNA, the amount of the PCSK9 protein is reduced by the antibody against PCSK9, or the PCSK9 and LDLR
  • Bokbunja is a medicinal herb made from unripe fruit of the rose family Bokbunja Strawberry. It is used for oil wells, dream tablets, and urinary tracts to boost kidney function. Blackens the hair and makes the skin soft and beautiful.
  • As a pharmacological action anti-inflammatory action, antioxidant action, anti-Helicobacter pylori action has been reported, mast cell activation inhibitory effect, weight control effect, blood lipid component change, antioxidant effect, blood pressure inhibitory effect, antibacterial effect and so on.
  • it is used for kidney function, infertility, vulgaris, well-being dream, and tonic. It is known to clear blood and eyes, protect liver, and have carbohydrate degrading enzyme inhibitory effect. It contains a lot of phosphorus, iron and potassium, especially organic acids and vitamin C, and camphorol and quercetin have been reported as phenolic compounds.
  • Conventional techniques related to bokbunja extract or fractions thereof include a technique related to a health functional food comprising bokbunja having blood lipid improving activity in Korean Patent No. 1320371, and the extract of Bokbunja immature fruit in Korean Patent Publication No. 2014-0031080
  • a composition for the prevention and treatment of metabolic syndrome disease, which contains as an active ingredient, is disclosed in Korean Patent No. 1401253 for the inhibition of the statin compound from increasing the expression of the PCSK9 gene.
  • compositions for inducing low density lipoprotein receptor increase through containing a bokbunja extract of the present invention or a fraction thereof as an active ingredient, a composition for reducing the expression of the PCSK9 gene or increasing the low density lipoprotein receptor (LDLR) No technology has been reported.
  • LDLR low density lipoprotein receptor
  • the present invention is derived from the above requirements, the present invention contains a bokbunja extract or a fraction thereof as an active ingredient, to provide a composition for reducing the expression of the PCSK9 gene, or to increase the low density lipoprotein receptor (LDLR)
  • the composition of the present invention was completed by confirming that the influx of low density lipoprotein (LDL) into hepatocytes was increased by increasing the low density lipoprotein receptor (LDLR) through inhibition of expression of the PCSK9 gene.
  • the present invention contains a bokbunja extract or a fraction thereof as an active ingredient, and provides a composition for reducing the expression of the PCSK9 gene, or to increase the low density lipoprotein receptor (LDLR).
  • a bokbunja extract or a fraction thereof as an active ingredient, and provides a composition for reducing the expression of the PCSK9 gene, or to increase the low density lipoprotein receptor (LDLR).
  • LDLR low density lipoprotein receptor
  • the present invention contains a bokbunja extract or a fraction thereof as an active ingredient, and provides a composition for reducing the expression of the increased PCSK9 gene by the statin-based compound, or to increase the low density lipoprotein receptor (LDLR).
  • a bokbunja extract or a fraction thereof as an active ingredient, and provides a composition for reducing the expression of the increased PCSK9 gene by the statin-based compound, or to increase the low density lipoprotein receptor (LDLR).
  • LDLR low density lipoprotein receptor
  • the present invention contains a bokbunja extract or a fraction thereof as an active ingredient, and provides a dietary supplement composition for reducing the expression of the PCSK9 gene or increasing the low density lipoprotein receptor (LDLR).
  • LDLR low density lipoprotein receptor
  • the present invention contains a statin-based compound and bokbunja extract or fractions thereof as an active ingredient, the prevention or treatment of hypercholesterolemia characterized by reducing the expression of the PCSK9 gene or increasing the low density lipoprotein receptor (LDLR) It provides a pharmaceutical composition.
  • a statin-based compound and bokbunja extract or fractions thereof as an active ingredient, the prevention or treatment of hypercholesterolemia characterized by reducing the expression of the PCSK9 gene or increasing the low density lipoprotein receptor (LDLR) It provides a pharmaceutical composition.
  • LDLR low density lipoprotein receptor
  • the present invention also provides a veterinary composition for preventing or treating hypercholesterolemia in animals other than humans, including bokbunja extract or a fraction thereof as an active ingredient.
  • the present invention also provides a feed additive for the prevention or improvement of hypercholesterolemia, including the bokbunja extract or a fraction thereof as an active ingredient.
  • the present invention contains a bokbunja extract or a fraction thereof as an active ingredient, and relates to a composition for reducing the expression of the PCSK9 gene or increasing the low density lipoprotein receptor (LDLR).
  • LDLR low density lipoprotein receptor
  • composition of the present invention is effective in inhibiting the expression of the increased PCSK9 gene by statin compounds and increasing the low density lipoprotein receptor, and can be utilized for the prevention, improvement or treatment of metabolic diseases by lowering blood levels of cholesterol. have.
  • FIG. 1 is a schematic diagram of a luciferase assay system.
  • (A) is a transient transduction into HepG2 cells after cloning the promoter of the human PCSK9 gene in the firefly Luciferase reporter (Firefly Luciferase reporter) transfection), and treated with Berberine (20 ⁇ M) known to inhibit the expression of the PCSK9 gene and chlorophyll hydrothermal extract (Coptis chinensis, 200 ⁇ g / ml) containing berberine, followed by luciferase assay. It was confirmed that the aze reporter activity is working normally.
  • (B) confirms that promoter activity is increased by co-transfection of the SREBP-1c expression vector, which is known to regulate human PCSK9 gene expression, with the reporter, thereby the luciferase reporter activity used in the present invention is operating normally. Check the drawing.
  • Figure 3 transient transduction of the promoter (about 1.5 kb) reporter of the human PCSK9 gene into HepG2 cells 24 hours later, the hydrothermal extract Chinese herbal medicine library (18 sweets) was treated for 24 hours at a concentration of 200 ⁇ g / ml And luciferase assay.
  • Berberin (20 ⁇ M) was used as a positive control, and was calibrated based on the negative control (Con) treated with the vehicle used to dissolve the material.
  • FIG. 5 is a diagram confirming the mRNA expression level of the PCSK9 gene in HepG2 cells after 1-200 ⁇ g / ml bokbunja hydrothermal extract was treated with 10 ⁇ M simvastatin for 24 hours or alone.
  • FIG. 6 mRNA of the low density lipoprotein receptor (LDLR) after HepG2 cells were treated with 1 to 200 ⁇ g / ml bokbunja hydrothermal extract for 24 hours with or without 10 ⁇ M simvastatin or alone. It is a figure which confirmed the expression amount.
  • LDLR low density lipoprotein receptor
  • FIG. 8 is a view showing the results of analysis of changes in LDL uptake of cells when bokbunja extract alone or in combination with statin compounds.
  • $$ means that the treatment of Bokbunja extract alone was statistically significant compared to the negative control treated with vehicle used to dissolve the material, and $$ means p value is less than 0.01.
  • FIG. 10 shows HepG2 cells, hexane fraction (Hx), chloroform fraction (CHCl 3 ), ethyl acetate fraction (EtAc), butanol fraction (BuOH), water fraction (H 2 O) and 100 ⁇ g / ml bokbunja hydrothermal extract Bokbunja hydrothermal extract (BRE) after treatment with 10 ⁇ M simvastatin (simvastatin) for 24 hours, it is a diagram confirming the mRNA expression level of the PCSK9 gene.
  • CON is a negative control group treated with vehicle instead of bokbunja hydrothermal extract or fractions thereof.
  • Bokbunja hydrothermal extract was treated with 10 ⁇ M simvastatin for 24 hours, and then the amount of expression of PCSK9 and LDLR protein was confirmed by Western blot.
  • CON is a negative control, treated with vehicle instead of bokbunja extract or fractions thereof,-is treated with only 10 ⁇ M simvastatin, ⁇ -actin is a loading control.
  • the present invention contains a bokbunja extract or a fraction thereof as an active ingredient, and relates to a composition for reducing the expression of the PCSK9 gene or increasing the low density lipoprotein receptor (LDLR). Since the composition increases the expression of low density lipoprotein receptor (LDLR) by reducing the amount of PCSK9 and consequently lowers the blood cholesterol level, the composition of metabolic diseases such as obesity, type 2 diabetes, hypertension, cardiovascular disease or hyperlipidemia It can be utilized as a health functional food composition for prevention or improvement.
  • LDLR low density lipoprotein receptor
  • the present invention contains a bokbunja extract or a fraction thereof as an active ingredient, to reduce the expression of the PCSK9 gene increased by the statin-based compound, or to increase the low density lipoprotein receptor (LDLR) reduced by the statin-based compound It relates to a composition.
  • a bokbunja extract or a fraction thereof as an active ingredient, to reduce the expression of the PCSK9 gene increased by the statin-based compound, or to increase the low density lipoprotein receptor (LDLR) reduced by the statin-based compound It relates to a composition.
  • LDLR low density lipoprotein receptor
  • the statin-based compound is not limited as long as it is a statin-based compound, but preferred examples thereof include simvastatin, atorvastatin, cerivastatin, cerivastatin, lovastatin, pitavastatin, and rosuvastatin. (rosuvastatin), pravastatin, fluvastatin and fluvastatin and mevastatin (mevastatin) may be one or more selected.
  • the bokbunja extract may use a conventional extraction solvent known in the art, but one example of a preferred extraction solvent is one or more selected from lower alcohols having 1 to 4 carbon atoms, water and mixtures thereof, more preferably water Or ethanol, even more preferably water.
  • a preferred extraction solvent is one or more selected from lower alcohols having 1 to 4 carbon atoms, water and mixtures thereof, more preferably water Or ethanol, even more preferably water.
  • water is added in a volume of 5 to 10 times the weight of the bokbunja, and extracted by heating at 90 to 110 ° C. for 2 to 3 hours.
  • the extraction solution is preferably filtered through filter paper and then lyophilized, but not always limited thereto.
  • 80% ethanol is added in a volume of 5 to 10 times the weight of bokbunja and extracted by dipping for 22 to 24 hours at a room temperature of 20 to 25 ° C.
  • the extraction solution is preferably filtered through filter paper and then lyophilized, but not always limited thereto.
  • the fraction of the bokbunja hydrothermal extract is preferably any one selected from hexane fraction (Hx), chloroform fraction (CHCl 3 ), ethyl acetate fraction (EtAc), butanol fraction (BuOH) and water fraction (H 2 O), more Preferably it is, but is not limited to, a chloroform fraction or an ethyl acetate fraction.
  • the present invention contains a bokbunja extract or a fraction thereof as an active ingredient, and relates to a dietary supplement composition for reducing the expression of the PCSK9 gene or increasing the low density lipoprotein receptor (LDLR).
  • LDLR low density lipoprotein receptor
  • the dietary supplement is not particularly limited, and may be any form of dietary supplement, dietary supplement, nutritional supplement, pharmafood, health food, nutraceutical, designer food, or food additive.
  • dietary supplement preferably, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, drinks, tea, drinks, alcoholic drinks and vitamin complexes Can be.
  • the method of addition is suitably used according to a conventional method, and the addition amount can be suitably determined according to a use purpose.
  • the composition may further include various components other than the active ingredient, and these various components are not particularly limited, but preferably various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and Salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks in order to add palatability or functionality, and these ingredients may be used independently or in combination.
  • the present invention also relates to a pharmaceutical composition for preventing or treating hypercholesterolemia, which comprises a statin-based compound and bokbunja extract or a fraction thereof as an active ingredient.
  • the bokbunja extract or fractions thereof are characterized by inhibiting the expression of the PCSK9 gene or increasing the expression of low density lipoprotein receptor (LDLR), and lowering the blood levels of cholesterol by inhibiting the expression of the PCSK9 gene or increasing the expression of the lipoprotein receptor, It can be used as a pharmaceutical composition for the prevention or treatment of metabolic diseases such as obesity, type 2 diabetes, hypertension, cardiovascular disease or hyperlipidemia.
  • metabolic diseases such as obesity, type 2 diabetes, hypertension, cardiovascular disease or hyperlipidemia.
  • the bokbunja extract may use a conventional extraction solvent known in the art, but one example of a preferred extraction solvent is one or more selected from lower alcohols having 1 to 4 carbon atoms, water and mixtures thereof, more preferably water Or ethanol, even more preferably water.
  • a preferred extraction solvent is one or more selected from lower alcohols having 1 to 4 carbon atoms, water and mixtures thereof, more preferably water Or ethanol, even more preferably water.
  • water is added in a volume of 5 to 10 times the weight of the bokbunja, and extracted by heating at 90 to 110 ° C. for 2 to 3 hours.
  • the extraction solution is preferably filtered through filter paper and then lyophilized, but not always limited thereto.
  • 80% ethanol is added in a volume of 5 to 10 times the weight of bokbunja and extracted by dipping for 22 to 24 hours at a room temperature of 20 to 25 ° C.
  • the extraction solution is preferably filtered through filter paper and then lyophilized, but not always limited thereto.
  • the fraction of the bokbunja extract is preferably any one selected from hexane fraction (Hx), chloroform fraction (CHCl 3 ), ethyl acetate fraction (EtAc), butanol fraction (BuOH) and water fraction (H 2 O), more preferably.
  • Hx hexane fraction
  • CHCl 3 chloroform fraction
  • EtAc ethyl acetate fraction
  • BuOH butanol fraction
  • water fraction H 2 O
  • it is, but is not limited to, a chloroform fraction or an ethyl acetate fraction.
  • the statin-based compound is not limited as long as it is a statin-based compound, but preferred examples thereof include simvastatin, atorvastatin, cerivastatin, cerivastatin, lovastatin, pitavastatin, and rosuvastatin. (rosuvastatin), pravastatin, fluvastatin and fluvastatin and mevastatin (mevastatin) may be one or more selected.
  • the pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are conventionally used in the preparation, saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, lactose, dex Straw, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, syrups, methyl cellulose, methylhydroxybenzoate, propyl Hydroxybenzoate, talc, magnesium stearate and mineral oil, and the like.
  • the pharmaceutical composition of the present invention may further include antioxidants, buffers, bacteriostatic agents, diluents, surfactants, binders, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents or preservatives, etc., in addition to the above components.
  • the pharmaceutical composition of the present invention may be administered orally or parenterally, and preferably applied by oral administration.
  • Suitable dosages of the pharmaceutical compositions of the present invention may be prescribed in various ways depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be.
  • compositions of the present invention may be prepared in unit dosage form or in multi-dose form by formulating with a pharmaceutically acceptable carrier or excipient according to methods which can be easily carried out by those skilled in the art. It may be prepared by incorporation into a container.
  • the formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media or in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise a dispersing or stabilizing agent.
  • the present invention also relates to a veterinary composition for preventing or treating hypercholesterolemia in animals other than humans, including bokbunja extract or fractions thereof as an active ingredient.
  • Veterinary compositions comprising the bokbunja extract or fractions thereof of the present invention may further comprise suitable excipients and diluents according to conventional methods.
  • Excipients and diluents that may be included in veterinary compositions comprising the bokbunja extract of the present invention or fractions thereof include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate , Gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, cetanol, stearyl alcohol , Liquid paraffin, sorbitan monostearate, polysorbate 60, methyl paraben, propyl paraben and mineral oil.
  • the veterinary composition comprising the bokbunja extract or fractions thereof according to the present invention may further comprise a filler, an anticoagulant, a lubricant, a humectant, a spice, an emulsifier, a preservative, and the like.
  • a filler an anticoagulant, a lubricant, a humectant, a spice, an emulsifier, a preservative, and the like.
  • the formulation can be in the form of powders, granules, tablets, capsules, suspensions, emulsions, solutions, syrups, Aerosols, soft or hard gelatin capsules, suppositories, sterile injectable solutions, sterile external preparations, and the like.
  • the veterinary composition according to the present invention may vary depending on the age, sex and weight of the animal, but may be administered in an amount of 0.1 to 100 mg / kg once or several times a day, and the dosage may be a route of administration, a degree of disease, It may increase or decrease depending on gender, weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.
  • the present invention also relates to a feed additive for the prevention or improvement of hypercholesterolemia comprising bokbunja extract or fractions thereof as an active ingredient.
  • the feed additive may be in the form of a high concentrate, powder or granules containing 20 to 90% by weight of bokbunja extract or fractions thereof.
  • the feed additive of the present invention includes organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate and polyphosphate (polyphosphate), polyphenols, catechins and alpha. It may further include one or more selected from natural antioxidants such as tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid.
  • organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate and polyphosphate (polyphosphate), polyphenols, catechins and alpha. It may further include one or more selected from natural antioxidants such as tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid.
  • Animal feed additives containing bokbunja extract or fractions thereof of the present invention and feed containing them are amino acids, inorganic salts, vitamins, antibiotics, antibacterial substances, antioxidants, antifungal enzymes, digestion and absorption enhancers, growth promoters, It can be used with substances such as disease prevention agents.
  • the feed additive may be administered to the animal alone in combination with other feed additives in an edible carrier.
  • the feed additives can be easily administered as a top dressing or directly mixed into animal feed or separately from the feed, in a separate oral formulation, by injection or transdermal or in combination with other ingredients.
  • single or divided daily dosages can be used as is well known in the art.
  • dosage forms of the extracts as is well known in the art, can be prepared in immediate release or sustained release formulations in combination with non-toxic pharmaceutically acceptable edible carriers.
  • Such edible carriers may be solid or liquid, for example corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol.
  • the dosage form of the extract may be tablets, capsules, powders, torokies or sugar-containing tablets or top dressings in microdisperse form.
  • a liquid carrier it may be in the form of soft gelatin capsules or syrups or liquid suspensions, emulsions or solutions.
  • the dosage form may contain auxiliaries such as preservatives, stabilizers, wetting or emulsifying agents, solution promoters and the like.
  • the animal feed in which the bokbunja extract or fractions thereof is included as a feed additive may be any protein-containing organic cereal meal commonly used to meet the dietary needs of the animal. Such protein-containing flours typically consist mainly of corn, soy flour, or corn / bean flour mixtures.
  • the feed additive may be used by adding to the animal feed by dipping, spraying or mixing.
  • the veterinary composition or feed additive of the present invention can be applied to the diet of a companion animal.
  • Liver cancer cell line (HepG2) was purchased from the American Type Culture Collection (ATCC). Liver cancer cell line (HepG2) was incubated with DMEM / F-12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) medium containing 10% FBS, 1% penicillin-streptomycin. The cells were cultured in a 37 ° C., 5% CO 2 incubator, and the cultured cells were tested by dispensing a predetermined number of cells (1 ⁇ 10 5 cells / 500 ⁇ l well) in a 24-well plate. After dispensing the cells in a 24-well plate, the cells were completely attached to the 24 wells and used to form when the cells reached a confluence of 75%.
  • DMEM / F-12 Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12
  • the promoter of the human PCSK9 gene (approximately 1.5 kb) was cloned into the firefly luciferase reporter, followed by transient transfection into HepG2 cells, and 20 ⁇ M of berberine and 200 ⁇ g / ml of berberine. Treatment was carried out for 24 hours by treating the containing hot water extract (Coptis chinensis). Thereafter, the degree of emission of firefly luciferase was measured and the results are shown in FIG. 2 (A).
  • Luciferase assays were performed to select herbal extracts that can reduce the expression level of the PCSK9 gene among 18 herbal library.
  • Luciferase reporter containing a human PCSK9 promoter (about 1.5 kb) was transfected into HepG2 cells, treated with 18 kinds of herbal extracts of 200 ⁇ g / ml and incubated overnight. Then, the herbal extracts were selected to reduce the expression of the PCSK9 gene by measuring the degree of firefly luciferase luminescence. 3 and 4 show that the selected Chinese herbal extract of # 24 effectively reduces the expression level of the PCSK9 gene in both hot water and ethanol extract.
  • the herbal extract of # 24 disclosed in FIGS. 3 and 4 was identified as a bokbunja extract.
  • RNA and 200 ⁇ g / ml bokbunja extracts were treated alone or 10 ⁇ M simvastatin and 0, 1, 10, 50, 100 and 200 ⁇ g / ml bokbunja extract or berberine (20 ⁇ M) and incubated for 24 hours. Then, total RNA is separated from the cells, cDNA is synthesized using Reverse Transcription Polymerase Chain Reaction (RT-PCR), and PCSK9 is obtained through real-time PCR using taqman probe. The changes in the expression levels of, LDLR and HMG-CoA reductase genes were analyzed.
  • RT-PCR Reverse Transcription Polymerase Chain Reaction
  • Example 2 From the results of Examples 4 and 5, it was confirmed that the effect of inhibiting the expression of PCSK9 and increasing the LDLR expression of Example 2 provided in the present invention, whether this effect affects the change in the level of uptake for extracellular LDL Fluorescently-labeled LDL (fluorescently-labeled LDL) was used to identify changes in LDL uptake of cells when treated with bokbunja extract alone or in combination with statin compounds.
  • the solvent fractionation method uses a partition between solvents that are not mixed with each other.
  • Examples of typical partitioning methods include water and hexane, ethyl acetate, butanol, water, and chloroform. It is a fractionation method which consists of.
  • n-hexane, chloroform, ethyl acetate, butanol and the like are added to the bokbunja hydrothermal extract in the same amount as water, and then fractionated systematically. Concentration under reduced pressure for each solvent.
  • Example 7 Each fraction obtained in Example 7 was used to evaluate the screening efficacy of PCSK9 promoter activity.
  • N-hexane (Hx) fraction, chloroform (CHCl 3 ) fraction, ethyl acetate (EtAc) fraction, butanol (BuOH) fraction and water fraction (H 2 O) of bokbunja hydrothermal extract of the present invention were used for human PCSK9 promoter activity. The effect was performed on a luciferase reporter assay.
  • fractions of the bokbunja hydrothermal extract and statin compounds were prepared using real-time PCR using Taqman-probe. Changes in the PCSK9 gene when combined were analyzed.
  • bokbunja hydrothermal extract (BRE) was used as a positive control, and it was confirmed that the combination of 10 ⁇ M simvastatin and bokbunja hydrothermal extract, CHCl 3 fraction and EtAc fraction was effective.
  • PCSK9 protein was increased by the treatment of 10 ⁇ M simvastatin, and a combination of simvastatin and chloroform fraction (CHCl 3 ), ethyl acetate fraction (EtAc) and water fraction (H 2 O) of the bokbunja hydrothermal extract of the present invention was used.
  • CHCl 3 simvastatin and chloroform fraction
  • EtAc ethyl acetate fraction
  • H 2 O water fraction

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Abstract

The present invention relates to a composition containing Rubus coreanus extract or a fraction thereof as an active ingredient for decreasing the PCSK9 gene expression increased by a statin-based compound or for increasing a low-density lipoprotein receptor (LDLR) decreased by the statin-based compound. The composition of the present invention can lower blood cholesterol level by improving the expression of a low-density lipoprotein receptor by means of inhibition of PCSK9 gene expression. In addition, by lowering the amount of cholesterol in blood, the composition can be utilized in prevention, alleviation or treatment of metabolic diseases such as obesity, diabetes mellitus type 2, hypertension, cardiovascular diseases and hyperlipidemia.

Description

복분자 추출물 또는 이의 분획물을 유효성분으로 함유하는 PCSK9 유전자의 발현 억제를 통한 저밀도 지단백 수용체 증가용 조성물 및 이의 용도Composition for increasing low density lipoprotein receptor through inhibition of expression of PCSK9 gene containing bokbunja extract or fractions thereof as an active ingredient and use thereof
본 발명은 복분자 추출물 또는 이의 분획물을 유효성분으로 함유하며, PCSK9 유전자의 발현 억제를 통한 저밀도 지단백 수용체(LDLR)의 발현을 증가시키기 위한 조성물에 관한 것으로, 특히 스타틴계 화합물에 의해 증가된 PCSK9 유전자 발현을 복분자 추출물 또는 이의 분획물에 의해 감소시키는 것을 특징으로 하는 조성물에 관한 것이다.The present invention relates to a composition for increasing the expression of low-density lipoprotein receptor (LDLR) by suppressing the expression of the PCSK9 gene, containing bokbunja extract or fractions thereof as an active ingredient, in particular increased PCSK9 gene expression by statin compounds To a bokbunja extract or fractions thereof.
경제 성장과 식생활의 서구화로 인하여 음식물에서 얻는 지방의 섭취량이 증가하고 있으며, 운동부족 등으로 인한 고지혈증, 당뇨병, 고혈압, 동맥 경화증 및 지방간과 같은 대사성 질환이 증가하고 있는 추세이다.Due to economic growth and westernization of diet, the intake of fat from food is increasing, and metabolic diseases such as hyperlipidemia, diabetes, hypertension, atherosclerosis and fatty liver are increasing due to lack of exercise.
고지혈증(Hyperlipidemia)은 혈액 내에 유리 콜레스테롤, 콜레스테롤 에스테르, 인지질, 중성 지방 등의 지방질이 비정상적으로 증가한 상태로, 고콜레스테롤혈증, 고중성지방혈증 및 조합 고지혈증(고콜레스테롤혈증 및 고중성지방혈증)의 3가지 형태로 나타날 수 있다. 고지혈증은 일반적으로 그 자체가 특정 증상을 나타내는 것은 아니나, 혈액 내에 과다한 지질은 혈관 벽에 달라붙어 혈관을 좁히며, 염증 반응을 통한 동맥경화(Atherosclerosis)를 일으킨다. 이로 인해 관상동맥 심장 질환이나 뇌혈관 질환, 말초 혈관의 폐쇄 등이 발생할 수 있다. 또한 과다한 혈액 내 지질은 간 조직에 축적되게 되며 이로 인해 지방간(Fatty liver)이 유발될 수 있다.Hyperlipidemia is an abnormal increase in lipids such as free cholesterol, cholesterol esters, phospholipids, and triglycerides in the blood. It can come in the form of branches. Hyperlipidemia generally does not show specific symptoms by itself, but excess lipids in the blood cling to the walls of blood vessels to narrow blood vessels and cause atherosclerosis through inflammatory reactions. This can lead to coronary heart disease, cerebrovascular disease, obstruction of peripheral blood vessels, and the like. Excessive blood lipids also accumulate in liver tissue, which can lead to fatty liver.
현재까지 혈중 지질 농도를 감소시키는 방법으로는 콜레스테롤이나 지방이 많이 함유되어 있는 음식물의 섭취를 줄이는 식이요법, 운동 요법 및 약물 요법 등이 사용되고 있다. 하지만, 식이 요법이나 운동 요법은 엄격한 관리 및 실시가 곤란하며, 그 치료 효과에 있어서도 한계가 있다. 현재까지 개발된 지질 농도를 감소시키는 약물로는 담즙산 결합수지, 콜레스테롤 생합성에 있어서 중요한 효소인 HMG-CoA 환원효소 억제제(HMG-CoA reductase inhibitors)와 같이 콜레스테롤의 양을 낮추는 약물, 피브린산 유도체, 니코틴산 등의 중성지방을 낮추는 약제들이 개발되었다. Until now, the method of reducing blood lipid levels has been used to reduce the intake of foods containing a lot of cholesterol or fat, diet, exercise therapy and drug therapy. However, diet and exercise therapy are difficult to carry out strict management and implementation, and there is a limit in the therapeutic effect. Drugs that reduce the concentration of lipids developed to date include bile acid-binding resins, drugs that lower cholesterol, such as HMG-CoA reductase inhibitors, which are important enzymes for cholesterol biosynthesis, fibrin derivatives, and nicotinic acid. Drugs that lower back triglycerides have been developed.
그 중에서 현재, 고지혈증 치료제로서 가장 흔히 사용되는 약물은 스타틴(statin) 계열의 화합물이다(Goldstein, J. L. and Brown,　 M. S. 2009 Arterioscler Thromb Vasc Biol 29: 431-438). 스타틴계 화합물은 세포 내 콜레스테롤 합성 과정의 속도를 조절하는 효소인 HMG-CoA 환원효소(reductase)의 활성을 억제하여 세포 내 콜레스테롤의 양을 감소시킴으로써 혈중으로 분비되는 콜레스테롤의 양이 줄어들어 혈중 콜레스테롤의 농도를 낮춘다. 스타틴계 화합물이 저밀도 지단백 수용체(low density lipoprotein receptor)의 양을 증가시키는 것으로 알려져 있지만, 반대로 PCSK9 유전자의 발현을 전사 수준에서 증가시키는 작용도 한다(Janice Mayne et al. Lipids in health and Disease 2008, 7:22).Among them, the most commonly used drugs for treating hyperlipidemia are statin compounds (Goldstein, J. L. and Brown, M. S. 2009 Arterioscler Thromb Vasc Biol 29: 431-438). Statin compounds inhibit the activity of HMG-CoA reductase, an enzyme that regulates the rate of cholesterol synthesis in the cell, thereby reducing the amount of cholesterol in the cell, thereby reducing the amount of cholesterol secreted into the blood, thereby reducing the concentration of cholesterol in the blood. Lowers. Statin compounds are known to increase the amount of low density lipoprotein receptors, but they also increase the expression of the PCSK9 gene at the transcription level (Janice Mayne et al. Lipids in health and Disease 2008, 7 : 22).
즉, 스타틴계 화합물이 매우 효과적인 고콜레스테롤혈증의 약물이기는 하지만, 임상적으로 상기 스타틴계 화합물의 용량을 두 배 증가시켜도 용량에 비례하여 콜레스테롤을 감소시키지 못하고, 최초 용량에 의하여 감소된 정도에서 추가로 약 5~6% 더 감소하는 정도에 그친다. 그러한 이유가 스타틴계 화합물이 상기한 바와 같이 PCSK9을 증가시키기 때문이다. That is, although a statin compound is a very effective drug of hypercholesterolemia, clinically doubling the dose of the statin compound does not reduce cholesterol in proportion to the dose, but additionally at a reduced level by the initial dose. It's only about 5-6% less. This is because the statin compound increases PCSK9 as described above.
한편, PCSK9(proprotein convertase subtilisin/kexin type 9)은 세포막에 존재하는 저밀도 지단백 수용체(low density lipoprotein receptor; LDLR)의 EGF-A 도메인과 결합하여 세포 내로 이동한 후, 세포 내의 라이소좀(lysosome)에서 LDLR가 분해될 수 있도록 하는 단백질이다(Horton, J. et al. 2007 Trends in Biochemical Sciences 32: 71-77). PCSK9 유전자의 발현이 증가할 경우, LDLR이 감소하여 혈중 콜레스테롤의 농도가 급격히 증가하며, PCSK9 활성이 증가하는 획득 형질 돌연변이(gain-of-function mutation)가 있는 환자는 상염색체 우성 가족성 과콜레스테롤혈증(autosomal dominant familial hypercholesterolemia)이 발병하고(Abifadel, M., et al. 2003 Nat. Genet. 34: 154-156), 반대로 PCSK9 유전자의 유전자 기능이 상실된 결실 기능 돌연변이(loss-of-function mutation)가 있는 사람에서 혈중 LDL-콜레스테롤의 농도가 정상인에 비하여 감소되어 있으며 관상동맥 질환의 발병 확률이 매우 낮은 사실이 보고되었다(Cohen, J., et al. 2005. Nat. Genet. 37: 161-165). 또한, PCSK9가 결실된 마우스에서 LDLR의 발현 증가가 나타나며, 혈중 LDL 콜레스테롤의 농도가 감소됨이 보고되었다 (Rashid, S., et al. 2005 Proc. Natl. Acad. Sci. U.S.A. 102: 5374-5379). 따라서 PCSK9 유전자의 발현을 억제할 경우 LDLR의 양을 증가시킬 수 있으며, 혈중 콜레스테롤 농도를 감소시키게 되어 관상동맥질환의 발병률을 현저히 낮출 수 있는 고콜레스테롤혈증의 새로운 치료 표적으로 밝혀지고 있다.On the other hand, PCSK9 (proprotein convertase subtilisin / kexin type 9) binds to the EGF-A domain of the low density lipoprotein receptor (LDLR) present in the cell membrane, moves into the cell, and then in the lysosome in the cell. A protein that allows LDLR to be degraded (Horton, J. et al. 2007 Trends in Biochemical Sciences 32: 71-77). Increasing the expression of the PCSK9 gene causes a rapid increase in blood cholesterol levels due to a decrease in LDLR, and an autosomal dominant familial hypercholesterolemia in patients with gain-of-function mutations that increase PCSK9 activity. (autosomal dominant familial hypercholesterolemia) (Abifadel, M., et al. 2003 Nat. Genet. 34: 154-156), and conversely, loss-of-function mutations in which the gene function of the PCSK9 gene is lost. In humans, the concentration of LDL-cholesterol in the blood was reduced compared to normal persons and the incidence of coronary artery disease was reported to be very low (Cohen, J., et al. 2005. Nat. Genet. 37: 161-165). . In addition, increased expression of LDLR is observed in mice lacking PCSK9, and a decrease in the concentration of LDL cholesterol in blood has been reported (Rashid, S., et al. 2005 Proc. Natl. Acad. Sci. USA 102: 5374-5379). . Therefore, the inhibition of the expression of the PCSK9 gene can increase the amount of LDLR and decrease the blood cholesterol level has been revealed as a new therapeutic target of hypercholesterolemia that can significantly lower the incidence of coronary artery disease.
현재까지 PCSK9 유전자의 조절을 통하여 혈중 콜레스테롤 농도의 감소를 유도하는 방법으로는 siRNA를 이용하여 PCSK9 유전자의 합성을 억제하는 방법, PCSK9에 대한 항체를 이용하여 PCSK9 단백질의 양을 감소시키거나 PCSK9과 LDLR간의 결합을 방해하는 펩티드를 이용하여 PCSK9 유전자의 활성을 억제하는 방법 및 소분자 화합물(small compound)을 이용하여 PCSK9 유전자의 활성을 억제하거나 발현 양의 감소를 유도하는 방법이 있었다.Until now, the method of inducing the decrease of blood cholesterol level through the regulation of the PCSK9 gene is to inhibit the synthesis of the PCSK9 gene using siRNA, the amount of the PCSK9 protein is reduced by the antibody against PCSK9, or the PCSK9 and LDLR There is a method of inhibiting the activity of the PCSK9 gene using a peptide that interferes with the binding of the liver and a method of inhibiting the activity of the PCSK9 gene or reducing the expression amount using a small compound.
한편, 복분자는 장미과의 복분자 딸기의 채 익지 않은 열매로 만든 약재로서 신(腎)기능을 북돋아 유정(遺精), 몽정(夢精), 유뇨(遺尿) 등에 사용하며, 시력약화에 쓰고 몸을 가볍게 하고 머리를 검게 하며 살결을 부드럽고 아름답게 한다. 약리작용으로 항염작용, 항산화작용, 항헬리코박터 파이로리 작용이 보고되고 있으며, 비만세포 활성화 억제효과, 체중 조절 효과, 혈중지질성분 변화에 효과, 항산화효과, 혈압저해효과, 항균효과 등이 보고되었다. 한방에서는 신장기능, 불임증, 음위증, 유정몽설, 강장제로 쓰였으며 혈액과 눈을 맑게 해주며, 간을 보호하는 효능이 있다고 알려져 있으며, 탄수화물 분해 효소저해작용이 다소 있고, 영양성분으로는 무기질의 인과 철, 칼륨을 많이 함유하고 있고 특히 유기산과 비타민C가 많이 포함되어 있으며, 페놀 화합물로 캠프페롤(kaempferol), 케르세틴(quercetin) 등이 보고되었다. On the other hand, Bokbunja is a medicinal herb made from unripe fruit of the rose family Bokbunja Strawberry. It is used for oil wells, dream tablets, and urinary tracts to boost kidney function. Blackens the hair and makes the skin soft and beautiful. As a pharmacological action, anti-inflammatory action, antioxidant action, anti-Helicobacter pylori action has been reported, mast cell activation inhibitory effect, weight control effect, blood lipid component change, antioxidant effect, blood pressure inhibitory effect, antibacterial effect and so on. In oriental medicine, it is used for kidney function, infertility, vulgaris, well-being dream, and tonic. It is known to clear blood and eyes, protect liver, and have carbohydrate degrading enzyme inhibitory effect. It contains a lot of phosphorus, iron and potassium, especially organic acids and vitamin C, and camphorol and quercetin have been reported as phenolic compounds.
복분자 추출물 또는 이의 분획물에 관련된 종래 기술로는 한국등록특허 제1320371호에 혈중지질개선 활성을 지니는 복분자를 포함하는 건강기능식품에 관한 기술이 있으며, 한국공개특허 제2014-0031080호에 복분자 미숙과 추출물을 유효성분으로 함유하는 대사증후군 질환의 예방 및 치료용 조성물에 관해 개시되어 있고, 스타틴계 화합물이 PCSK9 유전자의 발현을 증가시키는 것을 억제하는 것에 관한 기술로서 한국등록특허 제1401253호에 PCSK9 발현 억제를 통한 저밀도 지단백 수용체 증가 유도용 조성물이 개시되어 있으나, 본 발명의 복분자 추출물 또는 이의 분획물을 유효성분으로 함유하며, PCSK9 유전자의 발현을 감소시키거나, 저밀도 지단백 수용체(LDLR)를 증가시키기 위한 조성물에 관한 기술은 보고된 바 없다. Conventional techniques related to bokbunja extract or fractions thereof include a technique related to a health functional food comprising bokbunja having blood lipid improving activity in Korean Patent No. 1320371, and the extract of Bokbunja immature fruit in Korean Patent Publication No. 2014-0031080 A composition for the prevention and treatment of metabolic syndrome disease, which contains as an active ingredient, is disclosed in Korean Patent No. 1401253 for the inhibition of the statin compound from increasing the expression of the PCSK9 gene. Although a composition for inducing low density lipoprotein receptor increase through is disclosed, containing a bokbunja extract of the present invention or a fraction thereof as an active ingredient, a composition for reducing the expression of the PCSK9 gene or increasing the low density lipoprotein receptor (LDLR) No technology has been reported.
본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명은 복분자 추출물 또는 이의 분획물을 유효성분으로 함유하며, PCSK9 유전자의 발현을 감소시키거나, 저밀도 지단백 수용체(LDLR)를 증가시키기 위한 조성물을 제공하고, 상기 조성물이 PCSK9 유전자의 발현 억제를 통한 저밀도 지단백 수용체(LDLR)를 증가시켜 간세포 내로의 저밀도 지단백(LDL)의 유입이 증가되는 것을 확인함으로써, 본 발명을 완성하였다.The present invention is derived from the above requirements, the present invention contains a bokbunja extract or a fraction thereof as an active ingredient, to provide a composition for reducing the expression of the PCSK9 gene, or to increase the low density lipoprotein receptor (LDLR) In addition, the composition of the present invention was completed by confirming that the influx of low density lipoprotein (LDL) into hepatocytes was increased by increasing the low density lipoprotein receptor (LDLR) through inhibition of expression of the PCSK9 gene.
상기 목적을 달성하기 위하여, 본 발명은 복분자 추출물 또는 이의 분획물을 유효성분으로 함유하며, PCSK9 유전자의 발현을 감소시키거나, 저밀도 지단백 수용체(LDLR)를 증가시키기 위한 조성물을 제공한다.In order to achieve the above object, the present invention contains a bokbunja extract or a fraction thereof as an active ingredient, and provides a composition for reducing the expression of the PCSK9 gene, or to increase the low density lipoprotein receptor (LDLR).
또한, 본 발명은 복분자 추출물 또는 이의 분획물을 유효성분으로 함유하며, 스타틴계 화합물에 의해 증가된 PCSK9 유전자의 발현을 감소시키거나, 저밀도 지단백 수용체(LDLR)를 증가시키기 위한 조성물을 제공한다.In addition, the present invention contains a bokbunja extract or a fraction thereof as an active ingredient, and provides a composition for reducing the expression of the increased PCSK9 gene by the statin-based compound, or to increase the low density lipoprotein receptor (LDLR).
또한, 본 발명은 복분자 추출물 또는 이의 분획물을 유효성분으로 함유하며, PCSK9 유전자의 발현 감소 또는 저밀도 지단백 수용체(LDLR) 증가용 건강기능식품 조성물을 제공한다.In addition, the present invention contains a bokbunja extract or a fraction thereof as an active ingredient, and provides a dietary supplement composition for reducing the expression of the PCSK9 gene or increasing the low density lipoprotein receptor (LDLR).
또한, 본 발명은 스타틴계 화합물 및 복분자 추출물 또는 이의 분획물을 유효성분으로 함유하며, PCSK9 유전자의 발현을 감소시키거나, 저밀도 지단백 수용체(LDLR)를 증가시키는 것을 특징으로 하는 고콜레스테롤혈증의 예방 또는 치료용 약학 조성물을 제공한다.In addition, the present invention contains a statin-based compound and bokbunja extract or fractions thereof as an active ingredient, the prevention or treatment of hypercholesterolemia characterized by reducing the expression of the PCSK9 gene or increasing the low density lipoprotein receptor (LDLR) It provides a pharmaceutical composition.
또한, 본 발명은 복분자 추출물 또는 이의 분획물을 유효성분으로 포함하는 인간을 제외한 동물의 고콜레스테롤혈증의 예방 또는 치료용 수의학적 조성물을 제공한다.The present invention also provides a veterinary composition for preventing or treating hypercholesterolemia in animals other than humans, including bokbunja extract or a fraction thereof as an active ingredient.
또한, 본 발명은 복분자 추출물 또는 이의 분획물을 유효성분으로 포함하는 고콜레스테롤혈증의 예방 또는 개선용 사료 첨가제를 제공한다.The present invention also provides a feed additive for the prevention or improvement of hypercholesterolemia, including the bokbunja extract or a fraction thereof as an active ingredient.
본 발명은 복분자 추출물 또는 이의 분획물을 유효성분으로 함유하며, PCSK9 유전자의 발현을 감소시키거나, 저밀도 지단백 수용체(LDLR)를 증가시키기 위한 조성물에 관한 것이다. The present invention contains a bokbunja extract or a fraction thereof as an active ingredient, and relates to a composition for reducing the expression of the PCSK9 gene or increasing the low density lipoprotein receptor (LDLR).
본 발명의 조성물은 특히, 스타틴계 화합물에 의해 증가된 PCSK9 유전자의 발현을 억제시키고, 저밀도 지단백 수용체를 증가시키는 효과가 있는 것으로, 콜레스테롤의 혈중 수치를 낮춰 대사성 질환의 예방, 개선 또는 치료에 활용할 수 있다.In particular, the composition of the present invention is effective in inhibiting the expression of the increased PCSK9 gene by statin compounds and increasing the low density lipoprotein receptor, and can be utilized for the prevention, improvement or treatment of metabolic diseases by lowering blood levels of cholesterol. have.
도 1은 루시퍼라아제 어세이 시스템의 모식도이다. 1 is a schematic diagram of a luciferase assay system.
도 2는 루시퍼라아제 어세이 시스템의 작동 여부를 확인한 결과로서, (A)는 인간 PCSK9 유전자의 프로모터를 반딧불이 루시퍼라아제 리포터(Firefly Luciferase reporter)에 클로닝 한 후, HepG2 세포에 일과성 형질도입(transient transfection)하고, PCSK9 유전자 발현을 억제하는 것으로 알려져 있는 베르베린(berberine, 20μM) 및 베르베린을 포함하는 황련 열수 추출물(Coptis chinensis, 200㎍/㎖)을 처리한 후, 루시퍼라아제 어세이를 통해 루시퍼라아제 리포터 활성이 정상적으로 작동하는 것을 확인한 것이다. (B)는 인간 PCSK9 유전자 발현을 조절하는 것으로 알려져 있는 SREBP-1c 발현 벡터를 리포터와 co-transfection하여 프로모터 활성이 증가하는 것을 확인함으로써, 본 발명에 사용된 루시퍼라아제 리포터 활성이 정상적으로 작동하고 있음을 확인한 도면이다. 2 is a result confirming the operation of the luciferase assay system, (A) is a transient transduction into HepG2 cells after cloning the promoter of the human PCSK9 gene in the firefly Luciferase reporter (Firefly Luciferase reporter) transfection), and treated with Berberine (20 μM) known to inhibit the expression of the PCSK9 gene and chlorophyll hydrothermal extract (Coptis chinensis, 200 μg / ml) containing berberine, followed by luciferase assay. It was confirmed that the aze reporter activity is working normally. (B) confirms that promoter activity is increased by co-transfection of the SREBP-1c expression vector, which is known to regulate human PCSK9 gene expression, with the reporter, thereby the luciferase reporter activity used in the present invention is operating normally. Check the drawing.
도 3은 인간 PCSK9 유전자의 프로모터(약 1.5 kb) 리포터를 HepG2 세포에 일과성 형질도입하고 24시간 후, 열수 추출 한약재 라이브러리(18종의 단미재)를 200㎍/㎖의 농도로 24시간 동안 처리하고, 루시퍼라아제 어세이를 실시한 결과이다. 양성대조군으로 베르베린(berberin, 20μM)을 사용하였고, 물질을 녹이는데 사용한 vehicle을 처리한 음성대조군(Con)을 기준으로 보정하였다. Figure 3 transient transduction of the promoter (about 1.5 kb) reporter of the human PCSK9 gene into HepG2 cells 24 hours later, the hydrothermal extract Chinese herbal medicine library (18 sweets) was treated for 24 hours at a concentration of 200 ㎍ / ㎖ And luciferase assay. Berberin (20 μM) was used as a positive control, and was calibrated based on the negative control (Con) treated with the vehicle used to dissolve the material.
도 4는 인간 PCSK9 유전자의 프로모터(약 1.5 kb) 리포터를 HepG2 세포에 일과성 형질도입하고 24시간 후, 에탄올 추출 한약재 라이브러리(18종의 단미재)를 200㎍/㎖의 농도로 24시간 동안 처리하고, 루시퍼라아제 어세이를 실시한 결과이다. 양성대조군으로 베르베린(berberin, 20μM)을 사용하였고, 물질을 녹이는데 사용한 vehicle을 처리한 음성대조군(Con)을 기준으로 보정하였다. 4 is a transient transduction of the promoter (about 1.5 kb) reporter of the human PCSK9 gene into HepG2 cells, and 24 hours later, the ethanol extract Chinese herbal medicine library (18 sweets) was treated at a concentration of 200 µg / ml for 24 hours. And luciferase assay. Berberin (20 μM) was used as a positive control, and was calibrated based on the negative control (Con) treated with the vehicle used to dissolve the material.
도 5는 HepG2 세포에, 1 내지 200㎍/㎖의 복분자 열수 추출물을 24시간 동안 10μM의 심바스타틴(simvastatin)과 함께 처리하거나 단독으로 처리한 후, PCSK9 유전자의 mRNA 발현량을 확인한 도면이다. 5 is a diagram confirming the mRNA expression level of the PCSK9 gene in HepG2 cells after 1-200 μg / ml bokbunja hydrothermal extract was treated with 10 μM simvastatin for 24 hours or alone.
도 6은 HepG2 세포에, 1 내지 200㎍/㎖의 복분자 열수 추출물을 24시간 동안 10μM의 심바스타틴(simvastatin)과 함께 처리하거나 단독으로 처리한 후, 저밀도 지단백 수용체(low density lipoprotein receptor; LDLR)의 mRNA 발현량을 확인한 도면이다. Figure 6 mRNA of the low density lipoprotein receptor (LDLR) after HepG2 cells were treated with 1 to 200 µg / ml bokbunja hydrothermal extract for 24 hours with or without 10 μM simvastatin or alone. It is a figure which confirmed the expression amount.
도 7은 HepG2 세포에, 1 내지 200㎍/㎖의 복분자 열수 추출물을 24시간 동안 10μM의 심바스타틴(simvastatin)과 함께 처리하거나 단독으로 처리한 후, HMG-CoA 환원효소(HMG-CoA reductase)의 mRNA 발현량을 확인한 도면이다. Figure 7 mRNA of HMG-CoA reductase (HMG-CoA reductase) after treatment with HepG2 cells with 1-200 μg / ml bokbunja hydrothermal extract for 24 hours with 10 μM simvastatin or alone It is a figure which confirmed the expression amount.
도 8은 복분자 추출물 단독, 또는 이들을 스타틴계 화합물과 병용 처리하였을 때 세포의 LDL 유입(uptake)량 변화 분석결과를 나타낸 도면이다. *는 심바스타틴(simvastatin)을 단독 처리한 것에 대비한 심바스타틴 및 복분자 열수 추출물을 함께 처리한 것이 통계적으로 유의하다는 것을 의미하며, *는 p값이 0.05 미만임을 의미하고, #은 물질을 녹이는데 사용한 vehicle을 처리한 음성 대조군에 대비하여 복분자 추출물을 단독으로 처리한 것이 통계적으로 유의하다는 것을 의미하며, #은 p값이 0.05 미만임을 의미한다. $$는 물질을 녹이는데 사용한 vehicle을 처리한 음성 대조군에 대비하여 복분자 추출물을 단독으로 처리한 것이 통계적으로 유의하다는 것을 의미하며, $$는 p값이 0.01 미만임을 의미한다. 8 is a view showing the results of analysis of changes in LDL uptake of cells when bokbunja extract alone or in combination with statin compounds. * Indicates statistically significant treatment with simvastatin and bokbunja hydrothermal extract compared to simvastatin alone, * means p value is less than 0.05, # means vehicle used to dissolve Compared to the negative control treated with the bokbunja extract alone treatment means that the statistical significance, # means that the p value is less than 0.05. $$ means that the treatment of Bokbunja extract alone was statistically significant compared to the negative control treated with vehicle used to dissolve the material, and $$ means p value is less than 0.01.
도 9는 인간 PCSK9 유전자의 프로모터(약 1.5 kb) 리포터를 HepG2 세포에 일과성 형질도입하고 24시간 후, 복분자 열수 추출물의 헥산 분획물(Hx), 클로로포름 분획물(CHCl3), 에틸아세테이트 분획물(EtAc), 부탄올 분획물(BuOH), 물 분획물(H2O) 각각을 10, 50, 100㎍/㎖의 농도로 24시간 동안 처리하고, 루시퍼라아제 어세이를 실시한 결과이다. 양성대조군으로 베르베린(berberin, 20μM)을 사용하였고, 물질을 녹이는데 사용한 vehicle을 처리한 음성대조군(CON)을 기준으로 보정하였다. *, **은 물질을 녹이는데 사용한 vehicle을 처리한 음성 대조군에 대비하여 복분자 추출물의 각 분획물을 처리한 것이 통계적으로 유의하다는 것을 의미하며, *는 p값이 0.05 미만이고, **는 p값이 0.01 미만임을 의미한다.9 is a transient transduction of the promoter (about 1.5 kb) reporter of the human PCSK9 gene into HepG2 cells 24 hours, hexane fraction (Hx), chloroform fraction (CHCl 3 ), ethyl acetate fraction (EtAc), The butanol fraction (BuOH) and the water fraction (H 2 O) were each treated for 24 hours at concentrations of 10, 50, and 100 µg / ml, followed by a luciferase assay. Berberin (20 μM) was used as a positive control, and was corrected based on the negative control (CON) treated with the vehicle used to dissolve the material. *, ** indicates that the treatment of each fraction of Bokbunja extract was statistically significant compared to the negative control treated with vehicle used to dissolve the material. * Indicates p value less than 0.05 and ** indicates p value. Means less than 0.01.
도 10은 HepG2 세포에, 100㎍/㎖의 복분자 열수 추출물의 헥산 분획물(Hx), 클로로포름 분획물(CHCl3), 에틸아세테이트 분획물(EtAc), 부탄올 분획물(BuOH), 물 분획물(H2O) 및 복분자 열수 추출물(BRE)을 24시간 동안 10μM의 심바스타틴(simvastatin)과 함께 처리한 후, PCSK9 유전자의 mRNA 발현량을 확인한 도면이다. CON은 음성대조군으로, 복분자 열수 추출물 또는 이의 분획물 대신에 vehicle을 처리한 것이다.FIG. 10 shows HepG2 cells, hexane fraction (Hx), chloroform fraction (CHCl 3 ), ethyl acetate fraction (EtAc), butanol fraction (BuOH), water fraction (H 2 O) and 100 μg / ml bokbunja hydrothermal extract Bokbunja hydrothermal extract (BRE) after treatment with 10 μM simvastatin (simvastatin) for 24 hours, it is a diagram confirming the mRNA expression level of the PCSK9 gene. CON is a negative control group treated with vehicle instead of bokbunja hydrothermal extract or fractions thereof.
도 11은 HepG2 세포에, 100㎍/㎖의 복분자 열수 추출물의 헥산 분획물(Hx), 클로로포름 분획물(CHCl3), 에틸아세테이트 분획물(EtAc), 부탄올 분획물(BuOH), 물 분획물(H2O) 및 복분자 열수 추출물(BRE)을 24시간 동안 10μM의 심바스타틴(simvastatin)과 함께 처리한 후, PCSK9 및 LDLR 단백질의 발현량을 웨스턴 블랏으로 확인한 도면이다. CON은 음성대조군으로, 복분자 추출물 또는 이의 분획물 대신에 vehicle을 처리한 것이며, -는 10μM의 심바스타틴만 처리한 것이고, β-actin은 로딩 컨드롤이다.11 shows HexG2 cells, hexane fraction (Hx), chloroform fraction (CHCl 3 ), ethyl acetate fraction (EtAc), butanol fraction (BuOH), water fraction (H 2 O) Bokbunja hydrothermal extract (BRE) was treated with 10 μM simvastatin for 24 hours, and then the amount of expression of PCSK9 and LDLR protein was confirmed by Western blot. CON is a negative control, treated with vehicle instead of bokbunja extract or fractions thereof,-is treated with only 10 μM simvastatin, β-actin is a loading control.
본 발명은 복분자 추출물 또는 이의 분획물을 유효성분으로 함유하며, PCSK9 유전자의 발현을 감소시키거나, 저밀도 지단백 수용체(LDLR)를 증가시키기 위한 조성물에 관한 것이다. 상기 조성물은 PCSK9 발현량의 감소시킴으로써 저밀도 지단백 수용체(LDLR)의 발현을 증가시키고, 결과적으로 혈중의 콜레스테롤 수치를 낮추는 것이므로, 비만, 제2형 당뇨병, 고혈압, 심장혈관 질환 또는 고지혈증 등의 대사성 질환의 예방 또는 개선용 건강기능식품 조성물로 활용할 수 있다. The present invention contains a bokbunja extract or a fraction thereof as an active ingredient, and relates to a composition for reducing the expression of the PCSK9 gene or increasing the low density lipoprotein receptor (LDLR). Since the composition increases the expression of low density lipoprotein receptor (LDLR) by reducing the amount of PCSK9 and consequently lowers the blood cholesterol level, the composition of metabolic diseases such as obesity, type 2 diabetes, hypertension, cardiovascular disease or hyperlipidemia It can be utilized as a health functional food composition for prevention or improvement.
또한, 본 발명은 복분자 추출물 또는 이의 분획물을 유효성분으로 함유하며, 스타틴계 화합물에 의해 증가된 PCSK9 유전자의 발현을 감소시키거나, 스타틴계 화합물에 의해 감소된 저밀도 지단백 수용체(LDLR)를 증가시키기 위한 조성물에 관한 것이다.In addition, the present invention contains a bokbunja extract or a fraction thereof as an active ingredient, to reduce the expression of the PCSK9 gene increased by the statin-based compound, or to increase the low density lipoprotein receptor (LDLR) reduced by the statin-based compound It relates to a composition.
상기 스타틴계 화합물은 스타틴 계열의 화합물이라면 제한하지 않지만, 바람직한 일례로는 심바스타틴(simvastatin), 아토르바스타틴(atorvastatin), 세리바스타틴(cerivastatin), 로바스타틴(lovastatin), 피타바스타틴(pitavastatin), 로수바스타틴(rosuvastatin), 프라바스타틴(pravastatin), 플루바스타틴(fluvastatin) 및 메바스타틴(mevastatin) 중에서 선택된 하나 이상일 수 있다. The statin-based compound is not limited as long as it is a statin-based compound, but preferred examples thereof include simvastatin, atorvastatin, cerivastatin, cerivastatin, lovastatin, pitavastatin, and rosuvastatin. (rosuvastatin), pravastatin, fluvastatin and fluvastatin and mevastatin (mevastatin) may be one or more selected.
상기 복분자 추출물은 당 업계에 공지된 통상적인 추출용매를 사용할 수 있으나, 바람직한 추출용매의 일례로는 탄소 수 1 내지 4의 저급 알코올, 물 및 이들의 혼합물 중에서 선택된 하나 이상이며, 더 바람직하게는 물 또는 에탄올이고, 더욱더 바람직하게는 물이다. 상기 물을 이용하여 추출하는 열수 추출인 경우, 복분자 중량에 대하여 5 내지 10배 부피만큼 물을 넣고, 90 내지 110℃에서 2 내지 3시간 동안 가열하여 추출하는 것이다. 추출 용액을 여과지로 여과한 후, 동결건조하여 사용하는 것이 바람직하지만 이에 한정하지 않는다. 또한, 에탄올 추출을 하는 경우, 복분자 중량에 대하여 5 내지 10배 부피만큼 80% 에탄올을 넣고, 20 내지 25℃의 상온에서 22 내지 24시간 동안 침지하여 추출하는 것이다. 추출 용액을 여과지로 여과한 후, 동결건조하여 사용하는 것이 바람직하지만 이에 한정하지 않는다.The bokbunja extract may use a conventional extraction solvent known in the art, but one example of a preferred extraction solvent is one or more selected from lower alcohols having 1 to 4 carbon atoms, water and mixtures thereof, more preferably water Or ethanol, even more preferably water. In the case of hot water extraction using the water, water is added in a volume of 5 to 10 times the weight of the bokbunja, and extracted by heating at 90 to 110 ° C. for 2 to 3 hours. The extraction solution is preferably filtered through filter paper and then lyophilized, but not always limited thereto. In addition, in the case of ethanol extraction, 80% ethanol is added in a volume of 5 to 10 times the weight of bokbunja and extracted by dipping for 22 to 24 hours at a room temperature of 20 to 25 ° C. The extraction solution is preferably filtered through filter paper and then lyophilized, but not always limited thereto.
상기 복분자 열수 추출물의 분획물은 헥산 분획물(Hx), 클로로포름 분획물(CHCl3), 에틸아세테이트 분획물(EtAc), 부탄올 분획물(BuOH) 및 물 분획물(H2O) 중에서 선택한 어느 하나인 것이 바람직하며, 더 바람직하게는 클로로포름 분획물 또는 에틸아세테이트 분획물인 것이지만, 이에 한정하지 않는다. The fraction of the bokbunja hydrothermal extract is preferably any one selected from hexane fraction (Hx), chloroform fraction (CHCl 3 ), ethyl acetate fraction (EtAc), butanol fraction (BuOH) and water fraction (H 2 O), more Preferably it is, but is not limited to, a chloroform fraction or an ethyl acetate fraction.
또한, 본 발명은 복분자 추출물 또는 이의 분획물을 유효성분으로 함유하며, PCSK9 유전자의 발현 감소 또는 저밀도 지단백 수용체(LDLR) 증가용 건강기능식품 조성물에 관한 것이다. In addition, the present invention contains a bokbunja extract or a fraction thereof as an active ingredient, and relates to a dietary supplement composition for reducing the expression of the PCSK9 gene or increasing the low density lipoprotein receptor (LDLR).
상기 건강기능식품은 특별히 제한되지 않으며, 건강기능식품, 영양보조제, 영양제, 파머푸드(pharmafood), 건강식품, 뉴트라슈티칼(nutraceutical), 디자이너 푸드, 식품 첨가제 등의 모든 형태의 식품이 될 수 있는데, 바람직하게는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올 음료 및 비타민 복합제 등이 될 수 있다. 이때, 첨가하는 방법은 통상적인 방법에 따라 적절하게 사용되고, 첨가량은 사용 목적에 따라 적합하게 결정될 수 있다. The dietary supplement is not particularly limited, and may be any form of dietary supplement, dietary supplement, nutritional supplement, pharmafood, health food, nutraceutical, designer food, or food additive. Preferably, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, drinks, tea, drinks, alcoholic drinks and vitamin complexes Can be. At this time, the method of addition is suitably used according to a conventional method, and the addition amount can be suitably determined according to a use purpose.
상기 조성물은 유효성분 이외의 다양한 성분을 추가로 포함할 수도 있는데, 이들 다양한 성분은 특별히 제한되지 않으나, 바람직하게는 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등이 될 수 있다. 또한, 기호성 또는 기능성을 부가하기 위하여 천연 과일 주스, 과일 주스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있으며, 이들 성분들은 독립적으로 또는 조합하여 사용할 수 있다.The composition may further include various components other than the active ingredient, and these various components are not particularly limited, but preferably various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and Salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like. It may also contain flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks in order to add palatability or functionality, and these ingredients may be used independently or in combination.
또한, 본 발명은 스타틴계 화합물 및 복분자 추출물 또는 이의 분획물을 유효성분으로 함유하는 것을 특징으로 하는 고콜레스테롤혈증의 예방 또는 치료용 약학 조성물에 관한 것이다. 상기 복분자 추출물 또는 이의 분획물은 PCSK9 유전자의 발현을 억제하거나 저밀도 지단백 수용체(LDLR)의 발현을 증가시키는 것이 특징이며, PCSK9 유전자의 발현 억제 또는 지단백 수용체의 발현 증가에 의해 콜레스테롤의 혈중 수치를 낮추는 것이므로, 비만, 제2형 당뇨병, 고혈압, 심장혈관 질환 또는 고지혈증 등의 대사성 질환의 예방 또는 치료용 약학 조성물로 활용할 수 있다.The present invention also relates to a pharmaceutical composition for preventing or treating hypercholesterolemia, which comprises a statin-based compound and bokbunja extract or a fraction thereof as an active ingredient. The bokbunja extract or fractions thereof are characterized by inhibiting the expression of the PCSK9 gene or increasing the expression of low density lipoprotein receptor (LDLR), and lowering the blood levels of cholesterol by inhibiting the expression of the PCSK9 gene or increasing the expression of the lipoprotein receptor, It can be used as a pharmaceutical composition for the prevention or treatment of metabolic diseases such as obesity, type 2 diabetes, hypertension, cardiovascular disease or hyperlipidemia.
상기 복분자 추출물은 당 업계에 공지된 통상적인 추출용매를 사용할 수 있으나, 바람직한 추출용매의 일례로는 탄소 수 1 내지 4의 저급 알코올, 물 및 이들의 혼합물 중에서 선택된 하나 이상이며, 더 바람직하게는 물 또는 에탄올이고, 더욱더 바람직하게는 물이다. 상기 물을 이용하여 추출하는 열수 추출인 경우, 복분자 중량에 대하여 5 내지 10배 부피만큼 물을 넣고, 90 내지 110℃에서 2 내지 3시간 동안 가열하여 추출하는 것이다. 추출 용액을 여과지로 여과한 후, 동결건조하여 사용하는 것이 바람직하지만 이에 한정하지 않는다. 또한, 에탄올 추출을 하는 경우, 복분자 중량에 대하여 5 내지 10배 부피만큼 80% 에탄올을 넣고, 20 내지 25℃의 상온에서 22 내지 24시간 동안 침지하여 추출하는 것이다. 추출 용액을 여과지로 여과한 후, 동결건조하여 사용하는 것이 바람직하지만 이에 한정하지 않는다.The bokbunja extract may use a conventional extraction solvent known in the art, but one example of a preferred extraction solvent is one or more selected from lower alcohols having 1 to 4 carbon atoms, water and mixtures thereof, more preferably water Or ethanol, even more preferably water. In the case of hot water extraction using the water, water is added in a volume of 5 to 10 times the weight of the bokbunja, and extracted by heating at 90 to 110 ° C. for 2 to 3 hours. The extraction solution is preferably filtered through filter paper and then lyophilized, but not always limited thereto. In addition, in the case of ethanol extraction, 80% ethanol is added in a volume of 5 to 10 times the weight of bokbunja and extracted by dipping for 22 to 24 hours at a room temperature of 20 to 25 ° C. The extraction solution is preferably filtered through filter paper and then lyophilized, but not always limited thereto.
상기 복분자 추출물의 분획물은 헥산 분획물(Hx), 클로로포름 분획물(CHCl3), 에틸아세테이트 분획물(EtAc), 부탄올 분획물(BuOH) 및 물 분획물(H2O) 중에서 선택한 어느 하나인 것이 바람직하며, 더 바람직하게는 클로로포름 분획물 또는 에틸아세테이트 분획물인 것이지만, 이에 한정하지 않는다. The fraction of the bokbunja extract is preferably any one selected from hexane fraction (Hx), chloroform fraction (CHCl 3 ), ethyl acetate fraction (EtAc), butanol fraction (BuOH) and water fraction (H 2 O), more preferably. Preferably it is, but is not limited to, a chloroform fraction or an ethyl acetate fraction.
상기 스타틴계 화합물은 스타틴 계열의 화합물이라면 제한하지 않지만, 바람직한 일례로는 심바스타틴(simvastatin), 아토르바스타틴(atorvastatin), 세리바스타틴(cerivastatin), 로바스타틴(lovastatin), 피타바스타틴(pitavastatin), 로수바스타틴(rosuvastatin), 프라바스타틴(pravastatin), 플루바스타틴(fluvastatin) 및 메바스타틴(mevastatin) 중에서 선택된 하나 이상일 수 있다. The statin-based compound is not limited as long as it is a statin-based compound, but preferred examples thereof include simvastatin, atorvastatin, cerivastatin, cerivastatin, lovastatin, pitavastatin, and rosuvastatin. (rosuvastatin), pravastatin, fluvastatin and fluvastatin and mevastatin (mevastatin) may be one or more selected.
본 발명의 약학 조성물은 약학적으로 허용되는 담체를 포함할 수 있다. 본 발명의 약학 조성물에 포함되는 약학적으로 허용되는 담체는 제제 시에 통상적으로 이용되는 것으로서, 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로즈 용액, 말토덱스트린 용액, 글리세롤, 에탄올, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. The pharmaceutical composition of the present invention may comprise a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are conventionally used in the preparation, saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, lactose, dex Straw, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, syrups, methyl cellulose, methylhydroxybenzoate, propyl Hydroxybenzoate, talc, magnesium stearate and mineral oil, and the like.
본 발명의 약학 조성물은 상기 성분들 이외에 항산화제, 완충액, 정균제, 희석제, 계면활성제, 결합제, 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제 또는 보존제 등을 추가로 포함할 수 있다. The pharmaceutical composition of the present invention may further include antioxidants, buffers, bacteriostatic agents, diluents, surfactants, binders, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents or preservatives, etc., in addition to the above components.
본 발명의 약학 조성물은 경구 또는 비경구 투여할 수 있으며, 바람직하게는 경구 투여 방식으로 적용된다. 본 발명의 약학 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다. The pharmaceutical composition of the present invention may be administered orally or parenterally, and preferably applied by oral administration. Suitable dosages of the pharmaceutical compositions of the present invention may be prescribed in various ways depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be.
본 발명의 약학 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약학적으로 허용되는 담체 또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질 중의 용액, 현탁액, 시럽제 또는 유화액 형태이거나 엑스제, 산제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가로 포함할 수 있다.The pharmaceutical compositions of the present invention may be prepared in unit dosage form or in multi-dose form by formulating with a pharmaceutically acceptable carrier or excipient according to methods which can be easily carried out by those skilled in the art. It may be prepared by incorporation into a container. The formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media or in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise a dispersing or stabilizing agent.
또한, 본 발명은 복분자 추출물 또는 이의 분획물을 유효성분으로 포함하는 인간을 제외한 동물의 고콜레스테롤혈증의 예방 또는 치료용 수의학적 조성물에 관한 것이다. The present invention also relates to a veterinary composition for preventing or treating hypercholesterolemia in animals other than humans, including bokbunja extract or fractions thereof as an active ingredient.
본 발명의 복분자 추출물 또는 이의 분획물을 포함하는 수의학적 조성물은 통상의 방법에 따른 적절한 부형제 및 희석제를 더 포함할 수 있다. 본 발명의 복분자 추출물 또는 이의 분획물을 포함하는 수의학적 조성물에 포함될 수 있는 부형제 및 희석제로는, 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트, 세탄올, 스테아릴알콜, 유동파라핀, 솔비탄모노스테아레이트, 폴리소르베이트 60, 메틸파라벤, 프로필파라벤 및 광물유를 들 수 있다. Veterinary compositions comprising the bokbunja extract or fractions thereof of the present invention may further comprise suitable excipients and diluents according to conventional methods. Excipients and diluents that may be included in veterinary compositions comprising the bokbunja extract of the present invention or fractions thereof include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate , Gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, cetanol, stearyl alcohol , Liquid paraffin, sorbitan monostearate, polysorbate 60, methyl paraben, propyl paraben and mineral oil.
본 발명에 따른 복분자 추출물 또는 이의 분획물을 포함하는 수의학적 조성물은 충진제, 항응집제, 윤활제, 습윤제, 향신료, 유화제, 방부제 등을 추가로 포함할 수 있는데, 본 발명에 의한 수의학적 조성물은 동물에 투여된 후 활성 성분의 신속, 지속 또는 지연된 방출을 제공할 수 있도록 당업계에 잘 알려진 방법을 사용하여 제형화될 수 있고, 제형은 산제, 과립제, 정제, 캡슐제, 현탁액, 에멀젼, 용액, 시럽, 에어로졸, 연질 또는 경질 젤라틴 캅셀, 좌제, 멸균 주사용액, 멸균 외용제 등의 형태일 수 있다.The veterinary composition comprising the bokbunja extract or fractions thereof according to the present invention may further comprise a filler, an anticoagulant, a lubricant, a humectant, a spice, an emulsifier, a preservative, and the like. Can be formulated using methods well known in the art to provide rapid, sustained or delayed release of the active ingredient, the formulation can be in the form of powders, granules, tablets, capsules, suspensions, emulsions, solutions, syrups, Aerosols, soft or hard gelatin capsules, suppositories, sterile injectable solutions, sterile external preparations, and the like.
본 발명에 의한 수의학적 조성물은 동물의 나이, 성별, 체중에 따라 달라질 수 있으나, 0.1 내지 100mg/㎏의 양을 1일 1회 내지 수회 투여할 수 있고, 투여량은 투여경로, 질병의 정도, 성별, 체중, 나이 등에 따라서 증감될 수 있다. 따라서, 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다. The veterinary composition according to the present invention may vary depending on the age, sex and weight of the animal, but may be administered in an amount of 0.1 to 100 mg / kg once or several times a day, and the dosage may be a route of administration, a degree of disease, It may increase or decrease depending on gender, weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.
또한, 본 발명은 복분자 추출물 또는 이의 분획물을 유효성분으로 포함하는 고콜레스테롤혈증의 예방 또는 개선용 사료 첨가제에 관한 것이다.The present invention also relates to a feed additive for the prevention or improvement of hypercholesterolemia comprising bokbunja extract or fractions thereof as an active ingredient.
상기 사료 첨가제는 20 내지 90중량%의 복분자 추출물 또는 이의 분획물을 함유하는 고농축액, 분말 또는 과립형태일 수 있다. The feed additive may be in the form of a high concentrate, powder or granules containing 20 to 90% by weight of bokbunja extract or fractions thereof.
본 발명의 사료 첨가제는 구연산, 후말산, 아디픽산, 젖산, 사과산 등의 유기산이나 인산나트륨, 인산칼륨, 산성피로인산염, 폴리인산염(중합인산염) 등의 인산염이나 폴리페놀, 카테킨(catechin), 알파-토코페롤, 로즈메리 추출물(rosemary extract), 비타민 C, 녹차 추출물, 감초 추출물, 키토산, 탄닌산, 피틴산 등의 천연 항산화제 중에서 선택된 하나 이상을 추가로 포함할 수 있다.The feed additive of the present invention includes organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, malic acid, phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate and polyphosphate (polyphosphate), polyphenols, catechins and alpha. It may further include one or more selected from natural antioxidants such as tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, phytic acid.
본 발명의 복분자 추출물 또는 이의 분획물을 함유하는 동물사료 첨가제 및 이를 포함하는 사료는 보조성분으로 아미노산, 무기염류, 비타민, 항생물질, 항균물질, 항산화, 항곰팡이 효소, 소화 및 흡수향상제, 성장촉진제, 질병예방제 등과 같은 물질과 함께 사용될 수 있다.Animal feed additives containing bokbunja extract or fractions thereof of the present invention and feed containing them are amino acids, inorganic salts, vitamins, antibiotics, antibacterial substances, antioxidants, antifungal enzymes, digestion and absorption enhancers, growth promoters, It can be used with substances such as disease prevention agents.
상기 사료 첨가제는 동물에게 단독으로 식용 담체 중에서 다른 사료 첨가제와 조합되어 투여될 수 있다. 또한, 상기 사료첨가제는 탑 드레싱으로서 또는 이들을 동물 사료에 직접 혼합하거나 또는 사료와 별도로, 별도의 경구 제형으로, 주사 또는 경피로 또는 다른 성분과 조합하여 쉽게 투여할 수 있다. 통상적으로, 당 업계에 잘 알려진 바와 같이 단독 일일 투여량 또는 분할 일일 투여량을 사용할 수 있다. 상기 사료 첨가제를 동물 사료와 별도로 투여할 경우, 당 업계에 잘 알려진 바와 같이 추출물의 투여 형태는 이들을 비-독성 제약상 허용 가능한 식용 담체와 조합하여 즉석 방출 또는 서방성 제형으로 제조할 수 있다. 이러한 식용 담체는 고체 또는 액체, 예를 들어 옥수수 전분, 락토스, 수크로스, 콩 플레이크, 땅콩유, 올리브유, 참깨유 및 프로필렌 글리콜일 수 있다. 고체 담체가 사용될 경우, 추출물의 투여형은 정제, 캡슐제, 산제, 토로키제 또는 함당정제 또는 미분산성 형태의 탑 드레싱일 수 있다. 액체 담체가 사용될 경우, 연 젤라틴 캡슐제, 또는 시럽제 또는 액체 현탁액제, 에멀젼제 또는 용액제의 투여 형태일 수 있다. 또한, 투여 형태는 보조제, 예를 들어 보존제, 안정화제, 습윤제 또는 유화제, 용액 촉진제 등을 함유할 수 있다. 또한, 복분자 추출물 또는 이의 분획물이 사료 첨가제로 포함되는 동물사료는 동물의 식이 요구를 충족시키는데 통상적으로 사용되는 임의의 단백질-함유 유기 곡분일 수 있다. 이러한 단백질-함유 곡분은 통상적으로 옥수수, 콩 곡분 또는 옥수수/콩 곡분 믹스로 주로 구성되어 있다. 상기의 사료 첨가제는 침지, 분무 또는 혼합하여 상기 동물사료에 첨가하여 이용될 수 있다.The feed additive may be administered to the animal alone in combination with other feed additives in an edible carrier. In addition, the feed additives can be easily administered as a top dressing or directly mixed into animal feed or separately from the feed, in a separate oral formulation, by injection or transdermal or in combination with other ingredients. Typically, single or divided daily dosages can be used as is well known in the art. When the feed additives are administered separately from the animal feed, dosage forms of the extracts, as is well known in the art, can be prepared in immediate release or sustained release formulations in combination with non-toxic pharmaceutically acceptable edible carriers. Such edible carriers may be solid or liquid, for example corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol. When a solid carrier is used, the dosage form of the extract may be tablets, capsules, powders, torokies or sugar-containing tablets or top dressings in microdisperse form. If a liquid carrier is used, it may be in the form of soft gelatin capsules or syrups or liquid suspensions, emulsions or solutions. In addition, the dosage form may contain auxiliaries such as preservatives, stabilizers, wetting or emulsifying agents, solution promoters and the like. In addition, the animal feed in which the bokbunja extract or fractions thereof is included as a feed additive may be any protein-containing organic cereal meal commonly used to meet the dietary needs of the animal. Such protein-containing flours typically consist mainly of corn, soy flour, or corn / bean flour mixtures. The feed additive may be used by adding to the animal feed by dipping, spraying or mixing.
본 발명의 수의학적 조성물 또는 사료첨가제는 반려동물의 식이에 적용할 수 있다.The veterinary composition or feed additive of the present invention can be applied to the diet of a companion animal.
이하, 실시예를 이용하여 본 발명을 더욱 상세하게 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로 본 발명의 범위가 이들에 의해 제한되지 않는다는 것은 당해 기술분야에서 통상의 지식을 가진 자에게 있어 자명한 것이다. Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for explaining the present invention in more detail, it is obvious to those skilled in the art that the scope of the present invention is not limited by them.
실시예 1. 간암 세포주(HepG2)의 배양Example 1 Culture of Hepatocellular Carcinoma Cell Line (HepG2)
간암 세포주(HepG2)는 ATCC(American Type Culture Collection) 사에서 구입하여 사용하였다. 간암 세포주(HepG2)는 10% FBS, 1% 페니실린-스트렙토마이신이 포함된 DMEM/F-12(Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) 배지로 배양하였다. 상기 세포는 37℃, 5% CO2 배양기에서 배양하였고, 배양된 세포는 24웰 플레이트에 일정한 수의 세포(1×105 cells/ 500㎕ well)를 분주하여 실험을 하였다. 24웰 플레이트에 세포를 분주한 후, 세포가 24웰에 완전히 붙어 세포의 모양을 갖춘 후 세포가 75%의 포화도(confluence)에 도달했을 때 사용하였다. Liver cancer cell line (HepG2) was purchased from the American Type Culture Collection (ATCC). Liver cancer cell line (HepG2) was incubated with DMEM / F-12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12) medium containing 10% FBS, 1% penicillin-streptomycin. The cells were cultured in a 37 ° C., 5% CO 2 incubator, and the cultured cells were tested by dispensing a predetermined number of cells (1 × 10 5 cells / 500 μl well) in a 24-well plate. After dispensing the cells in a 24-well plate, the cells were completely attached to the 24 wells and used to form when the cells reached a confluence of 75%.
실시예 2. 복분자 추출물의 제조Example 2. Preparation of Bokbunja Extract
1) 복분자 열수 추출물의 제조1) Preparation of Bokbunja Hydrothermal Extract
복분자 200g에 대하여 2ℓ의 물을 가하고, 90 내지 100℃의 온도에서 2 내지 3시간 동안 가열 후, 추출물을 여과하였다. 이후에 감압농축기로 농축하고, 동결건조기를 이용하여 감압 건조하여 수득한 복분자 열수 추출물을 실험에 사용하기 위하여 10%(v/v) DMSO에 녹여 사용하였다.2 L of water was added to 200 g of bokbunja, and heated at a temperature of 90 to 100 ° C. for 2 to 3 hours, and then the extract was filtered. Thereafter, the resultant was concentrated in a vacuum concentrator, and the bokbunja hot water extract obtained by drying under reduced pressure using a lyophilizer was used by dissolving in 10% (v / v) DMSO.
2) 복분자 에탄올 추출물의 제조2) Preparation of Bokbunja Ethanol Extract
복분자 200g에 대하여 2ℓ의 80% 에탄올을 가하고, 20 내지 25℃의 상온에서 22 내지 24시간 동안 침지한 후, 추출물을 여과하였다. 이후에 감압 농축기로 농축하고 동결건조기를 이용하여 감압 건조하여 수득한 복분자 에탄올 추출물을 실험에 사용하기 위하여 10%(v/v) DMSO에 녹여 사용하였다.2 L of 80% ethanol was added to 200 g of bokbunja, immersed for 22 to 24 hours at room temperature of 20 to 25 ℃, and then the extract was filtered. Thereafter, the bokbunja ethanol extract obtained by concentrating with a vacuum condenser and drying under reduced pressure using a lyophilizer was used by dissolving in 10% (v / v) DMSO.
실시예 3. 루시퍼라아제 어세이 시스템 구축Example 3 Luciferase Assay System Construction
도 1에 개시한 바와 같이, PCSK9 유전자의 발현량 변화를 확인하기 위해, 루시퍼라아제 어세이 시스템을 구축하고, 정상적으로 작동하는지 여부를 확인하였다.As shown in FIG. 1, in order to confirm the expression level change of the PCSK9 gene, a luciferase assay system was constructed, and it was confirmed whether or not it operated normally.
인간 PCSK9 유전자의 프로모터(약 1.5kb)를 반딧불이 루시퍼라아제 리포터에 클로닝한 후, HepG2 세포에 일과성 형질도입(transient transfection)하고, 20μM의 베르베린(berberine)과 200㎍/㎖의 베르베린(berberine)을 포함하는 황련 열수추출물(Coptis chinensis)를 처리하여 24시간 동안 배양하였다. 이후, 반딧불이 루시퍼라아제의 발광정도를 측정하여 결과를 도 2(A)에 개시하였다. PCSK9 유전자 발현을 억제하는 것으로 알려져 있는 베르베린(berberine, 20μM) 및 베르베린을 포함하는 황련 열수 추출물(Coptis chinensis, 200㎍/㎖)을 처리한 경우, PCSK9 유전자의 발현량이 물질을 녹이는데 사용한 vehicle을 처리한 음성대조군에 비해 현저하게 감소하는 것을 확인하였다. The promoter of the human PCSK9 gene (approximately 1.5 kb) was cloned into the firefly luciferase reporter, followed by transient transfection into HepG2 cells, and 20 μM of berberine and 200 μg / ml of berberine. Treatment was carried out for 24 hours by treating the containing hot water extract (Coptis chinensis). Thereafter, the degree of emission of firefly luciferase was measured and the results are shown in FIG. 2 (A). When treated with berberine (20 μM) known to inhibit PCSK9 gene expression and coarse water extract (Coptis chinensis, 200 μg / ml) containing berberine, the vehicle used for dissolving the amount of PCSK9 gene was treated. It was confirmed that the decrease significantly compared to one negative control group.
또한, 인간 PCSK9 유전자의 유전자 발현을 조절한다고 알려져 있는 SREBP-1c 발현벡터를 리포터와 co-transfection하여 프로모터 활성이 증가하는 것을 확인함으로써, 본 발명에 사용된 루시퍼라아제 리포터 활성이 정상적으로 작동하고 있음을 알 수 있었다(도 2(B)).In addition, co-transfection of the SREBP-1c expression vector, which is known to regulate gene expression of the human PCSK9 gene, with the reporter confirmed that the promoter activity was increased, indicating that the luciferase reporter activity used in the present invention is operating normally. It was found (Fig. 2 (B)).
이하 실시예에서는 본 실시예에서 구축한 루시퍼라아제 어세이 시스템을 이용하였다.In the following examples, the luciferase assay system constructed in this example was used.
실시예 4. PCSK9 유전자의 프로모터 부위의 전사 활성의 변화를 이용한 스크리닝Example 4 Screening Using Changes in Transcriptional Activity of Promoter Sites of PCSK9 Gene
18종의 한약재 라이브러리 중에서 PCSK9 유전자의 발현량을 감소시킬 수 있는 한약재 추출물을 선별하기 위하여 루시퍼라아제 어세이를 실시하였다. Luciferase assays were performed to select herbal extracts that can reduce the expression level of the PCSK9 gene among 18 herbal library.
인간 PCSK9 프로모터(약 1.5kb)를 포함하는 루시퍼라아제 리포터(luciferase reporter)를 HepG2 세포에 일과성 형질도입(transient transfection) 후, 200㎍/㎖의 한약재 추출물 18종을 처리하고 하룻밤 동안 배양하였다. 이후, 반딧불이 루시퍼라아제 발광 정도를 측정하여 PCSK9 유전자의 발현을 감소시키는 한약재 추출물을 선정하였다. 선정된 #24의 한약재 추출물은 열수 및 에탄올 추출물 둘 다에서 효과적으로 PCSK9 유전자의 발현량을 감소시킨다는 것을 도 3 및 도 4에 개시하였다. Luciferase reporter containing a human PCSK9 promoter (about 1.5 kb) was transfected into HepG2 cells, treated with 18 kinds of herbal extracts of 200 µg / ml and incubated overnight. Then, the herbal extracts were selected to reduce the expression of the PCSK9 gene by measuring the degree of firefly luciferase luminescence. 3 and 4 show that the selected Chinese herbal extract of # 24 effectively reduces the expression level of the PCSK9 gene in both hot water and ethanol extract.
도 3 및 도 4에 개시한 #24의 한약재 추출물은 복분자 추출물로 확인되었다. The herbal extract of # 24 disclosed in FIGS. 3 and 4 was identified as a bokbunja extract.
실시예 5. 복분자 추출물 단독, 또는 스타틴계 화합물과 병용 처리되었을 때의 PCSK9, LDLR 및 HMG-CoA reductase 유전자의 발현량 변화 분석Example 5 Analysis of Expression Changes of PCSK9, LDLR and HMG-CoA Reductase Genes When Treated with Bokbunja Extract Alone or in Combination with Statin Compounds
실시예 4에서 실시한 루시퍼라아제 어세이 스크리닝 결과를 확인하기 위하여 taqman probe를 활용한 실시간 중합효소연쇄반응(real-time PCR)를 이용하여 복분자 추출물 단독 또는 스타틴계 화합물과 복분자 열수 추출물이 처리되었을 때의 PCSK9, LDLR 및 HMG-CoA reductase 유전자의 발현량 변화를 분석하였다.When confirming the results of luciferase assay screening carried out in Example 4 when the bokbunja extract alone or statin-based compound and bokbunja hot water extract was treated by using real-time PCR using a taqman probe The expression changes of PCSK9, LDLR and HMG-CoA reductase genes were analyzed.
100 및 200㎍/㎖의 복분자 추출물을 단독 혹은 10μM의 심바스타틴과 0, 1, 10, 50, 100 및 200㎍/㎖의 복분자 추출물 혹은 베르베린(berberine, 20μM)을 처리하고 24시간 동안 배양하였다. 이후 세포로부터 total RNA를 분리하고 역전사 중합효소 연쇄반응(Reverse Transcription Polymerase Chain Reaction, RT-PCR)을 이용해 cDNA를 합성하고, taqman probe를 활용한 실시간 중합효소연쇄반응(real-time PCR)을 통해 PCSK9, LDLR 및 HMG-CoA reductase 유전자의 발현량 변화를 분석하였다.100 and 200 μg / ml bokbunja extracts were treated alone or 10 μM simvastatin and 0, 1, 10, 50, 100 and 200 μg / ml bokbunja extract or berberine (20 μM) and incubated for 24 hours. Then, total RNA is separated from the cells, cDNA is synthesized using Reverse Transcription Polymerase Chain Reaction (RT-PCR), and PCSK9 is obtained through real-time PCR using taqman probe. The changes in the expression levels of, LDLR and HMG-CoA reductase genes were analyzed.
10μM의 심바스타틴만을 처리하였을 때, PCSK9 유전자의 발현이 증가하였고(도 5), HMG-CoA 환원효소 유전자의 발현이 증가한 것을 확인하였다. 하지만 복분자 추출물 단독 혹은 심바스타틴과 복분자 추출물을 병용 처리했을 때는 PCSK9 유전자의 발현은 감소하였고(도 5), LDLR의 발현은 증가하였으며(도 6), HMG-CoA 환원효소 유전자의 발현은 감소하는 것을 확인하였다(도 7). 본 실시예에서의 양성 대조군은 20μM의 베르베린(berberine)을 사용하였으며, 본 발명의 심바스타틴과 복분자 추출물을 병용처리했을 때가 베르베린 처리보다 효과적이라는 것을 확인할 수 있었다.When only 10 μM of simvastatin was treated, the expression of the PCSK9 gene was increased (FIG. 5), and the expression of the HMG-CoA reductase gene was confirmed to be increased. However, when bokbunja extract alone or simvastatin and bokbunja extract was treated in combination, the expression of PCSK9 gene was reduced (Fig. 5), LDLR expression was increased (Fig. 6), HMG-CoA reductase gene expression was confirmed to decrease (FIG. 7). 20 μM of berberine was used as a positive control in this example, and the combination of simvastatin and bokbunja extract of the present invention was more effective than berberine treatment.
실시예 6. 복분자 추출물 단독, 또는 스타틴계 화합물과 병용 처리되었을 때의 세포의 LDL 유입(uptake)량 변화 분석Example 6 Analysis of Changes in LDL Uptake of Cells When Treated with Bokbunja Extract Alone or in Combination with Statin Compounds
실시예 4와 5의 결과로부터, 본 발명에서 제공하는 실시예 2의 PCSK9의 발현 억제와 LDLR 발현 증가 효과를 나타냄을 확인하였으므로, 이러한 효과가 세포외 LDL에 대한 흡수 수준의 변화에 영향을 미치는지를 확인하고자 형광-표지된 LDL(fluorescently-labeled LDL)을 활용하여 복분자 추출물 단독 혹은 스타틴계 화합물과 병용 처리되었을 때의 세포의 LDL uptake량 변화 분석을 실시하였다.From the results of Examples 4 and 5, it was confirmed that the effect of inhibiting the expression of PCSK9 and increasing the LDLR expression of Example 2 provided in the present invention, whether this effect affects the change in the level of uptake for extracellular LDL Fluorescently-labeled LDL (fluorescently-labeled LDL) was used to identify changes in LDL uptake of cells when treated with bokbunja extract alone or in combination with statin compounds.
구체적으로, 96웰 흰색 투명바닥 세포배양 플레이트(white clear-bottom cell culture plate)에 3~4×104 cells/웰로 분주하고(FBS 포함 media), 24시간 배양 후, Serum-free media로 교환하여 24시간 기아상태(starvation)로 나뒀다. 기아상태(starvation) 후, media 제거하고 세척을 (어세이 완충용액으로 웰 당 100㎕씩) 3회 실시하였다. 각 웰당 10㎕의 형광 표지된(fluorescently-labeled) LDL 및 90㎕의 serum-free media를 분주하고, 24시간 동안 처리하였다. LDL 흡수의 특이성을 확인하기 위해, 10㎕의 형광 표지하지 않은(unlabled; UN) LDL을 웰에 처리하였다(UL-LDL, compete out the signal). 24시간 추가배양 후, 세척을 4회 실시하였고, 이후 어세이 완충용액을 웰당 100㎕씩 분주하고, 유세포 분석기에 적용하여 각 세포에서 발현하는 형광을 측정(Ex/Em=540nm/575nm)하였고, 그 결과를 도 8에 나타내었다.Specifically, aliquots of 3-4 × 10 4 cells / well into 96-well white clear-bottom cell culture plates (media containing FBS), followed by incubation for 24 hours, exchanged with Serum-free media. It was left starvation for 24 hours. After starvation, the media was removed and washed three times (100 μl per well with assay buffer). 10 μl of fluorescently-labeled LDL and 90 μl of serum-free media were dispensed per well and treated for 24 hours. To confirm the specificity of LDL uptake, 10 μl of unlabled (UN) LDL was treated in the wells (UL-LDL, compete out the signal). After further incubation for 24 hours, washing was performed 4 times, and then assay assay solution was dispensed 100 μl per well, and applied to flow cytometry to measure the fluorescence expressed in each cell (Ex / Em = 540 nm / 575 nm). The results are shown in FIG.
그 결과, 도 8에 나타낸 바와 같이, HepG2 세포에서 복분자 추출물 단독 혹은 심바스타틴과 복분자 추출물을 병용 처리했을 때는 LDL의 유입을 통계적으로 유의하게 증가시키는 결과를 나타내었다. As a result, as shown in Figure 8, when the bokbunja extract alone or simbastatin and bokbunja extract in combination treatment in HepG2 cells showed a significant increase in the influx of LDL.
실시예 7. 용매의 극성에 따른 복분자 열수 추출물의 분획물 제조Example 7 Preparation of Fractions of Bokbunja Hydrothermal Extract According to Polarity of Solvent
복분자 열수 추출물에 존재하는 다량의 화합물 중에서 물리화학적 성질이 유사한 화합물 군끼리 분류하기 위하여 보편적으로 이용되는 분획방법인 용매분획법을 이용하여 분획하였다.Among a large amount of compounds present in the bokbunja hydrothermal extract was fractionated using a solvent fractionation method, which is a commonly used fractionation method for classifying compounds having similar physicochemical properties.
용매분획법은 서로 섞이지 않는 용매 사이의 분배(solvent partition)를 이용한 방법으로 대표적인 분배의 예로는 물과 헥산(hexane), 에틸아세테이트(ethyl acetate), 부탄올(butanol), 물과 클로로포름(chloroform)으로 이루어지는 분획법이다. The solvent fractionation method uses a partition between solvents that are not mixed with each other. Examples of typical partitioning methods include water and hexane, ethyl acetate, butanol, water, and chloroform. It is a fractionation method which consists of.
이에, 본 발명에서는 복분자 열수 추출물에 n-헥산(n-hexane), 클로로포름(chloroform), 에틸아세테이트(ethyl acetate), 부탄올(butanol) 용매를 물과 동량으로 첨가한 후, 계통적으로 분획하여 각각의 용매별로 감압 농축하였다.Accordingly, in the present invention, n-hexane, chloroform, ethyl acetate, butanol and the like are added to the bokbunja hydrothermal extract in the same amount as water, and then fractionated systematically. Concentration under reduced pressure for each solvent.
그 결과, 0.66g의 n-헥산(n-hexane) 분획물, 0.08g의 클로로포름chloroform) 분획물, 0.3g의 에틸아세테이트(ethyl acetate) 분획물, 5.0g의 부탄올(butanol) 분획물 및 7.88g의 물 분획물을 획득하였다. As a result, 0.66 g of n-hexane fraction, 0.08 g of chloroform fraction, 0.3 g of ethyl acetate fraction, 5.0 g of butanol fraction and 7.88 g of water fraction Obtained.
실시예 8. 복분자 열수 추출물의 분획물을 이용한 PCSK9 유전자의 프로모터 부위의 전사 활성 확인Example 8. Confirmation of transcriptional activity of promoter region of PCSK9 gene using fractions of bokbunja hydrothermal extract
상기 실시예 7에서 획득한 각 분획물을 이용하여 PCSK9 프로모터(promoter) 활성 스크리닝 효능평가를 실시하였다. Each fraction obtained in Example 7 was used to evaluate the screening efficacy of PCSK9 promoter activity.
본 발명의 복분자 열수 추출물의 n-헥산(Hx) 분획물, 클로로포름(CHCl3) 분획물, 에틸아세테이트(EtAc) 분획물, 부탄올(BuOH) 분획물 및 물 분획물(H2O)을 이용하여 인간 PCSK9 프로모터 활성에 미치는 영향을 루시퍼라아제 리포터 어세이를 수행하였다. N-hexane (Hx) fraction, chloroform (CHCl 3 ) fraction, ethyl acetate (EtAc) fraction, butanol (BuOH) fraction and water fraction (H 2 O) of bokbunja hydrothermal extract of the present invention were used for human PCSK9 promoter activity. The effect was performed on a luciferase reporter assay.
상기 각각의 분획물을 농도별로 처리하였을 때, 다른 분획물에 비해 클로로포름 분획물과 에틸아세테이트 분획물에서 조금 더 낮은 농도에서 인간 PCSK9 프로모터 리포터의 활성을 억제하는 것을 확인하였다(도 9).When each fraction was treated by concentration, it was confirmed that the activity of the human PCSK9 promoter reporter at a slightly lower concentration in the chloroform fraction and ethyl acetate fraction compared to the other fractions (Fig. 9).
실시예 9. 복분자 열수 추출물의 분획물을 이용한 스타틴계 화합물과 병용 처리 시, PCSK9 유전자의 발현량 변화 분석Example 9 Analysis of Expression Change of PCSK9 Gene in Combination Treatment with Statin Compound Using Fractions of Bokbunja Hot-water Extract
상기 실시예 7에서 실시한 루시퍼라아제 어세이를 이용한 스크리닝 결과를 확인하기 위하여, 택맨-프로브를 활용한 실시간 중합효소연쇄반응(real-time PCR)를 이용하여 복분자 열수 추출물의 분획물과 스타틴계 화합물을 병용처리했을 때의 PCSK9 유전자의 변화를 분석하였다.In order to confirm the screening results using the luciferase assay performed in Example 7, fractions of the bokbunja hydrothermal extract and statin compounds were prepared using real-time PCR using Taqman-probe. Changes in the PCSK9 gene when combined were analyzed.
10μM의 심바스타틴과 100㎍/㎖의 복분자 열수 추출물의 분획물을 각각 처리하고 24시간 동안 배양하였다. 이후 세포로부터 total RNA를 분리하고 역전사 중합효소 연쇄반응(Reverse transcription polymerase chain reaction, RT-PCR)을 이용해 cDNA를 합성하고, 택맨 프로브(taqman probe)를 활용한 실시간 중합효소연쇄반응(real-time PCR)을 수행하여 PCSK9 유전자의 발현량을 확인하였다.Each fraction of 10 μM simvastatin and 100 μg / ml bokbunja hydrothermal extract was treated and incubated for 24 hours. Then, total RNA was separated from the cells, cDNA was synthesized using reverse transcription polymerase chain reaction (RT-PCR), and real-time PCR using a taqman probe. ) To confirm the expression level of the PCSK9 gene.
10μM의 심바스타틴만을 처리하였을 때, PCSK9 유전자의 발현이 증가하였지만, 복분자 열수 추출물 분획물 중에서 CHCl3 분획물과 EtAc 분획물을 심바스타틴과 병용 처리했을 때는 PCSK9 유전자의 발현이 감소하였다(도 10). 본 실시예 9에서는 양성 대조군으로 복분자 열수 추출물(BRE)을 사용하였으며, 10μM의 심바스타틴과 복분자 열수 추출물, CHCl3 분획물 및 EtAc 분획물을 병용처리했을 때가 효과적이라는 것을 확인할 수 있었다.When only 10 μM of simvastatin was treated, the expression of the PCSK9 gene was increased. However, when the CHCl 3 fraction and the EtAc fraction were co-treated with simvastatin, the expression of the PCSK9 gene was decreased (Fig. 10). In Example 9, bokbunja hydrothermal extract (BRE) was used as a positive control, and it was confirmed that the combination of 10 μM simvastatin and bokbunja hydrothermal extract, CHCl 3 fraction and EtAc fraction was effective.
실시예 10. 복분자 열수 추출물의 분획물을 이용한 스타틴계 화합물과 병용 처리 시, PCSK9 및 LDLR 단백질의 발현량 변화 분석Example 10 Analysis of Expression Changes of PCSK9 and LDLR Proteins in Combination Treatment with Statin Compounds Using Fractions of Bokbunja Hot-water Extracts
복분자 열수 추출물의 분획물 및 복분자 열수 추출물(BRE)의 처리에 따른 mRNA의 발현양상이 단백질의 발현 수준에서도 조절되는지를 확인하기 위하여, 웨스턴 블랏 분석를 통해 10μM의 심바스타틴과 복분자 열수 추출물의 분획물의 병용처리에 의한 PCSK9 및 LDLR의 단백질 발현 양상을 확인하였다.In order to confirm whether the expression pattern of mRNA according to the treatment of the fraction of Bokbunja hydrothermal extract and the treatment of Bokbunja hydrothermal extract (BRE) is regulated even at the level of protein expression, Western blot analysis was performed in the combination treatment of the fraction of 10 μM simvastatin and the fraction of Bokbunja hydrothermal extract. The protein expression pattern of PCSK9 and LDLR was confirmed.
10μM의 심바스타틴의 처리에 의해 PCSK9 단백질의 발현이 증가되는 것을 확인하였고, 심바스타틴과 본 발명의 복분자 열수 추출물의 클로로포름 분획물(CHCl3) 및 에틸아세테이트 분획물(EtAc) 및 물 분획물(H2O)을 병용처리한 경우, PCSK9의 발현량을 감소하였으며, LDLR 단백질의 발현은 증가된 것을 확인하였다(도 11).It was confirmed that the expression of PCSK9 protein was increased by the treatment of 10 μM simvastatin, and a combination of simvastatin and chloroform fraction (CHCl 3 ), ethyl acetate fraction (EtAc) and water fraction (H 2 O) of the bokbunja hydrothermal extract of the present invention was used. In the case of treatment, the expression level of PCSK9 was decreased, and the expression of LDLR protein was increased (FIG. 11).

Claims (13)

  1. 복분자 추출물 또는 이의 분획물을 유효성분으로 함유하며, PCSK9 유전자의 발현을 감소시키거나, 저밀도 지단백 수용체(LDLR)를 증가시키기 위한 조성물.A composition for containing a bokbunja extract or a fraction thereof as an active ingredient, to reduce the expression of the PCSK9 gene or to increase the low density lipoprotein receptor (LDLR).
  2. 복분자 추출물 또는 이의 분획물을 유효성분으로 함유하며, 스타틴계 화합물에 의해 증가된 PCSK9 유전자의 발현을 감소시키거나, 저밀도 지단백 수용체(LDLR)를 증가시키기 위한 조성물.A composition for containing a bokbunja extract or a fraction thereof as an active ingredient, to reduce the expression of the PCSK9 gene increased by the statin-based compound, or to increase the low density lipoprotein receptor (LDLR).
  3. 복분자 추출물 또는 이의 분획물을 유효성분으로 함유하며, PCSK9 유전자의 발현 감소 또는 저밀도 지단백 수용체(LDLR) 증가용 건강기능식품 조성물.Health functional food composition for reducing the expression of PCSK9 gene or increasing the low density lipoprotein receptor (LDLR) containing bokbunja extract or fractions thereof as an active ingredient.
  4. 제3항에 있어서, 상기 복분자 추출물의 용매는 탄소 수 1 내지 4의 저급 알코올, 물 및 이들의 혼합물 중에서 선택된 어느 하나인 것을 특징으로 하는 건강기능식품 조성물.The dietary supplement composition of claim 3, wherein the solvent of the bokbunja extract is any one selected from lower alcohols having 1 to 4 carbon atoms, water, and mixtures thereof.
  5. 제3항에 있어서, 상기 복분자 추출물의 분획물은 에틸아세테이트 분획물 또는 클로로포름 분획물인 것을 특징으로 하는 건강기능 식품 조성물.The health functional food composition according to claim 3, wherein the fraction of the bokbunja extract is an ethyl acetate fraction or a chloroform fraction.
  6. 제3항에 있어서, 상기 유효성분 이외에 추가로 영양제, 비타민, 전해질, 풍미제, 착색제, 중진제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올 및 탄산 음료에 사용되는 탄산화제 중에서 선택된 하나 이상을 포함하는 것을 특징으로 하는 건강기능식품 조성물.According to claim 3, In addition to the active ingredient nutritional supplements, vitamins, electrolytes, flavors, coloring agents, neutralizing agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH regulators, stabilizers, A dietary supplement comprising at least one selected from preservatives, glycerin, alcohols and carbonation agents used in carbonated beverages.
  7. 스타틴계 화합물 및 복분자 추출물 또는 이의 분획물을 유효성분으로 함유하며, PCSK9 유전자의 발현을 감소시키거나, 저밀도 지단백 수용체(LDLR)를 증가시키는 것을 특징으로 하는 고콜레스테롤혈증의 예방 또는 치료용 약학 조성물.A pharmaceutical composition for preventing or treating hypercholesterolemia, comprising a statin-based compound and a bokbunja extract or a fraction thereof as an active ingredient, reducing the expression of the PCSK9 gene or increasing the low density lipoprotein receptor (LDLR).
  8. 제7항에 있어서, 상기 스타틴계 화합물은 심바스타틴(simvastatin), 아토르바스타틴(atorvastatin), 세리바스타틴(cerivastatin), 로바스타틴(lovastatin), 피타바스타틴(pitavastatin), 로수바스타틴(rosuvastatin), 프라바스타틴(pravastatin), 플루바스타틴(fluvastatin) 및 메바스타틴(mevastatin) 중에서 선택된 하나 이상인 것을 특징으로 하는 고콜레스테롤혈증의 예방 또는 치료용 약학 조성물.According to claim 7, The statin compound is simvastatin (simvastatin), atorvastatin (atorvastatin), cerivastatin (cerivastatin), lovastatin (lovastatin), pitavastatin, rosuvastatin, rovavastatin (pravastatin) ), Fluvastatin and mevastatin (mevastatin) is a pharmaceutical composition for the prevention or treatment of hypercholesterolemia, characterized in that at least one.
  9. 제7항에 있어서, 상기 복분자 추출물의 용매는 탄소 수 1 내지 4의 저급 알코올, 물 및 이들의 혼합물 중에서 선택된 어느 하나인 것을 특징으로 하는 고콜레스테롤혈증의 예방 또는 치료용 약학 조성물.The pharmaceutical composition for preventing or treating hypercholesterolemia according to claim 7, wherein the solvent of the bokbunja extract is any one selected from lower alcohols having 1 to 4 carbon atoms, water, and mixtures thereof.
  10. 제7항에 있어서, 상기 복분자 추출물의 분획물은 에틸아세테이트 분획물 또는 클로로포름 분획물인 것을 특징으로 하는 고콜레스테롤혈증의 예방 또는 치료용 약학 조성물.8. The pharmaceutical composition for preventing or treating hypercholesterolemia according to claim 7, wherein the fraction of the bokbunja extract is an ethyl acetate fraction or a chloroform fraction.
  11. 제7항에 있어서, 상기 유효성분 이외에 추가로 담체, 부형제 또는 희석제를 포함하는 것을 특징으로 하는 고콜레스테롤혈증의 예방 또는 치료용 약학 조성물.8. The pharmaceutical composition for preventing or treating hypercholesterolemia according to claim 7, further comprising a carrier, an excipient, or a diluent in addition to the active ingredient.
  12. 복분자 추출물 또는 이의 분획물을 유효성분으로 포함하는 인간을 제외한 동물의 고콜레스테롤혈증의 예방 또는 치료용 수의학적 조성물.Veterinary composition for the prevention or treatment of hypercholesterolemia in animals other than humans, including bokbunja extract or fractions thereof as an active ingredient.
  13. 복분자 추출물 또는 이의 분획물을 유효성분으로 포함하는 고콜레스테롤혈증의 예방 또는 개선용 사료 첨가제.Feed additive for the prevention or improvement of hypercholesterolemia comprising the bokbunja extract or fractions thereof as an active ingredient.
PCT/KR2015/013365 2015-05-11 2015-12-08 Composition containing rubus coreanus extract or fraction thereof as active ingredient for increasing low-density lipoprotein receptor by inhibiting pcsk9 gene expression, and use thereof WO2016182161A1 (en)

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