WO2013129769A1 - Composition for treating or preventing hypertension - Google Patents

Composition for treating or preventing hypertension Download PDF

Info

Publication number
WO2013129769A1
WO2013129769A1 PCT/KR2012/011835 KR2012011835W WO2013129769A1 WO 2013129769 A1 WO2013129769 A1 WO 2013129769A1 KR 2012011835 W KR2012011835 W KR 2012011835W WO 2013129769 A1 WO2013129769 A1 WO 2013129769A1
Authority
WO
WIPO (PCT)
Prior art keywords
extract
compound
formula
epoxide hydrolase
composition
Prior art date
Application number
PCT/KR2012/011835
Other languages
French (fr)
Korean (ko)
Inventor
마승진
천길용
류동영
Original Assignee
목포대학교 산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from KR1020120021519A external-priority patent/KR101819606B1/en
Priority claimed from KR1020120031444A external-priority patent/KR101400735B1/en
Application filed by 목포대학교 산학협력단 filed Critical 목포대학교 산학협력단
Publication of WO2013129769A1 publication Critical patent/WO2013129769A1/en

Links

Images

Classifications

    • 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)
    • A61K36/84—Valerianaceae (Valerian family), e.g. valerian
    • 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
    • A23L33/40—Complete food formulations for specific consumer groups or specific purposes, e.g. infant formula
    • 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
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • 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
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/343—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide condensed with a carbocyclic ring, e.g. coumaran, bufuralol, befunolol, clobenfurol, amiodarone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives

Definitions

  • the present invention relates to a composition for treating or preventing hypertension.
  • Hypertension is the most frequent disease among chronic circulatory diseases, and it is relatively asymptomatic, but it can cause fatal complications such as stroke, heart failure, and coronary artery disease.
  • essential hypertension corresponds to 80% of hypertension and is a complex product of genetic and environmental factors, including family history of hypertension, race, salt intake, insulin resistance, obesity, and excessive drinking and aging. It is thought.
  • the epoxide hydrolase plays an important role in the metabolism of harmful foreign compound such as hormones, chemotherapy drugs, calcinegen, environmental pollutants, mycotoxins.
  • the mammalian soluble epoxide hydrolase is a homodimer consisting of two domains. Among them, the C-terminal domain catalyzes the epoxide hydrolysis, the N-terminal domain acts as the phosphatase, and the water-soluble epoxide hydrolase is the epoxyeicosatrienoic acids associated with hypertension. It is known to hydrolyze epoxy fatty acid intermediates such as).
  • the epoxide hydrolase is classified into microsomal epoxide hydrolase (mEH) and soluble epoxide hydrolase (sEH) according to its location and substrate selectivity, and is a plant natural product. It has a complementary function in the decomposition of.
  • the soluble epoxide hydrolase is mainly involved in metabolism of lipid oxides such as arachidonic acid and linoleic acid. Hydrolysis of the epoxyeicosatrienoic acid (EET) of arachidonic acid shows that they can regulate endothelial function by soluble epoxide hydrolase (sEH) by controlling their fusion to coronary endothelial phospholipids.
  • lipid oxides such as arachidonic acid and linoleic acid.
  • EET epoxyeicosatrienoic acid
  • SEH soluble epoxide hydrolase
  • SHR spontaneous hypertensive rats
  • male knockout sEH mice showed significantly lower blood pressure than wild-type mice, demonstrating that soluble epoxide hydrolase can regulate blood pressure.
  • the present invention effectively inhibits the water-soluble epoxide hydrolase, has a blood pressure lowering ability, is useful for the treatment, prevention or improvement of hypertension and does not cause side effects including natural products.
  • the antihypertensive action is excellent, and thus, the substance that inhibits the epoxide hydrolase is used for the treatment, improvement or prevention of cardiovascular diseases or as an active ingredient of the composition for the purpose. Since it can be used as an object, to provide an active ingredient of the composition for inhibiting the epoxide hydrolase.
  • the present invention provides a food composition for improving or preventing cardiovascular diseases, including the extract persimmon extract to achieve the above object as an active ingredient.
  • the present invention also provides a pharmaceutical composition for the treatment or prevention of cardiovascular diseases comprising a compound or a pharmaceutically acceptable salt thereof isolated from the extract of Scent.
  • the present invention provides a food composition for improving or preventing cardiovascular diseases comprising a compound or a pharmaceutically acceptable salt thereof isolated from the extract of Scent.
  • the cardiovascular disease is a disease including heart disease and vascular disease, and in a crowd consisting of hypertension, hypertensive heart disease, heart disease, arrhythmia, stroke, thrombosis, angina pectoris, heart failure, myocardial infarction, atherosclerosis and arteriosclerosis It may be any one selected, preferably hypertension or hypertensive heart disease.
  • blood pressure refers to the force that blood exerts on the vessel wall. When reading blood pressure, it is divided into systolic blood pressure (highest blood pressure) and diastolic blood pressure (lowest blood pressure).
  • systolic blood pressure is the pressure exerted on blood vessels as the heart contracts and releases blood
  • diastolic blood pressure is the pressure the blood vessel receives when the heart receives blood as it expands (relaxes).
  • hypertension is a disease in which the systolic blood pressure is 140 mmHg or more or the diastolic blood pressure is 90 mmHg or more in adults 18 years or older, and the cause is not found. Cases occur, and about 95% of all hypertension patients are essential hypertension. Diseases caused by the hypertension include hypertensive heart disease (hypertensive heart disease).
  • Atherosclerosis refers to a disease in which the walls of the arteries become thick and the elasticity of the arteries is reduced.
  • the blood supply to the organs that received the blood from the hardened arteries is reduced, so that additional diseases may develop.
  • An example of a typical disease related to this is coronary artery disease in which atherosclerosis changes in the coronary arteries that supply blood to the muscles of the heart.
  • the inhibitor of the soluble epoxide hydrolase since the inhibitor of the soluble epoxide hydrolase has an anti-inflammatory or antihypertensive effect, the inhibitory effect of the soluble epoxide hydrolase has the effect of treating, preventing or improving cardiovascular diseases such as hypertension. It is expected to be.
  • the antihypertensive action is excellent, and thus, the substance inhibiting the epoxide hydrolase is used for the treatment, improvement or prevention of a cardiovascular disease, or Can be used as an active ingredient.
  • the present invention relates to a pharmaceutical composition for the treatment or prevention of cardiovascular diseases comprising a compound isolated from the extract of sensation or pharmaceutically acceptable salt thereof as an active ingredient.
  • the present invention provides a food composition for improving or preventing cardiovascular diseases comprising a compound or a pharmaceutically acceptable salt thereof isolated from the extract of Scent.
  • examples of the food composition of the present invention include food, food additives, beverages or beverage additives.
  • the present invention provides a composition for inhibiting epoxide hydrolase (EH) comprising a compound isolated from the extract of Persimmon Scent as an active ingredient.
  • EH epoxide hydrolase
  • the epoxide hydrolase may be a soluble epoxide hydrolase (sEH).
  • the inhibitory composition refers to a composition capable of inhibiting the activity of the epoxide hydrolase or inhibiting the activity of the enzyme.
  • the compound is a pharmaceutically acceptable or pharmacological agent, including salts, prodrug conjugates, such as esters and amides, metabolites, hydrates and solvates, etc., as well as the entirety of the specified molecule. Meaning an active derivative.
  • composition is intended to include not only products comprising specified components in specified amounts, but also any products which are produced directly or indirectly from combinations of specified components in specified amounts.
  • pharmaceutically acceptable means that the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not be detrimental to its recipients.
  • the soluble epoxide hydrolase is a hydroxyeicosatrienoic acid (EETs) in the endothelial cells, smooth muscle and other cell types DHET) is an enzyme that converts to dihydroxy derivatives called.
  • the soluble epoxide hydrolase may be, for example, a human soluble epoxide hydrolase.
  • the inventors of the present invention study the anti-hypertensive substance derived from natural substance or the substance for the treatment or prevention of cardiovascular disease, which is much less likely to be related to the chemically synthesized drug and the possibility of safety related problems such as side effects or resistance is easy.
  • the sapson extract has excellent epoxide hydrolase inhibitory activity
  • the saccharin extract is a safety material used as a food.
  • the methanol extract and the hexane fraction of the methanol extract in the extract of the persimmon extract were more excellent in water-soluble epoxide hydrolase inhibitory effect than when the solvent or fraction solvent.
  • the present invention has been completed based on the above confirmed results.
  • the present invention relates to a food composition for improving or preventing cardiovascular diseases, including the extract of Nardostachyschinensis as an active ingredient.
  • the cardiovascular disease may be hypertension or hypertensive heart disease.
  • the extract may be prepared by a conventional extraction method used in connection with the preparation of plant extracts such as solvent extraction, supercritical extraction, subcritical extraction, soxhlet extraction, SDE extraction.
  • the solvent extraction method means a method of extracting one or two or more of the components in a solid or liquid sample using a solvent in order to extract a specific substance from the plant to be extracted.
  • the supercritical extraction is an extraction method using a supercritical fluid, also called a supercritical fluid extraction (SFE).
  • SFE supercritical fluid extraction
  • the supercritical extraction method uses a supercritical fluid that is a fluid in a state exceeding a critical temperature and a boundary pressure.
  • the supercritical fluid has a high density and has a low viscosity and a remarkably higher diffusion ability than a liquid. Compared to the above, there is an advantage of high efficiency, and carbon dioxide is mainly used as the supercritical fluid.
  • the subcritical extraction method is an extraction method using a fluid similar to a supercritical condition, that is, a critical temperature or a pressure lower than the critical pressure.
  • Subcritical Water Extraction (SWE) using a solvent is mainly used.
  • the steam distillation extraction (SDE) extraction method is an extraction method using a simultaneous distillation extraction apparatus, called a steam distillation method, and is the extraction method most commonly used to extract essential oils. It is a method of extracting essential oils from plants by passing hot steam through the extract and cooling the steam again.
  • the soxhlet extraction method is an extraction method using an soxhlet extraction device, which is mainly used for extracting oils and fats.
  • the persimmon extract is preferably prepared by solvent extraction, supercritical extraction, Soxhlet extraction or SDE extraction.
  • the extract of persimmon is at least one extraction solvent selected from the group consisting of water, alcohol having 1 to 4 carbon atoms of 50% to 100%, ethyl acetate, hexane, chloroform, methylene chloride and dichloromethane, preferably methanol It may be an extract extracted using.
  • the persimmon extract is extracted with methanol, that is, at least one fraction solvent selected from the group consisting of water, alcohol having 1 to 4 carbon atoms, ethyl acetate, hexane and dichloromethane in the extract persimmon methanol, preferably hexane It may be fractionated by.
  • methanol that is, at least one fraction solvent selected from the group consisting of water, alcohol having 1 to 4 carbon atoms, ethyl acetate, hexane and dichloromethane in the extract persimmon methanol, preferably hexane It may be fractionated by.
  • the present invention may be a pharmaceutical composition for the treatment or prevention of cardiovascular diseases comprising a compound or a pharmaceutically acceptable salt thereof isolated from the extract of Persimmon.
  • the present invention is a cardiovascular disease treatment comprising any one compound selected from the group consisting of a compound having a structure of formula (1) and a compound having a structure of formula (2) or a pharmaceutically acceptable salt thereof as an active ingredient Or a prophylactic pharmaceutical composition.
  • the cardiovascular disease may be hypertension or hypertensive heart disease.
  • the present invention may be a composition for inhibiting epoxide hydrolase comprising a compound or a pharmaceutically acceptable salt thereof isolated from the persimmon extract as an active ingredient.
  • the present invention is an epoxide hydrolysis comprising any one compound selected from the group consisting of a compound having a structure of Formula 1 and a compound having a structure of Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient It may be a composition for inhibiting enzyme.
  • the epoxide hydrolase may be a soluble epoxide hydrolase (sEH).
  • the present invention may be the use of the compound isolated from the extract of the Persimmon scent, specifically for the use of epoxide hydrolase inhibition or for the treatment, improvement or prevention of hypertension.
  • the present invention in this aspect is a compound having the structure of Formula 1 or It may be the use of a compound having a structure of Formula 2.
  • the use of the compound having the structure of Formula 1 or the compound having the structure of Formula 2 may be used for the treatment, prevention or improvement of cardiovascular diseases including anti-inflammatory use or anti-hypertensive use.
  • the extract of Gamsong an active ingredient of the food composition for improving or preventing the cardiovascular disease of the present invention, inhibits the decomposition of epoxy fatty acids such as EETs by inhibiting water-soluble epoxide hydrolase. It can be effective in the improvement or prevention of cardiovascular diseases, and the extract is a natural plant-derived substance unlike the existing cardiovascular disease treatment agent of artificial compound, so the side effects are not a problem, the present invention
  • the extract derived from the extract of the present invention may inhibit NO production and inhibit epoxide hydrolase, the prophylactic or therapeutic effect of cardiovascular disease is recognized, and not only has an anti-inflammatory effect, Since it is a substance derived from a safe plant extract, safety can be ensured with respect to side effects and the like, and thus, it can be used for the treatment, improvement or prevention of cardiovascular diseases, especially hypertension.
  • FIG. 1 is a schematic diagram showing a process for preparing the extract and fractions of the persimmon scent in accordance with an embodiment of the present invention.
  • Figure 2 is a graph showing the yield according to the extraction solvent of the extract persimmon flavor according to an embodiment of the present invention, the horizontal axis represents the extraction solvent, the vertical axis represents the extraction yield.
  • FIG. 3 is a graph confirming the cell viability (%) for confirming the cytotoxicity of the extract persimmon according to an embodiment of the present invention
  • the horizontal axis of the graph means the content of the extract ( ⁇ g / mL)
  • the vertical axis of the graph means cell viability
  • G ⁇ means hydrothermal extract
  • GM means methanol extract
  • GE7 means 70% ethanol aqueous extract
  • GE 95% Mean ethanol extract
  • GH means n-hexane extract
  • GEt means ethyl acetate extract.
  • Figure 4 is a graph (% of control) showing the NO generating ability of the extract according to an embodiment of the present invention as an activity compared to the control, the horizontal axis of the graph means the type of extract, the vertical axis of the graph is NO generating ability
  • GE means 95% ethanol aqueous extract
  • GE7 means 70% ethanol aqueous extract
  • GEt means ethyl acetate extract
  • GH means n-hexane extract
  • G ⁇ stands for hot water extract
  • GM stands for methanol extract.
  • FIG. 5 is a graph showing the water-soluble epoxide hydrolase of the extract persimmon extract according to an embodiment of the present invention (IC 50 ), the horizontal axis of the graph means the type of extract, the vertical axis of the graph is a water-soluble epoxide Mean amount (IC 50 , ⁇ g / mL) to inhibit 50% hydrolase, in each graph, GM means methanol extract, GE means 95% ethanol aqueous solution, GE7 70% Mean ethanol extract, GEt means ethyl acetate extract, GH means n-hexane extract, G ⁇ means hot water extract.
  • IC 50 water-soluble epoxide hydrolase of the extract persimmon extract according to an embodiment of the present invention
  • n-hexane fr Means hexane fraction, chloroform fr.
  • chloroform fr Means chloroform fraction, ethyl acetate fr.
  • butanol Fractions, water fr. Means water fraction.
  • Figure 7 is a photograph showing the TLC results of the hexane fraction of the extract of the persimmon flavor according to an embodiment of the present invention
  • 1 of the horizontal axis of the graph means hexane fraction
  • 3 means sub-fraction 2 of the hexane fraction
  • 4 means sub-fraction 3 of the hexane fraction
  • 4 means sub-fraction 5 of the hexane fraction.
  • Figure 8 shows the epoxide hydrolase inhibitory activity of the column fraction of the sub-fraction 2 of the hexane fraction of the extract of the persimmon extract according to an embodiment of the present invention
  • the horizontal axis of the graph is fraction according to the solvent elution amount of the column It means the type of the result
  • the vertical axis of the graph means the amount of use (IC 50 , ⁇ g / mL) for inhibiting 50% of the water-soluble epoxide hydrolase.
  • Figure 9 is a sub-fraction of No. 2 of the hexane fraction of the extract persimmon extract according to an embodiment of the present invention. Sephadex LH-20 Chromatography of the Elution Fraction Result.
  • FIG. 10 is a photograph showing the Sephadex LH-20 chromatography results of the sub-fraction 3 of the hexane fraction of the extract of Persimmon flavor according to one embodiment of the present invention.
  • FIG 11 is a No. 2 fraction of the hexane fraction of the hexane fraction of extract persimmon according to one embodiment of the present invention.
  • Figure 16 shows the GC-MS spectrum of Compound 1 separated from the eluted fraction.
  • FIG. 12 and 13 are photographs showing the NMR spectral output of Compound 2 separated from sub-fraction 3 of the hexane fraction of the extract of Persimmon Fragrance according to one embodiment of the present invention, and FIG. 12 shows the 1 H-NMR spectral output. It is a photograph, Figure 13 is a photograph showing the result of the 13 C-NMR spectrum.
  • NMR spectrum product of Compound 1 separated from the eluted fraction product Figure 14 is a picture showing the 1 H-NMR spectrum results
  • Figure 15 is a picture showing the 13 C-NMR spectrum results
  • Figure 16 is the EI -MS spectral photograph.
  • the present invention relates to a food composition for improving or preventing cardiovascular diseases, including the extract as an active ingredient.
  • the cardiovascular disease may preferably be hypertension.
  • the persimmon extract may be a persimmon methanol extract, the persimmon extract may be a hexane fraction of the persimmon methanol extract.
  • the food composition may be a functional food composition.
  • the sense songhyang (Nardostachyschinensis) bulriwoomyeo is also known as a perennial plant belonging to the mountains to exchange valerianaceae originating in India or Nepal, fragrant spikenard Reply to this because its taste, spikenard, and Mitch, also known as pine fragrance.
  • the persimmon fragrance is cylindrical in shape with a bow shape, and the outer surface is glossy to yellowish brown to brown, and the cut surface is yellowish brown.
  • the outer surface has 5 to 6 longitudinal folds and irregular folds therebetween, which is hard, long and thick with roots, with beard roots and soft and easy to bend. Cortex divided into dark brown to form pieces, and neck is yellowish white.
  • the sweet scent is said to give a refreshing feeling, bitter taste, spicy and sweet, and in the private sector, it is said to be effective for tooth decay and hemorrhoids. It has been used as an analgesic material since ancient times, and Jews are known as nad fragrance, one of the balms used for weddings.
  • the persimmon incense may be above ground or underground, and may be any one selected from the group consisting of leaves, stems, roots, flowers, fruits, and mixtures thereof.
  • Extract of the present invention may be prepared according to a conventional land plant, for example, root, leaf, stem, flower, fruit or a method for preparing an extract to extract a mixture thereof, solvent extraction method, supercritical extraction method, sub It can be prepared by conventional extraction methods used in connection with the preparation of plant extracts, such as critical extraction, soxhlet extraction, SDE extraction.
  • the solvent extraction method means a method of extracting one or two or more of the components in a solid or liquid sample using a solvent in order to extract a specific substance from the plant to be extracted.
  • the solvent extraction may be cold extraction, hot extraction or thermal extraction, and may be performed using a conventional extraction device, an ultrasonic mill extractor or a fractionator.
  • the supercritical extraction is an extraction method using a supercritical fluid, also called a supercritical fluid extraction (SFE).
  • SFE supercritical fluid extraction
  • the supercritical extraction method uses a supercritical fluid that is a fluid in a state exceeding a critical temperature and a boundary pressure.
  • the supercritical fluid has a high density and has a low viscosity and a remarkably higher diffusion ability than a liquid.
  • the subcritical extraction method is an extraction method using a fluid similar to a supercritical condition, that is, a critical temperature or a pressure lower than the critical pressure.
  • Subcritical Water Extraction (SWE) using a solvent is mainly used.
  • the steam distillation extraction (SDE) extraction method is an extraction method using a simultaneous distillation extraction apparatus, called a steam distillation method, and is the extraction method most commonly used to extract essential oils. It is a method of extracting essential oils from plants by passing hot steam through the extract and cooling the steam again.
  • the soxhlet extraction method is an extraction method using an soxhlet extraction device, which is mainly used for extracting oils and fats.
  • the persimmon extract is preferably prepared by solvent extraction, supercritical extraction, Soxhlet extraction or SDE extraction.
  • the solvent extraction method may be performed by extracting with an extraction solvent or by adding a fractional solvent to the crude extract prepared by extraction with an extraction solvent.
  • the extraction solvent may be at least one selected from the group consisting of water and an organic solvent.
  • the organic solvent may be a polar solvent such as alcohol having 1 to 5 carbon atoms such as methanol or ethanol, ethyl acetate or acetone, and a nonpolar solvent of ether, chloroform, benzene, hexane or dichloromethane, or a mixed solvent thereof.
  • the extraction solvent of the present invention may preferably be at least one solvent selected from the group consisting of water and alcohols having 1 to 4 carbon atoms, and more preferably at least one selected from the group consisting of alcohols having 1 to 4 carbon atoms and hexane. More preferably, ethanol or methanol, most preferably methanol.
  • the extract of the present invention may be a fraction further subjected to the fractionation process after adding a fractional solvent to the crude extract extracted with the solvent.
  • the fractional solvent is a solvent selected from the group consisting of ethyl acetate, ether, chloroform, benzene, hexane, methylene chloride, butanol, water and mixed solvents thereof, preferably hexane, chloroform and a mixture thereof. More preferably, it may be n-hexane.
  • the prepared extract or the fraction obtained by performing the fractionation process can be filtered or concentrated or dried to remove the solvent, it can be carried out both filtration, concentration and drying.
  • the filtration may be performed using a filter paper or a reduced pressure filter
  • the concentration may be concentrated under reduced pressure using a reduced pressure concentrator, for example, a rotary evaporator
  • the drying may be performed by, for example, a lyophilization method.
  • the extract may be obtained by the following method.
  • the extract may be prepared by adding methanol to the root of the extract persimmon, filtered and concentrated under reduced pressure, and then lyophilized.
  • the saenghyang fragrance fraction may be prepared by sequentially adding the fraction solvent of hexane, chloroform, ethyl acetate, butanol and water to the saenghyang extract, and then separating each solvent fraction.
  • the extract of Methanol fragrance extract is not only excellent in inhibiting ability of the water-soluble epoxide hydrolase, but also less likely to cause side effects or resistance as a substance derived from natural products, and has excellent safety. It can be used as an active ingredient of a food composition for the improvement or prevention of cardiovascular diseases, including.
  • the present invention relates to a cardiovascular disease improvement or prevention composition
  • a cardiovascular disease improvement or prevention composition comprising the extract as an active ingredient.
  • the cardiovascular disease is a disease including heart disease and vascular disease, any one selected from the group consisting of hypertension, hypertensive heart disease, heart disease, arrhythmia, stroke, thrombosis, angina pectoris, heart failure, myocardial infarction, atherosclerosis and arteriosclerosis And preferably hypertension or hypertensive heart disease.
  • the hypertension is a blood vessel-related disease, and may be arterial hypertension with high arterial blood pressure.
  • the hypertension may cause arteriosclerosis or hypertensive heart disease.
  • the extract of S. extract is able to effectively inhibit the water-soluble epoxide hydrolase, the blood pressure lowering ability of the extract was confirmed, the extract of S. extract is a cardiovascular disease such as hypertension and heart disease, arteriosclerosis or cerebral hemorrhage. It is evaluated to have a therapeutic, prophylactic or ameliorating effect.
  • the cardiovascular disease improvement or prevention composition comprising the extract of Persimmon as an active ingredient of the present invention may include the extract of 0.001 to 99.99% by weight, preferably 0.1 to 99% by weight based on the total weight of the composition. .
  • the cardiovascular disease improvement or prevention composition comprising the extract as an active ingredient has an excellent effect in that there is no problem of side effects in that it is an extract of a natural substance, and there is an improvement or prevention effect of the cardiovascular disease. .
  • composition for improving or preventing cardiovascular diseases comprising the extract of Persimmon Fragrance of the present invention as an active ingredient may be directly applied to humans.
  • the cardiovascular disease improvement or prevention composition comprising the extract as an active ingredient may include the extract as an active ingredient alone, and according to the other formulation, method and purpose of use additional ingredients, that is, pharmaceutical Or further nutritionally acceptable carriers, excipients, diluents or accessory ingredients.
  • the term "food” means a natural product or processed product containing one or more nutrients, and preferably means a state in which it can be directly eaten through some processing process. It is intended to include all dietary supplements, beverages, food additives and beverage additives.
  • Examples of the food of the present invention include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.
  • the food in the present invention includes special nutritional products (e.g., prepared oils, infants, baby food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g. ramen, noodles, etc.), health supplements, seasoned foods ( For example, soy sauce, miso, red pepper paste, mixed soy sauce), sauces, confectionery (e.g. snacks), dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, pickles (various kimchi, pickles, etc.), beverages ( Examples include, but are not limited to, fruits, vegetable drinks, soy milk, fermented beverages, and the like, and natural seasonings (eg, ramen soup).
  • the food, health functional food, beverages, food additives and beverage additives may be prepared by conventional manufacturing methods.
  • the health functional food is a biological defense rhythm control, disease prevention and the like having a food group or a food composition that has added value to the food by using physical, biochemical, biotechnological techniques, etc. It means a food that is designed and processed to fully express the gymnastics function on recovery.
  • the dietary supplement may include food acceptable food additives, and may further include appropriate carriers, excipients and diluents commonly used in the manufacture of dietary supplements.
  • the drink refers to a generic term for drinking to quench thirst or to enjoy the taste and is intended to include a functional beverage.
  • the beverage is not particularly limited to other ingredients other than including the sweet persimmon extract as an active ingredient in the indicated ratio as an active ingredient, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. have.
  • natural carbohydrates examples include monosaccharides such as glucose, fructose and other disaccharides such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like, and Sugar alcohols such as xylitol, sorbitol, and erythritol.
  • natural flavoring agents tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. .
  • the ratio of the natural carbohydrate may be generally about 1 to 20 g, preferably 5 to 12 g per 100 ml of the food composition of the present invention.
  • the composition of the present invention may further contain a pulp for the production of natural fruit juices, fruit juice drinks, vegetable drinks.
  • the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and Salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like.
  • These components can be used independently or in combination.
  • the proportion of such additives is not so critical, but may be selected in the range of 0 to 2,000 parts by weight per 100 parts by weight of the persimmon extract of the present invention.
  • the health functional beverage is a biological defense rhythm control or disease prevention of a beverage group or beverage composition which has added value to the beverage by using physical, biochemical and biotechnological techniques to act and express the function of the beverage for a specific purpose.
  • the health functional beverage is not particularly limited to other ingredients except for containing the persimmon extract of the present invention as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. .
  • natural carbohydrates examples include monosaccharides such as glucose, fructose and other disaccharides such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like, and xylitol Sugar alcohols such as sorbitol and erythritol.
  • natural flavoring agents tacumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.
  • the proportion of natural carbohydrates is generally about 1 to 20 g, preferably 5 to 12 g per 100 ml of the composition of the present invention.
  • the amount of the extract persimmon may be included in 0.01 to 15% by weight of the total food weight, the beverage composition is 100 ml It may be included in a ratio of 0.02 to 5 g, preferably 0.3 to 1 g as a reference.
  • Persimmon extract of the present invention inhibits the water-soluble epoxide hydrolase, it is considered that it can be useful in patients with cardiovascular diseases, specifically hypertension or hypertensive heart disease or when there is such a risk.
  • the present invention provides a pharmaceutical composition for treating or preventing cardiovascular diseases comprising a compound or a pharmaceutically acceptable salt thereof isolated from the extract of Persimmon Scent.
  • the cardiovascular disease may be hypertension or hypertensive heart disease.
  • the present invention provides a food composition for improving or preventing cardiovascular diseases comprising a compound or a pharmaceutically acceptable salt thereof isolated from the extract of Scent.
  • the cardiovascular disease may be hypertension or hypertensive heart disease.
  • the food composition may be a health functional food composition.
  • the substance that inhibits the epoxide hydrolase is used for the treatment, improvement or prevention of cardiovascular diseases, or the composition for the purpose. It can be used as an active ingredient of.
  • the present invention may be the use of the compound isolated from the extract of the persimmon flavor, specifically for the use of epoxide hydrolase inhibition or for the treatment, improvement or prevention of hypertension.
  • the use of the compound isolated from the extract of the sweet persimmon may be for the treatment, prevention or improvement of cardiovascular diseases, including anti-inflammatory use or antihypertensive use.
  • the present invention is a cardiovascular disease treatment comprising any one compound selected from the group consisting of a compound having a structure of formula (1) and a compound having a structure of formula (2) or a pharmaceutically acceptable salt thereof as an active ingredient Or to preventive pharmaceutical compositions.
  • the cardiovascular disease may be any one selected from the group consisting of hypertension, hypertensive heart disease, heart disease, stroke, thrombosis, angina pectoris, heart failure, myocardial infarction, atherosclerosis and arteriosclerosis, preferably hypertension or hypertensive heart disease More preferably hypertension.
  • the hypertension is a blood vessel-related disease, and means arterial hypertension with high arterial pressure.
  • the hypertension may cause arteriosclerosis or hypertensive heart disease.
  • the atherosclerosis refers to a disease in which the walls of the arteries are thickened and the elasticity of the arteries is reduced. When the atherosclerosis develops, the blood supply to the organs that received the blood from the hardened arteries is reduced, so that additional diseases may develop.
  • An example of a typical disease related to this is coronary artery disease in which atherosclerosis changes in the coronary arteries that supply blood to the muscles of the heart.
  • a pharmaceutical composition for treating or preventing cardiovascular diseases comprising any one compound selected from the group consisting of the compound having the structure of Formula 1 and the compound having the structure of Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient It may include one or more of the pharmaceutical diluents selected from saline, buffered saline, dextrose, water, glycerol and ethanol, diluents are not limited thereto.
  • the pharmaceutical composition for treating or preventing the cardiovascular disease may be applied differently depending on the purpose of administration and the disease.
  • the amount of active ingredient administered substantially depends on a variety of related factors, such as the disease to be treated, the extent of the patient's condition, whether it is co-administered with other drugs (eg chemotactic drugs), the patient's age, gender, weight, food, time of administration, It should be determined in consideration of the route of administration and the ratio of the composition.
  • the pharmaceutical composition for treating or preventing the cardiovascular disease may be adjusted according to the dosage form and the severity of the disease, and may be administered once or once or three times a day, but is not limited thereto.
  • the pharmaceutical composition for treating or preventing the cardiovascular disease of the present invention may be administered orally or parenterally.
  • Parenteral administration refers to administration of medications other than oral, namely rectal, intravenous, peritoneal and muscular, arterial, transdermal, nasal, inhaled, ocular, and subcutaneous.
  • the pharmaceutical composition for treating or preventing cardiovascular diseases may be formulated in any form such as oral dosage form, injectable solution or topical preparation.
  • Formulations are preferably prepared for oral and injectable administration (true solution, suspension or emulsion), most preferably in oral form such as tablets, capsules, soft capsules, aqueous pharmaceuticals, pills, granules and the like. desirable.
  • the peptide of the present invention may be filled into a soft capsule without an excipient, and may be made into a suitable formulation after being mixed or diluted with the carrier.
  • suitable carriers include starch, water, saline, Ringer's solution, dextrose and the like.
  • the pharmaceutical composition for treating or preventing the cardiovascular disease may be a pharmaceutical composition.
  • the pharmaceutical composition may be directly applied to animals including humans.
  • the animal is a biological group corresponding to a plant, and mainly ingests organic substances as nutrients, and means that digestion, excretion, and respiratory organs are differentiated, and preferably, vertebrates, more preferably mammals.
  • the mammal may be human, pig, cow or goat, etc., preferably human.
  • the pharmaceutical composition may include any one compound selected from the group consisting of the compound having the structure of Formula 1 and the compound having the structure of Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient, and Depending on the formulation, the method of use and the purpose of use, it may further comprise additional ingredients, ie, pharmaceutically acceptable or nutritionally acceptable carriers, excipients, diluents or accessory ingredients.
  • the pharmaceutical composition may be added to the nutrients, vitamins, electrolytes, flavors, coloring agents, neutralizing agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH regulators, and stabilizers in addition to the active ingredient. It may further contain an agent, a preservative, glycerin, alcohol, a carbonation agent used in the carbonated beverage, and the like.
  • the carrier, excipient or diluent may be lactose, dextrose, sucrose, sorbitol, mannitol, ziitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose Selected from the group consisting of loose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin, saline It may be one or more, but is not limited to any conventional carrier, excipient or diluent can be used.
  • the components may be added independently or in combination to any one compound selected from the group consisting of a compound having the structure of Formula 1 and a compound having the structure of Formula 2 or a pharmaceutically acceptable salt thereof as the active ingredient. Can be.
  • the pharmaceutical composition is 0.001% to 99.9% by weight of any one compound selected from the group consisting of a compound having the structure of Formula 1 and a compound having the structure of Formula 2 or a pharmaceutically acceptable salt thereof based on the total weight of the composition %, Preferably 0.1% to 99% by weight, more preferably 1% to 50% by weight.
  • the pharmaceutical composition may further include conventional fillers, extenders, binders, disintegrants, surfactants, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers or preservatives when formulated, and oral or parenteral.
  • conventional fillers extenders, binders, disintegrants, surfactants, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers or preservatives when formulated, and oral or parenteral.
  • solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, water, or the like in the active ingredient. It may be prepared by mixing cross or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used.
  • Liquid preparations for oral use include suspensions, solvents, emulsions, and syrups.In addition to the commonly used simple diluents, water and liquid paraffin, various excipients may include, for example, wetting agents, sweeteners, fragrances, and preservatives. have.
  • the formulation of the pharmaceutical composition of the present invention may be in a preferred form depending on the method of use, in particular by adopting methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. It can be formulated. Examples of specific formulations include warnings, granules, lotions, linings, limonades, powders, syrups, ointments, liquids, aerosols, EXTRACTS, elixirs, ointments, liquid extracts, emulsions, Suspensions, premises, acupuncture, eye drops, tablets, suppositories, injections, spirits, capsules, creams, pills, soft or hard gelatin capsules.
  • compositions of the present invention are preferably formulated using appropriate methods known in the art or using methods disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA. Can be converted.
  • the dosage of the pharmaceutical composition according to the present invention may be appropriately selected by those skilled in the art in consideration of the method of administration, the age, sex and weight of the recipient, the severity of the disease, and the like.
  • the pharmaceutical composition of the present invention is 0.000001 mg / kg / day to 1000 mg based on any one compound selected from the group consisting of a compound having a structure of Formula 1 and a compound having a structure of Formula 2 It can be administered as / kg / day. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.
  • the pharmaceutical composition of the present invention in addition to any one compound selected from the group consisting of a compound having a structure of the formula (1) and a compound having the structure of the formula (2) or a pharmaceutically acceptable salt thereof is a known cardiovascular system
  • the compound may further include a compound having a therapeutic effect on the disease, and may be included in an amount of 5 parts by weight to 100 parts by weight based on 100 parts by weight of the active ingredient.
  • the present invention also provides a food composition for improving or preventing cardiovascular diseases comprising any one compound selected from the group consisting of a compound having the structure of Formula 1 and a compound having the structure of Formula 2 or a salt thereof as an active ingredient. do.
  • a food composition for improving or preventing cardiovascular diseases comprising any one compound selected from the group consisting of a compound having the structure of Formula 1 and a compound having the structure of Formula 2 or a salt thereof as an active ingredient. do.
  • Examples of the food composition of the present invention include food, food additives, beverages or beverage additives.
  • a food composition comprising any one compound selected from the group consisting of a compound having the structure of Formula 1 and a compound having the structure of Formula 2 or a salt thereof as an active ingredient is an appropriate carrier, excipient, and the like commonly used in the preparation thereof. Diluents may be further included.
  • the term "food” means a natural product or processed product containing one or more nutrients, and preferably means a state in which it can be directly eaten through some processing process. It is intended to include all foods, food additives, health functional foods and beverages, preferably gum or candy.
  • any compound selected from the group consisting of a compound having the structure of Formula 1 and a compound having the structure of Formula 2 or a salt thereof of the present invention may be added, for example, various foods, drinks, gum , Candy, tea, vitamin complexes, and functional foods.
  • the food in the present invention includes special nutritional products (e.g., crude oil, young, baby food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g. ramen, noodles, etc.), health supplements, seasonings ( For example, soy sauce, miso, red pepper paste, mixed soy sauce), sauces, confectionery (e.g. snacks), dairy products (e.g.
  • fermented milk, cheese, etc. other processed foods
  • kimchi, pickles various kimchi, pickles, etc.
  • beverages examples include, but are not limited to, fruits, vegetable drinks, soy milk, fermented beverages, ice cream, etc., natural seasonings (eg, ramen soup, etc.), vitamin complexes, alcoholic beverages, alcoholic beverages, and other dietary supplements.
  • the food, beverage or food additives may be prepared by a conventional manufacturing method.
  • Functional food in the present invention is the control of biological defense rhythm, disease prevention and recovery of food groups or food compositions that have added value to the food by using physical, biochemical, biotechnological techniques, etc.
  • the functional food may include food acceptable food additives, and may further include appropriate carriers, excipients and diluents commonly used in the manufacture of functional foods.
  • the present invention provides a composition for inhibiting epoxide hydrolase (EH) comprising a compound isolated from the extract of Persimmon Scent as an active ingredient.
  • EH epoxide hydrolase
  • the epoxide hydrolase may be a soluble epoxide hydrolase (sEH).
  • the inhibitory composition refers to a composition capable of inhibiting the activity of the epoxide hydrolase or inhibiting the activity of the enzyme.
  • the present invention is an epoxide hydrolase comprising any one or a pharmaceutically acceptable salt thereof selected from the group consisting of a compound having a structure of Formula 1 and a compound having the structure of Formula 2 as an active ingredient It may be a composition for inhibiting (epoxide hydrolase, EH).
  • the epoxide hydrolase may be a soluble epoxide hydrolase (sEH).
  • the present invention provides a compound having the structure of Formula 1 or structure of Formula 2 It may be the use of the compound having a. The use may be for the inhibition of epoxide hydrolase.
  • the compound having the structure of Formula 1 or the compound having the structure of Formula 2 may be used for the prevention or treatment of cardiovascular diseases including anti-inflammatory use or anti-hypertensive use.
  • Any one compound selected from the group consisting of a compound having the structure of Formula 1 and a compound having the structure of Formula 2 may be used alone in the composition for inhibiting epoxide hydrolase, and other pharmacologically acceptable Carriers, excipients, diluents or accessory ingredients may further be included.
  • composition comprising any one compound selected from the group consisting of a compound having a structure of Formula 1 and a compound having the structure of Formula 2 is used as a medicament, or used in medicine or pharmaceutical use
  • Any one compound selected from the group consisting of a compound having the structure of Formula 1 and a compound having the structure of Formula 2 may be mixed with a pharmaceutically acceptable carrier or excipient or diluted with a diluent according to a conventional method. Can be.
  • the content of any one compound selected from the group consisting of the compound having the structure of Formula 1 and the compound of Formula 2 in the composition is 0.001% to 99.9%, 0.1% to 99% by weight Or it may be 1% to 20% by weight, but is not limited thereto, the content of the compound may be used to appropriately adjust to the desired content according to the mode of use and method of use of the composition.
  • Examples of the pharmaceutically acceptable carrier, excipient or diluent 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 and mineral oil, propylhydroxybenzoate, talc, magnesium stearate and mineral oil , Dextrin, calcium carbonate, propylene glycol, liquid paraffin, and one or more selected from the group consisting of physiological saline, but is not limited thereto, and any conventional carrier, excipient or diluent may be used.
  • compositions of the present invention can be formulated using any suitable method known in the art or using methods disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA).
  • the dosage of the composition may be determined in consideration of the method of administration, age, sex, severity, condition of the patient, absorption of the active ingredient in the body, inactivation rate and the drug used in combination, for example, based on the daily effective ingredient 0.1 mg / kg (body weight) to 500 mg / kg (body weight), 0.1 mg / kg (body weight) to 400 mg / kg body weight or 1 mg / kg body weight to 300 mg / kg body weight It can be administered, and can be administered once or divided into several times.
  • the dosage does not limit the scope of the invention in any aspect.
  • Persimmon Scent Nardostachyschinensis
  • the root dried dried persimmon scent was purchased as a medicinal herb from Power Ginseng Dang Pharmaceutical Co., Ltd., Sangdong, Mokpo-si, Jeonnam, Korea.
  • the prepared extract was Whatman NO. Filtration under reduced pressure with 2 (Whatman, England) yielded each of filtrate and residue.
  • the obtained residue was repeated twice in the same manner as described above to perform the extraction and filtration process.
  • the hot water extract in the obtained filtrate was freeze-dried (Freezing dryer, Samwon, Korea), and finally a saccharin hot water extract was obtained, and the remaining organic solvent extract was equipped with a cooling aspirator (CA-11, Eyela, Tokyo, Japan). Extract was prepared by vacuum concentration at 35 ⁇ 2 °C using a vacuum evaporator (N-2N, Eyela, Tokyo, Japan).
  • the extraction yield of the extract was measured and shown in FIG. 2.
  • the extraction yield was determined by the weight ratio (w / w,%) of the extract obtained relative to the sweet scent administered in the preparation of the extract.
  • the extraction yield for each solvent of the persimmon aroma was 14.1% for methanol extract (GM), 12.3% for the 95% ethanol aqueous solution extract (GE), and 70% for the extract. 11.7% of the ethanol aqueous solution extract (GE7), 7.1% of the ethyl acetate extract (GEt), 7.2% of the n-hexane extract (GH).
  • the yield of the hydrothermal extract was 8.7%.
  • a Nardostachyschinensis fraction In order to prepare a Nardostachyschinensis fraction, first, a methanol extract was prepared.
  • Example 1-1 20 L of methanol was added to 3.0 kg of the persimmon incense roots in the cold storage of Example 1-1, and ground with a homogenizer (BM-2 Nissei bio-mixer, Nihonseiki Kaiseiki LTD, Japan), as in Example 1-1, 3 Extraction was repeated several times to obtain a total of 60 L of extract of persimmon root methanol.
  • BM-2 Nissei bio-mixer Nihonseiki Kaiseiki LTD, Japan
  • the methanol extract obtained above was Whatman NO. After filtering under reduced pressure to 2, the filtrate was prepared by extracting the filtrate under a reduced pressure at 35 ⁇ 2 ° C. using a vacuum evaporator equipped with a cooling aspirator to obtain 425 g of extract. The extract was stored at -20 ° C.
  • the solvent fraction of the extract was first dissolved in distilled water 20.0 g (141.2 g dry wt. Eq.) Of methanol extract, then n-hexane (5.9 g), chloroform (10.3 g), ethyl acetate (0.3 g), butanol (1.7 g) and water (0.8 g) were sequentially added to the fractional solvent, followed by solvent fractionation.
  • the obtained fraction was concentrated under reduced pressure in the case of a fraction layer using an organic solvent to obtain a fraction, and the water fraction was lyophilized to obtain a fraction.
  • Fraction yield of the fraction was determined by the weight ratio (w / w,%) of the fraction obtained relative to the sweet smell administered in the extraction step.
  • the fraction of persimmon was obtained 5.9 g for n-hexane, 10.3 g for chloroform, 0.3 g for ethyl acetate, and 1.7 g for butanol.
  • nitric oxide (NO) production for each extract solvent of the extract of Persimmon extract prepared in Example 1, a RAW 264.7 macropharge (mouse) cell line as a cell line was purchased and cultured at ATCC.
  • the RAW 264.7 macropharge (mouse) cell line contains 10% FBS (GIBCO, 16000-044) and 1% penicillin-streptomycin (GIBCO, 15140-122) in an incubator at 5% CO 2 , 95% humidity and 37 ° C. Cultured using a 75 cm 2 T-flask containing the DMEM medium (GIBCO, 12100-046).
  • the cultured cells were used for the experiment while subcultured every two or three days.
  • the cell number of the RAW 264.7 was measured using a hemocytometer made of a single cell.
  • Cytotoxicity of the extract of the persimmon extract prepared in Example 1 was confirmed by the method of cell viability (%) using the RAW 264.7 cells through the MTT assay.
  • the RAW 264.7 cells were suspended in a monolayer using 0.25% trypsin-EDTA, and precultured by dispensing 10% FBS MEM medium to 1 x 10 5 / 200 ⁇ L per well of a 96well plate. After 24 hours, the sample was replaced with fresh medium, and the final sample concentration was 10 ⁇ g / mL, 20 ⁇ g / mL, 50 ⁇ g / mL and 100 ⁇ g / mL, and the total volume was 200 ⁇ L for 72 hours. Incubated.
  • MTT formazan was formed by adding 20 ⁇ L of 2 mg / mL MTT (2,5-diphenyl-2H-tetrazoliumbromide, Sigma, USA) solution (in PBS) and incubating for 3 to 4 hours.
  • the purple MTT formazan was correctly dissolved in 1 mL of DMSO solvent, and absorbance was measured at 540 nm with an ELISA microplate reader (680, BIO-RAD, JAPAN). The measurement results are shown in FIG. 3.
  • the hydrothermal extract (GW), 95% ethanol aqueous solution extract (GE), and 70% ethanol aqueous solution extract (GE7) of the extracts did not show toxicity to cells up to a concentration of 100 ⁇ g / mL
  • methanol extract (GM) it showed 96.8% cell viability at a concentration of 50 ⁇ g / mL, and it was confirmed that it was safe up to a concentration of 50 ⁇ g / mL
  • n-hexane extract (GH) and ethyl acetate extract (GEt) The case showed low virulence with 83% cell viability at a concentration of 100 ⁇ g / mL.
  • the sample treatment concentration of the extract for each extract solvent was determined to be 50 ⁇ g / mL in the experiment to confirm the NO production inhibitory ability and the inhibitory ability of the water-soluble epoxide hydrolase of the extract according to each extract solvent. It was.
  • the extract showed overall NO production inhibiting ability, and the NO production of the relatively polar solvent extract of the hydrothermal extract (GW) and the 70% ethanol aqueous solution extract (GE7)
  • the inhibitory activity was 80.2% and 78.3%, showing a lower inhibitory activity than the other extracts.
  • the non-polar solvent extracts of n-hexane extract (GH), ethyl acetate extract (GEt), methanol extract (GM) NO production was 61.3%, 62.4% and 56.3%, respectively, indicating a high inhibitory activity.
  • the measuring method is specifically as follows. First, 150 ⁇ L of 25 mM bis / TrisHCl buffer (pH 7.0, containing 0.1 mg / mL of BSA) solution was added to a 96 well plate, and 2 ⁇ L of DMSO in the blank well and the DMSO solution prepared in Example 1 were prepared. Each 2 ⁇ L was dispensed.
  • CMNPC solution which is a substrate
  • a fluorescent microplate reader excitation 340 nm, emission 460 nm
  • the IC 50 value of the methanol extract (GM) among the extracts of Saengsonghyang showed the best inhibitory activity at 4.5 ⁇ g / mL, 95% ethanol aqueous solution extract (GE) and n-hexane extract (GH).
  • the IC 50 value of) was 7.0 ⁇ g / mL or 7.5 ⁇ g / mL, respectively, and the IC 50 value of the hydrothermal extract (GW) was 123.5 ⁇ g / mL, respectively, showing the lowest inhibitory activity.
  • Example 2 In order to measure the inhibitory ability of nitric oxide (NO) generation by fractional solvent of the extract of the saenghyanghyang methanol using the optimum extraction solvent identified in Example 2, it was performed in the same manner as in Example 2-2. The measurement results are shown in FIG. 6.
  • the NO production inhibitory activity was highest in the n-hexane fraction, 49.7%, 38.7% in the chloroform fraction, 21.1% in the ethyl acetate fraction, and 11.6% in the butanol fraction. It was found that the water fraction was 1.9%, which was the weakest inhibitor of NO production.
  • Table 1 shows the results of measuring the inhibitory activity of the water-soluble epoxide hydrolase (sEH) by fractional solvents of the extracts of saenghyang-methanol using methanol as the optimal extraction solvent identified in Example 2.
  • SEH water-soluble epoxide hydrolase
  • the IC 50 value of the n-hexane fraction showed the strongest inhibitory activity as 3.5 ⁇ g / mL, the next is the chloroform fraction IC 50 value of 7.0 ⁇ g / mL, while the ethyl acetate fraction
  • the IC 50 values were 21.7 ⁇ g / Ml and 50.3 ⁇ g / mL in the and butanol fractions, respectively.
  • the extract of saenghyanghyang preferably the extract of saenghyanghyang
  • cardiovascular system including blood pressure lowering ability and antihypertensive activity It is suggested that it has a treatment, prevention, or amelioration effect on a related disease.
  • the hexane fraction since the hexane fraction has the highest inhibitory activity of the soluble epoxide hydrolase among the fractions of the extract of Gamsong-hyang, the cardiovascular system including the blood pressure lowering ability and the antihypertensive activity related to the soluble epoxide hydrolase inhibition activity from the above results. It can be confirmed that the disease has a therapeutic effect.
  • the silica gel adsorption column is made of slurry by mixing silica gel (40-63 ⁇ m 60, SILICYCLE, Canada) equivalent to 200 times of the sample with n-hexane: ethyl acetate 99: 1 (v / v). It was prepared by filling in a column (35 mm ⁇ 700 mm). Subsequently, the sample was dissolved in n-hexane: ethyl acetate 99: 1 (v / v) mixed solvent and filled in a column, and then eluted with 1 L of n-hexane: ethyl acetate 99: 1 (v / v) mixed solvent.
  • the Sephadex LH-20 column is prepared by mixing Sephadex LH-20 (17-0090-01, GE Healthcare, Sweden) with chloroform: methanol 1: 1 (v / v) to make slurry, and after 24 hr swelling (30 mm ⁇ 700 mm) was prepared by filling up to 500 mm.
  • the sample fractions were loaded and eluted with 700 mL with a mixed solvent system of chloroform: methanol 1: 1 (v / v).
  • Each eluted sub-fraction was aliquoted into 25 mL test tubes, 15 mL, concentrated to 0.6 mg / dry weight, and concentrated under reduced pressure to measure hsEH inhibitory activity. The measurement results are shown in FIG. 8.
  • the ratio (Ve / Vt) of the elution volumn to the bed volumn is in the range of 0.60 to 0.68.
  • 14 to No. 16 showed 34.6%, 51.1%, and 80.8% of hsEH inhibitory activity, respectively.
  • No. 14 to No. 16 eluted fractionation layer was confirmed by TLC.
  • 16 elution fractions were identified as a single spot. The 16 eluting fractions were selected as fractions for purification by high performance liquid chromatography (HPLC).
  • the purification by HPLC was specifically carried out in the following manner.
  • the column used for HPL was C18 column (10 ⁇ 250 mm, 5C18-AR-II, Nacalai, JAPAN), and 210 nm detection at a flow rate of 4.72 mL / min (Model 980E, JASCO, Japan). It was performed at a wavelength (MD-2010 Multiwavelength detector, JASCO, Japan).
  • the No. The resulting eluted fraction was separated by ODS-HPLC using 50% acetonitrile as a mobile phase, and the main peak was repeatedly extracted from the elution fraction of t R 25.86 minutes to obtain the active compound 1.
  • sub-fraction 3 was repeatedly fractionated from the elution fraction at t R 12.85 minutes by ODS-HPLC using 50% acetonitrile as a mobile phase to obtain compound 2 as an active substance.
  • Example 4-1 In order to confirm the structures of the two effective compounds identified in Example 4-1, GC-MS analysis, LC-MS analysis, and FT-NMR analysis were performed.
  • GC-MS analysis was analyzed by GC-MS (5975C, Agilent Tech., USA) method. Specifically, the analysis conditions were maintained for 3 minutes at an initial temperature of 60 °C, and then raised to 230 °C by 5 °C by 1 minute, and then maintained at this temperature for 20 minutes. Mass spectrometry was performed by ionizing the components separated from GC at 70 eV. The fragmentation patterns were qualitatively analyzed using the Wiely / NIST library. LC-MS analysis, specifically LC-ESI-MS analysis was performed using LC (Agilent 1100 HPLC) equipped with MS (ESI Ion Trap LC-MS System, Bruker, Germany).
  • 2D-NMR shift correlated spectroscopy (COSY), heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple bond correlation (HMBC) was analyzed.
  • COSY shift correlated spectroscopy
  • HMQC heteronuclear multiple quantum coherence
  • HMBC heteronuclear multiple bond correlation
  • GC-MS analysis for the structural analysis of compound 1 showed 222.2 m / z , 207.2 m / z , 189.2 m / z , 179.2 m / z , 161.1 m at 21.86 min at t R.
  • the 15 signals identified in compound 1 is consistent with the signal of the existing patchouli alcohol, it was confirmed that the compound is patchouli alcohol.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Animal Behavior & Ethology (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Medicinal Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Mycology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Microbiology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Organic Chemistry (AREA)
  • Cardiology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Botany (AREA)
  • Medical Informatics (AREA)
  • Biotechnology (AREA)
  • Alternative & Traditional Medicine (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pediatric Medicine (AREA)
  • Nutrition Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Coloring Foods And Improving Nutritive Qualities (AREA)
  • Medicines Containing Plant Substances (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The present invention relates to a composition for treating, alleviating, or preventing cardiovascular diseases. The cardiovascular diseases can be hypertension or hypertensive cardiac disorders. In addition, the present invention relates to a composition for inhibiting epoxide hydrolase. A Rhizoma Nardostachysis extract, which is one of active ingredients of the present invention, remarkably alleviates or prevents cardiovascular diseases due to excellent soluble epoxide hydrolase inhibitory effects and remarkable NO generation inhibitory effects, and is excellent for having no side effects since a natural substance is contained as an active ingredient. Further, a compound isolated from a Rhizoma Nardostachysis extract among active ingredients of the present invention shows remarkable NO generation inhibitory effects and inhibits epoxide hydrolase to be able to inhibit the hydrolysis of an epoxy fatty acid intermediate such as epoxyeicosatrienoic acids (EETs) which is associated with hypertension, and thus can exhibit effectiveness for treating or preventing cardiovascular diseases, including an antihypertensive effect.

Description

고혈압 치료 또는 예방용 조성물Composition for treating or preventing hypertension
본 발명은 고혈압 치료 또는 예방용 조성물에 관한 것이다.The present invention relates to a composition for treating or preventing hypertension.
급속한 경제발전과 더불어 질병양상도 크게 변하여 고혈압, 허혈성 심질환 및 뇌혈관질환 등 순환기계 질환의 중요성이 더욱 커지고 있다. 최근 통계청이 발표한 자료에 의하면, 2005년 총인구 중에서 65세 이상 노령인구의 비율은 9.1%를 차지하고 있고, 14세 이하 유년인구의 비중은 점차 줄어들고 있으며, 65세 이상 인구의 비중은 지난 2000년에 7.2%를 기록하여 우리 사회는 이미 고령화 사회로 진입한 것으로 평가된다. 또한, 2003년 기준으로 우리나라 국민의 평균수명은 77.5세로 노령인구가 급격하게 증가되고 있는 추세이다. 한편, 통계청이 발표한 우리나라 1인당 GNI는 2004년 14,162달러였으며, 매년 10% 이상 증가하고 있어 삶의 질은 점차 나아지고 있다고 할 수 있다.With the rapid economic development, the disease pattern has changed greatly, and the importance of circulatory diseases such as hypertension, ischemic heart disease and cerebrovascular disease is increasing. According to data released recently by the National Statistical Office, the proportion of the elderly aged 65 and over accounted for 9.1% of the total population in 2005, and the proportion of young people aged 14 and younger is gradually decreasing. 7.2% recorded that our society has already entered an aging society. In addition, as of 2003, the average life span of Korean people is 77.5 years old, and the elderly population is rapidly increasing. Meanwhile, Korea's GNI per capita announced by the National Statistical Office was $ 14,162 in 2004, and it is increasing by more than 10% every year.
이러한 사회적 조건의 변화에 의해, 과거 주요한 사망원인인 전염성 질병에 의한 사망자 수는 급격하게 줄어들고 있는 반면에 암, 고혈압, 뇌혈관질환, 당뇨병 등의 퇴행성 질환에 의한 사망자 수는 꾸준히 증가하고 있다.Due to such changes in social conditions, the number of deaths from infectious diseases, which are the major causes of death, is rapidly decreasing, while the number of deaths from degenerative diseases such as cancer, hypertension, cerebrovascular disease, and diabetes is steadily increasing.
고혈압은 만성 순환기계 질환 중 발생빈도가 가장 높은 질환으로 비교적 증상이 없는 편이지만 뇌졸증, 심부전, 관상동맥질환 등 치명적인 합병증을 유발할 수 있기 때문에 보다 적극적인 관리와 치료가 요구된다. 특히, 본태성 고혈압은 고혈압의 80%에 해당하며 유전적 인자와 환경적 인자의 복합적인 산물로서 고혈압의 가족력, 인종, 염분의 섭취량, 인슐린의 저항성, 비만 그리고 과도한 음주 및 노화 등이 관여하는 것으로 생각되고 있다.Hypertension is the most frequent disease among chronic circulatory diseases, and it is relatively asymptomatic, but it can cause fatal complications such as stroke, heart failure, and coronary artery disease. In particular, essential hypertension corresponds to 80% of hypertension and is a complex product of genetic and environmental factors, including family history of hypertension, race, salt intake, insulin resistance, obesity, and excessive drinking and aging. It is thought.
최근 고혈압에 대한 일련의 연구결과에 의하면, 수용성 에폭사이드 가수분해효소(soluble epoxide hydrolase, sEH)의 저해제(sEH inhibitor, sEHIs)가 혈압강하 등의 효과를 갖고 있음이 보고되고 있다.Recently, a series of studies on hypertension have reported that inhibitors of soluble epoxide hydrolase (sEH) (sEH inhibitor, sEHIs) have an effect of lowering blood pressure.
상기 에폭사이드 가수분해효소는 호르몬, 화학요법용 약물, 칼시노겐, 환경 오염물질, 진균독 등의 유해한 이물질 화합물의 대사에서 중요한 역할을 한다.The epoxide hydrolase plays an important role in the metabolism of harmful foreign compound such as hormones, chemotherapy drugs, calcinegen, environmental pollutants, mycotoxins.
보다 구체적으로, 포유류의 수용성 에폭사이드 가수분해효소는 두 개의 domain으로 구성된 homodimer이다. 이 중, C-terminal domain은 에폭사이드 가수분해에 촉매작용을 하는 역할을 하며, N-terminal domain은 인산가수분해효소 역할을 하며, 수용성 에폭사이드 가수분해효소는 고혈압과 관련이 있는 epoxyeicosatrienoic acids(EETs)와 같은 에폭사이드 지방산(epoxy fatty acid) 중간물질을 가수분해하는 것으로 알려져 있다.More specifically, the mammalian soluble epoxide hydrolase is a homodimer consisting of two domains. Among them, the C-terminal domain catalyzes the epoxide hydrolysis, the N-terminal domain acts as the phosphatase, and the water-soluble epoxide hydrolase is the epoxyeicosatrienoic acids associated with hypertension. It is known to hydrolyze epoxy fatty acid intermediates such as).
상기 에폭사이드 가수분해효소는 세포 내 위치와 기질 선택성 등에 따라 마이크로좀 에폭사이드 가수분해효소(mEH: microsomal epoxide hydrolase)와 가용성 에폭사이드 가수분해효소(sEH: soluble epoxide hydrolase)로 구분되며, 식물성 천연 생성물의 분해에 있어서 상보적인 작용을 한다.The epoxide hydrolase is classified into microsomal epoxide hydrolase (mEH) and soluble epoxide hydrolase (sEH) according to its location and substrate selectivity, and is a plant natural product. It has a complementary function in the decomposition of.
상기 가용성 에폭사이드 가수분해효소는 주로 아라키돈산, 리놀레산 등 지질 산화물의 대사에 관여한다. 아라키돈산의 에폭사이드(EET: epoxyeicosatrienoic acid)의 가수분해는 이들이 관상 내피 인지질로 융합되는 것을 조절함으로써, 가용성 에폭사이드 가수분해효소(sEH)에 의한 내피 기능을 조절할 수 있다는 것을 보여준다.The soluble epoxide hydrolase is mainly involved in metabolism of lipid oxides such as arachidonic acid and linoleic acid. Hydrolysis of the epoxyeicosatrienoic acid (EET) of arachidonic acid shows that they can regulate endothelial function by soluble epoxide hydrolase (sEH) by controlling their fusion to coronary endothelial phospholipids.
상기 수용성 에폭사이드 가수분해효소에 의해 EETs에서 dihydroxyeicosatrienoic acids(DHETs)로 분해되면 그 생화학적 활성을 잃게 되므로, 이로 인하여, 수용성 에폭사이드 가수분해효소 저해에 의해 EETs의 농도를 유지하게 되면 질병을 개선하는데 높은 효과를 보일 것으로 알려져 있다.When the water-soluble epoxide hydrolase is decomposed from EETs to dihydroxyeicosatrienoic acids (DHETs), its biochemical activity is lost. Thus, maintaining the concentration of EETs by inhibiting water-soluble epoxide hydrolase improves disease. It is known to show a high effect.
최근 자연발생 고혈압 쥐(SHR: spontaneous hypertensive rat)를 선택적 가용성 에폭사이드 가수분해효소 억제제로 처리하여 혈압이 현저히 저하되었다는 것이 밝혀졌다. 또한, 수컷 녹아웃 sEH 쥐는 야생형 쥐에 비해 현저히 낮은 혈압을 나타내어, 가용성 에폭사이드 가수분해효소가 혈압을 조절할 수 있다는 것을 보여주었다.Recently, spontaneous hypertensive rats (SHR) were treated with selective soluble epoxide hydrolase inhibitors, indicating a significant decrease in blood pressure. In addition, male knockout sEH mice showed significantly lower blood pressure than wild-type mice, demonstrating that soluble epoxide hydrolase can regulate blood pressure.
기존에 고혈압 치료제로 알려진 물질은 전신에 힘이 빠지거나, 백혈구 감소, 혈관 부종, 간기능 이상, 구토, 마른 기침, 두통, 식욕부진, 및 미각이상을 일으키는 등 부작용과 관련된 문제가 있는 것으로 보고되고 있다.Substances previously known to treat high blood pressure have been reported to have side effects such as weakness throughout the body, reduced white blood cells, angioedema, liver failure, vomiting, dry cough, headache, anorexia, and taste disorders. have.
따라서, 이러한 문제점을 해결하기 위하여, 부작용이 문제되지 아니하고 안전성이 확보되는 천연물질 유래 수용성 에폭사이드 가수분해효소 저해제의 개발이 시급히 요구되고 있다. 특히, 식품으로 사용되는 천연물 유래의 수용성 에폭사이드 가수분해효소 저해제의 경우, 안전성 확보가 용이하므로, 이에 대한 연구의 필요성이 증가되고 있다.Therefore, in order to solve such a problem, there is an urgent need to develop a water-soluble epoxide hydrolase inhibitor derived from a natural substance which does not cause side effects and ensures safety. In particular, in the case of water-soluble epoxide hydrolase inhibitors derived from natural products used as food, it is easy to ensure safety, the need for research on them is increasing.
상기와 같은 종래기술의 문제점을 해결하기 위하여, 본 발명은 수용성 에폭사이드 가수분해효소를 효과적으로 저해하고, 혈압 강하능이 있어, 고혈압의 치료, 예방 또는 개선에 유용하고 천연물에서 비롯하여 부작용 등이 문제되지 아니하며, 안정성이 우수한 고혈압 치료 또는 예방용 조성물의 유효성분을 제공하는 것을 목적으로 한다.In order to solve the problems of the prior art as described above, the present invention effectively inhibits the water-soluble epoxide hydrolase, has a blood pressure lowering ability, is useful for the treatment, prevention or improvement of hypertension and does not cause side effects including natural products. To provide an effective ingredient of the composition for treating or preventing hypertension having excellent stability.
또한, 에폭사이드 가수분해효소를 저해하는 경우, 항고혈압 작용이 뛰어나므로, 에폭사이드 가수분해효소를 저해하는 물질의 경우 심혈관계 질환의 치료, 개선 또는 예방을 위해 사용되거나 상기 목적의 조성물의 유효성분으로 사용될 수 있으므로, 상기 에폭사이드 가수분해효소 저해용 조성물의 유효성분을 제공하는 것을 목적으로 한다.In addition, when the epoxide hydrolase is inhibited, the antihypertensive action is excellent, and thus, the substance that inhibits the epoxide hydrolase is used for the treatment, improvement or prevention of cardiovascular diseases or as an active ingredient of the composition for the purpose. Since it can be used as an object, to provide an active ingredient of the composition for inhibiting the epoxide hydrolase.
본 발명은 상기 목적을 달성하기 위하여 감송향 추출물을 유효성분으로 포함하는 심혈관계 질환 개선 또는 예방용 식품조성물을 제공한다.The present invention provides a food composition for improving or preventing cardiovascular diseases, including the extract persimmon extract to achieve the above object as an active ingredient.
또한, 본 발명은 감송향 추출물로부터 분리된 화합물 또는 그의 약학적으로 허용되는 염을 유효성분으로 포함하는 심혈관계 질환 치료 또는 예방용 의약조성물을 제공한다.The present invention also provides a pharmaceutical composition for the treatment or prevention of cardiovascular diseases comprising a compound or a pharmaceutically acceptable salt thereof isolated from the extract of Scent.
또한, 본 발명은 감송향 추출물로부터 분리된 화합물 또는 그의 약학적으로 허용되는 염을 유효성분으로 포함하는 심혈관계 질환 개선 또는 예방용 식품조성물을 제공한다.In another aspect, the present invention provides a food composition for improving or preventing cardiovascular diseases comprising a compound or a pharmaceutically acceptable salt thereof isolated from the extract of Scent.
본 발명에 있어서, 심혈관계 질환이란 심장질환과 혈관질환을 포함한 질환으로, 고혈압, 고혈압성 심장질환, 심장병, 부정맥, 뇌졸중, 혈전증, 협심증, 심부전, 심근경색, 죽상경화증 및 동맥경화로 이루어진 군중에서 선택된 어느 하나일 수 있으며, 바람직하게는 고혈압 또는 고혈압성 심장질환일 수 있다.In the present invention, the cardiovascular disease is a disease including heart disease and vascular disease, and in a crowd consisting of hypertension, hypertensive heart disease, heart disease, arrhythmia, stroke, thrombosis, angina pectoris, heart failure, myocardial infarction, atherosclerosis and arteriosclerosis It may be any one selected, preferably hypertension or hypertensive heart disease.
본 발명에서, 혈압이란 혈액이 혈관 벽에 가하는 힘을 말한다. 혈압을 읽을 때에는 수축기 혈압(최고혈압)과 확장기 혈압(최저혈압)으로 나누어서 읽는다. 수축기 혈압은 심장이 수축하면서 혈액을 내보낼 때 혈관에 가해지는 압력이고, 확장기 혈압은 심장이 확장(이완)하면서 혈액을 받아들일 때 혈관이 받는 압력이다.In the present invention, blood pressure refers to the force that blood exerts on the vessel wall. When reading blood pressure, it is divided into systolic blood pressure (highest blood pressure) and diastolic blood pressure (lowest blood pressure). Systolic blood pressure is the pressure exerted on blood vessels as the heart contracts and releases blood, while diastolic blood pressure is the pressure the blood vessel receives when the heart receives blood as it expands (relaxes).
본 발명에서, 고혈압이란 18세 이상의 성인에서 수축기 혈압이 140 mmHg 이상이거나 확장기 혈압이 90 mmHg이상인 질환으로, 원인이 발견되지 않는 경우를 본태성(일차성) 고혈압과 원인이 밝혀져 있고 이에 의해 고혈압이 발생하는 경우으로 구분되며, 전체 고혈압 환자의 약 95%는 본태성 고혈압이다. 상기 고혈압에 의해 유발되는 질병으로는 고혈압심장병(hypertensive heart disease) 등이 있다.In the present invention, hypertension is a disease in which the systolic blood pressure is 140 mmHg or more or the diastolic blood pressure is 90 mmHg or more in adults 18 years or older, and the cause is not found. Cases occur, and about 95% of all hypertension patients are essential hypertension. Diseases caused by the hypertension include hypertensive heart disease (hypertensive heart disease).
본 발명에서, 동맥경화는 동맥의 벽이 두꺼워져 동맥의 탄력이 저하되는 질환을 의미한다. 상기 동맥경화가 발병되면 경화된 동맥으로부터 혈액을 공급받던 장기에 대한 혈액 공급이 감소하게 되어, 추가적인 질병이 발병될 수 있다. 이와 관련된 대표적인 질병의 예는 심장의 근육에 혈액을 공급하는 심장동맥에 동맥경화의 변화가 오는 심장동맥병 등이 있다.In the present invention, atherosclerosis refers to a disease in which the walls of the arteries become thick and the elasticity of the arteries is reduced. When the atherosclerosis develops, the blood supply to the organs that received the blood from the hardened arteries is reduced, so that additional diseases may develop. An example of a typical disease related to this is coronary artery disease in which atherosclerosis changes in the coronary arteries that supply blood to the muscles of the heart.
보고된 자료에 의하면, 수용성 에폭사이드 가수분해효소의 저해제는 항염증 또는 항고혈압 효과가 있으므로, 상기 수용성 에폭사이드 가수분해효소의 저해효과에 의하여 고혈압 등의 심혈관계 질환의 치료, 예방 또는 개선 효과가 있을 것으로 예상된다.According to the reported data, since the inhibitor of the soluble epoxide hydrolase has an anti-inflammatory or antihypertensive effect, the inhibitory effect of the soluble epoxide hydrolase has the effect of treating, preventing or improving cardiovascular diseases such as hypertension. It is expected to be.
구체적으로, 상기 에폭사이드 가수분해효소를 저해하는 경우, 항고혈압 작용이 뛰어나므로, 에폭사이드 가수분해효소를 저해하는 물질의 경우 심혈관계 질환의 치료, 개선 또는 예방을 위해 사용되거나 상기 목적의 조성물의 유효성분으로 사용될 수 있다.Specifically, when the epoxide hydrolase is inhibited, the antihypertensive action is excellent, and thus, the substance inhibiting the epoxide hydrolase is used for the treatment, improvement or prevention of a cardiovascular disease, or Can be used as an active ingredient.
이러한 측면에서, 본 발명은 감송향 추출물로부터 분리된 화합물 또는 그의 약학적으로 허용되는 염을 유효성분으로 포함하는 심혈관계 질환 치료 또는 예방용 의약조성물에 관한 것이다.In this aspect, the present invention relates to a pharmaceutical composition for the treatment or prevention of cardiovascular diseases comprising a compound isolated from the extract of sensation or pharmaceutically acceptable salt thereof as an active ingredient.
또한, 본 발명은 감송향 추출물로부터 분리된 화합물 또는 그의 약학적으로 허용되는 염을 유효성분으로 포함하는 심혈관계 질환 개선 또는 예방용 식품조성물을 제공한다. 본 발명의 식품 조성물의 예로는 식품, 식품첨가제, 음료 또는 음료첨가제를 들 수 있다.In another aspect, the present invention provides a food composition for improving or preventing cardiovascular diseases comprising a compound or a pharmaceutically acceptable salt thereof isolated from the extract of Scent. Examples of the food composition of the present invention include food, food additives, beverages or beverage additives.
또한, 본 발명은 다른 측면에 있어서, 감송향 추출물로부터 분리된 화합물을 유효성분으로 포함하는 에폭사이드 가수분해효소(epoxide hydrolase, EH) 저해용 조성물을 제공한다. 상기 에폭사이드 가수분해효소는 가용성 에폭사이드 가수분해효소(soluble epoxide hydrolase, sEH)일 수 있다.In another aspect, the present invention provides a composition for inhibiting epoxide hydrolase (EH) comprising a compound isolated from the extract of Persimmon Scent as an active ingredient. The epoxide hydrolase may be a soluble epoxide hydrolase (sEH).
상기 저해용 조성물은 상기 에폭사이드 가수분해효소의 활성을 저해하거나 상기 효소의 활성을 억제할 수 있는 조성물을 의미한다.The inhibitory composition refers to a composition capable of inhibiting the activity of the epoxide hydrolase or inhibiting the activity of the enzyme.
본 발명에 있어서, 화합물은 특정된 분자 전체뿐만 아니라, 상기 특정된 분자의 염, 프로드럭 컨쥬게이트, 예컨데, 에스테르 및 아미드, 대사물, 수화물 및 용매화물 등을 포함한 약제학적으로 허용되는 것 또는 약리학적 활성 유도체를 포함하는 의미이다.In the present invention, the compound is a pharmaceutically acceptable or pharmacological agent, including salts, prodrug conjugates, such as esters and amides, metabolites, hydrates and solvates, etc., as well as the entirety of the specified molecule. Meaning an active derivative.
본 발명에 있어서, 조성물은 특정된 성분을 특정된 양으로 포함하는 생성물뿐만 아니라, 특정된 양의 특정된 성분의 조합으로부터 직접적으로 또는 간접적으로 생성되는 어떠한 생성물도 포함하는 것으로 의도된다.In the present invention, the composition is intended to include not only products comprising specified components in specified amounts, but also any products which are produced directly or indirectly from combinations of specified components in specified amounts.
본 발명에 있어서, 약제학적으로 허용되는 의 의미는 담체, 희석제 또는 부형제가 제형의 다른 성분과 조화 가능해야 하며 이의 수용자에게 유해하지 않아야 됨을 의미한다.In the present invention, pharmaceutically acceptable means that the carrier, diluent or excipient must be compatible with the other ingredients of the formulation and not be detrimental to its recipients.
본 발명에 있어서, 가용성 에폭사이드 가수분해효소(soluble epoxide hydrolase, sEH)는 내피세포, 평활근 및 그 밖의 세포 유형에서 에폭시에이코사트리엔 산(epoxyeicosatrienoic acids, EETs)을 히드록시에이코사트리엔 산(DHET)이라 일컬어지는 디히드록시 유도체로 전환시키는 효소이다. 상기 가용성 에폭사이드 가수분해효소는 일 예로 인간 가용성 에폭사이드 가수분해효소일 수 있다.In the present invention, the soluble epoxide hydrolase (sEH) is a hydroxyeicosatrienoic acid (EETs) in the endothelial cells, smooth muscle and other cell types DHET) is an enzyme that converts to dihydroxy derivatives called. The soluble epoxide hydrolase may be, for example, a human soluble epoxide hydrolase.
이하, 본 발명을 보다 상세하게 설명한다.Hereinafter, the present invention will be described in more detail.
본 발명의 발명자들은 부작용이나 내성 등과 관련된 안전성이 문제될 가능성이 기존 화학적으로 합성된 약물에 비하여 훨씬 적고 투약이 쉬운 천연물질 유래 항고혈압 소재 또는 심혈관계 질환의 치료 또는 예방을 위한 물질에 대하여 연구하기 위하여, 다양한 천연물 유래 성분에 대한 수용성 에폭사이드 가수분해효소 저해능을 확인하던 중, 감송향 추출물이 에폭사이드 가수분해효소 저해 활성이 뛰어날 뿐만 아니라, 상기 감송향 추출물은 기존에 식품으로 사용되던 재료로 안전성도 우수하며, 나아가 감송향 추출물 중 메탄올 추출물과 상기 메탄올 추출물의 헥산 분획물이 다른 용매 또는 분획용매에 의한 경우보다 수용성 에폭사이드 가수분해효소 저해 효과가 더욱 우수하다는 것을 확인하였다.The inventors of the present invention study the anti-hypertensive substance derived from natural substance or the substance for the treatment or prevention of cardiovascular disease, which is much less likely to be related to the chemically synthesized drug and the possibility of safety related problems such as side effects or resistance is easy. In order to confirm the water-soluble epoxide hydrolase inhibitory ability against various natural ingredients, not only the sapson extract has excellent epoxide hydrolase inhibitory activity, but also the saccharin extract is a safety material used as a food. In addition, it was confirmed that the methanol extract and the hexane fraction of the methanol extract in the extract of the persimmon extract were more excellent in water-soluble epoxide hydrolase inhibitory effect than when the solvent or fraction solvent.
또한, 추가적인 연구에 의하여, 감송향 추출물로부터 분리된 본 발명의 화합물이 NO 생성을 억제할 수 있을 뿐만 아니라, 에폭사이드 가수분해효소를 저해하여 고혈압과 관련이 있는 에폭시에이코사트리엔 산(epoxyeicosatrienoic acids, EETs)과 같은 에폭시 지방산(epoxy fatty acid) 중간물질의 가수분해를 저해할 수 있다는 결과를 통해, 상기 화합물이 항고혈압 작용을 포함한 심혈관계 질환의 치료, 개선 또는 예방 효과가 우수하다는 것을 확인하였다.Further studies have shown that the compounds of the present invention isolated from the extracts of persimmon can not only inhibit NO production, but also inhibit epoxide hydrolase, which is associated with hypertension and epoxyeicosatrienoic acids. As a result of inhibiting the hydrolysis of epoxy fatty acid intermediates such as EETs, it was confirmed that the compound is excellent in the treatment, improvement or prevention of cardiovascular diseases including antihypertensive action. .
상기 확인 결과를 토대로 본 발명을 완성하게 되었다.The present invention has been completed based on the above confirmed results.
본 발명은 감송향(Nardostachyschinensis)추출물을 유효성분으로 포함하는 심혈관계 질환 개선 또는 예방용 식품 조성물에 관한 것이다.The present invention relates to a food composition for improving or preventing cardiovascular diseases, including the extract of Nardostachyschinensis as an active ingredient.
상기 심혈관계 질환은 고혈압 또는 고혈압성 심장질환일 수 있다.The cardiovascular disease may be hypertension or hypertensive heart disease.
상기 감송향 추출물은 용매 추출법, 초임계 추출법, 아임계 추출법, 속실렛 추출법, SDE 추출법 등 식물 추출물의 제조와 관련하여 사용되는 통상의 추출법으로 제조될 수 있다.The extract may be prepared by a conventional extraction method used in connection with the preparation of plant extracts such as solvent extraction, supercritical extraction, subcritical extraction, soxhlet extraction, SDE extraction.
상기 용매 추출법(Solvent extraction method)은 추출대상인 식물로부터 특정 물질을 추출하기 위하여 용매를 써서 고체 또는 액체시료 중에서 성분물질의 1종 또는 2종 이상을 추출하는 방법을 의미한다. 상기 초임계 추출법(supercritical extraction)은 초임계 유체를 이용한 추출법으로, 초임계 유체 추출법(supercritical fluid extraction, SFE)이라고도 한다. 상기 초임계 추출법은 임계온도 및 경계압력을 초과하는 상태의 유체인 초임계 유체를 이용한 것으로, 상기 초임계 유체는 높은 밀도가 있으면서 낮은 점성과 액체 보다 현저하게 높은 확산능력이 있기 때문에 기존 용매 추출법에 비하여 효율이 높은 장점이 있으며, 상기 초임계 유체로는 주로 이산화탄소 등이 사용된다. 상기 아임계 추출법(Subcritical Extraction)은 초임계 조건과 유사한 조건 즉, 임계온도 또는 임계압력보다 낮은 조건의 유체를 이용한 추출법으로, 주로 수용매를 이용한 아임계수 추출법(Subcritical Water Extraction, SWE)이 사용된다. 상기 SDE(Steam distillation extraction) 추출법은 동시증류추출장치를 이용한 추출법으로, 증기증류법이라고 불리우며, 정유를 추출하기 위해 가장 흔하게 사용되는 추출법이다. 뜨거운 증기를 추출대상물에 통과시킨 후, 다시 그 증기를 식히는 방법을 통해, 식물의 정유를 추출해내는 방법이다. 상기 속실렛 추출법은 속실렛 추출장치를 이용하여 추출하는 추출법으로, 주로 유지를 추출하기 위해 사용되는 방법이다.The solvent extraction method (Solvent extraction method) means a method of extracting one or two or more of the components in a solid or liquid sample using a solvent in order to extract a specific substance from the plant to be extracted. The supercritical extraction is an extraction method using a supercritical fluid, also called a supercritical fluid extraction (SFE). The supercritical extraction method uses a supercritical fluid that is a fluid in a state exceeding a critical temperature and a boundary pressure. The supercritical fluid has a high density and has a low viscosity and a remarkably higher diffusion ability than a liquid. Compared to the above, there is an advantage of high efficiency, and carbon dioxide is mainly used as the supercritical fluid. The subcritical extraction method is an extraction method using a fluid similar to a supercritical condition, that is, a critical temperature or a pressure lower than the critical pressure. Subcritical Water Extraction (SWE) using a solvent is mainly used. . The steam distillation extraction (SDE) extraction method is an extraction method using a simultaneous distillation extraction apparatus, called a steam distillation method, and is the extraction method most commonly used to extract essential oils. It is a method of extracting essential oils from plants by passing hot steam through the extract and cooling the steam again. The soxhlet extraction method is an extraction method using an soxhlet extraction device, which is mainly used for extracting oils and fats.
상기 감송향 추출물은 바람직하게는 용매 추출법, 초임계 추출법, 속실렛 추출법 또는 SDE 추출법으로 제조될 수 있다.The persimmon extract is preferably prepared by solvent extraction, supercritical extraction, Soxhlet extraction or SDE extraction.
상기 감송향 추출물은 물, 50% 내지 100%의 탄소수 1 내지 4의 알콜, 에틸아세테이트, 헥산, 클로로포름, 메틸렌 클로라이드 및 디클로로메탄으로 이루어진 군 중에서 1종 이상 선택된 추출용매, 바람직하게는 메탄올을 추출용매로 이용하여 추출한 추출물일 수 있다.The extract of persimmon is at least one extraction solvent selected from the group consisting of water, alcohol having 1 to 4 carbon atoms of 50% to 100%, ethyl acetate, hexane, chloroform, methylene chloride and dichloromethane, preferably methanol It may be an extract extracted using.
또한, 상기 감송향 추출물은 상기 메탄올로 추출한 것 즉, 감송향 메탄올 추출물에 물, 탄소수 1 내지 4의 알콜, 에틸아세테이트, 헥산 및 디클로로메탄으로 이루어진 군 중에서 1종 이상 선택된 분획용매, 바람직하게는 헥산으로 분획한 것일 수 있다.In addition, the persimmon extract is extracted with methanol, that is, at least one fraction solvent selected from the group consisting of water, alcohol having 1 to 4 carbon atoms, ethyl acetate, hexane and dichloromethane in the extract persimmon methanol, preferably hexane It may be fractionated by.
또한, 본 발명은 감송향 추출물로부터 분리된 화합물 또는 그의 약학적으로 허용되는 염을 유효성분으로 포함하는 심혈관계 질환 치료 또는 예방용 의약조성물일 수 있다.In addition, the present invention may be a pharmaceutical composition for the treatment or prevention of cardiovascular diseases comprising a compound or a pharmaceutically acceptable salt thereof isolated from the extract of Persimmon.
구체적으로, 본 발명은 하기 화학식 1의 구조를 갖는 화합물 및 하기 화학식 2의 구조를 갖는 화합물로 이루어진 군 중에서 선택된 어느 하나의 화합물 또는 그의 약학적으로 허용되는 염을 유효성분으로 포함하는 심혈관계 질환 치료 또는 예방용 의약조성물일 수 있다.Specifically, the present invention is a cardiovascular disease treatment comprising any one compound selected from the group consisting of a compound having a structure of formula (1) and a compound having a structure of formula (2) or a pharmaceutically acceptable salt thereof as an active ingredient Or a prophylactic pharmaceutical composition.
[화학식 1][Formula 1]
[규칙 제91조에 의한 정정 05.03.2013]　
Figure WO-DOC-FIGURE-47
[Revision under Rule 91 05.03.2013]
Figure WO-DOC-FIGURE-47
[화학식 2][Formula 2]
[규칙 제91조에 의한 정정 05.03.2013]　
Figure WO-DOC-FIGURE-49
[Revision under Rule 91 05.03.2013]
Figure WO-DOC-FIGURE-49
상기 심혈관계 질환은 고혈압 또는 고혈압성 심장질환일 수 있다.The cardiovascular disease may be hypertension or hypertensive heart disease.
또한, 본 발명은 감송향 추출물로부터 분리된 화합물 또는 그의 약학적으로 허용되는 염을 유효성분으로 포함하는 에폭사이드 가수분해효소 저해용 조성물일 수 있다.In addition, the present invention may be a composition for inhibiting epoxide hydrolase comprising a compound or a pharmaceutically acceptable salt thereof isolated from the persimmon extract as an active ingredient.
구체적으로, 본 발명은 하기 화학식 1의 구조를 갖는 화합물 및 하기 화학식 2의 구조를 갖는 화합물로 이루어진 군 중에서 선택된 어느 하나의 화합물 또는 그의 약학적으로 허용되는 염을 유효성분으로 포함하는 에폭사이드 가수분해효소 저해용 조성물일 수 있다.Specifically, the present invention is an epoxide hydrolysis comprising any one compound selected from the group consisting of a compound having a structure of Formula 1 and a compound having a structure of Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient It may be a composition for inhibiting enzyme.
[화학식 1][Formula 1]
[규칙 제91조에 의한 정정 05.03.2013]　
Figure WO-DOC-FIGURE-54
[Revision under Rule 91 05.03.2013]
Figure WO-DOC-FIGURE-54
[화학식 2][Formula 2]
[규칙 제91조에 의한 정정 05.03.2013]　
Figure WO-DOC-FIGURE-56
[Revision under Rule 91 05.03.2013]
Figure WO-DOC-FIGURE-56
상기 에폭사이드 가수분해효소는 가용성 에폭사이드 가수분해효소(soluble epoxide hydrolase, sEH)일 수 있다.The epoxide hydrolase may be a soluble epoxide hydrolase (sEH).
또한, 본 발명은 감송향 추출물로부터 분리된 화합물의 용도, 구체적으로 에폭사이드 가수분해효소 저해용 용도 또는 고혈압 치료, 개선 또는 예방용 용도일 수 있다.In addition, the present invention may be the use of the compound isolated from the extract of the Persimmon scent, specifically for the use of epoxide hydrolase inhibition or for the treatment, improvement or prevention of hypertension.
상기한 바와 같이, 하기 화학식 1의 구조를 갖는 화합물 또는 하기 화학식 2의 구조를 갖는 화합물은 에폭사이드 가수분해효소의 저해 효과를 갖는 것이므로, 이러한 측면에서 본 발명은 상기 화학식 1의 구조를 갖는 화합물 또는 상기 화학식 2의 구조를 갖는 화합물의 용도일 수 있다.As described above, since the compound having the structure of Formula 1 or the compound having the structure of Formula 2 has an inhibitory effect of epoxide hydrolase, the present invention in this aspect is a compound having the structure of Formula 1 or It may be the use of a compound having a structure of Formula 2.
[화학식 1][Formula 1]
[규칙 제91조에 의한 정정 05.03.2013]　
Figure WO-DOC-FIGURE-61
[Revision under Rule 91 05.03.2013]
Figure WO-DOC-FIGURE-61
[화학식 2][Formula 2]
[규칙 제91조에 의한 정정 05.03.2013]　
Figure WO-DOC-FIGURE-63
[Revision under Rule 91 05.03.2013]
Figure WO-DOC-FIGURE-63
구체적으로, 상기 에폭사이드 가수분해효소를 저해하는 경우, 항염 효과 또는 항고혈압을 포함한 심혈관계 질환의 예방 또는 치료 효과가 있다. 따라서, 상기 화학식 1의 구조를 갖는 화합물 또는 상기 화학식 2의 구조를 갖는 화합물의 용도는 항염 용도 또는 항고혈압 치료용 용도를 포함한 심혈관계 질환의 치료, 예방 또는 개선용 용도일 수 있다.Specifically, when inhibiting the epoxide hydrolase, there is an anti-inflammatory effect or a prophylactic or therapeutic effect of cardiovascular diseases including antihypertensive. Therefore, the use of the compound having the structure of Formula 1 or the compound having the structure of Formula 2 may be used for the treatment, prevention or improvement of cardiovascular diseases including anti-inflammatory use or anti-hypertensive use.
상술한 바와 같이, 본 발명의 심혈관계 질환 개선 또는 예방용 식품조성물의 유효성분인 감송향 추출물은 수용성 에폭사이드 가수분해효소를 저해하여 EETs와 같은 에폭사이드 지방산(epoxy fatty acid)의 분해를 억제할 수 있어서, 심혈관계 질환의 개선 또는 예방에 효과가 있으며, 상기 감송향 추출물은 인공 화합물질인 기존의 심혈관계 질환 치료제와 달리 천연물질인 식물유래 물질로 부작용 등이 문제되지 아니하므로, 본 발명의 감송향 추출물을 유효성분으로 포함하는 심혈관계 질환 개선 또는 예방용 식품조성물은 산업적으로 그 효과가 매우 크다 할 것이다.As described above, the extract of Gamsong, an active ingredient of the food composition for improving or preventing the cardiovascular disease of the present invention, inhibits the decomposition of epoxy fatty acids such as EETs by inhibiting water-soluble epoxide hydrolase. It can be effective in the improvement or prevention of cardiovascular diseases, and the extract is a natural plant-derived substance unlike the existing cardiovascular disease treatment agent of artificial compound, so the side effects are not a problem, the present invention A food composition for improving or preventing cardiovascular diseases, including the extract of Persimmon flavor as an active ingredient, the effect will be very large industrially.
또한, 본 발명의 감송향 추출물 유래 화합물은 NO 생성을 억제하고, 에폭사이드 가수분해효소를 저해할 수 있으므로, 심혈관계 질환의 예방 또는 치료 효과가 인정되고, 항염 효과를 가질 것으로 인정될 뿐만 아니라, 안전한 식물 추출물 유래의 물질이므로, 부작용 등과 관련하여 안전성도 보장될 수 있으므로, 심혈관계 질환, 특히 고혈압의 치료, 개선 또는 예방 등을 위해 사용될 수 있다.In addition, since the extract derived from the extract of the present invention may inhibit NO production and inhibit epoxide hydrolase, the prophylactic or therapeutic effect of cardiovascular disease is recognized, and not only has an anti-inflammatory effect, Since it is a substance derived from a safe plant extract, safety can be ensured with respect to side effects and the like, and thus, it can be used for the treatment, improvement or prevention of cardiovascular diseases, especially hypertension.
도 1은 본 발명의 일 실시예에 따른 감송향의 추출물 및 분획물을 제조하는 과정을 나타내는 모식도이다.1 is a schematic diagram showing a process for preparing the extract and fractions of the persimmon scent in accordance with an embodiment of the present invention.
도 2는 본 발명의 일 실시예에 따른 감송향 추출물의 추출용매에 따른 수율을 나타낸 그래프로, 가로축은 추출용매를 나타내고, 세로축은 추출수율을 나타낸다.Figure 2 is a graph showing the yield according to the extraction solvent of the extract persimmon flavor according to an embodiment of the present invention, the horizontal axis represents the extraction solvent, the vertical axis represents the extraction yield.
도 3은 본 발명의 일 실시예에 따른 감송향 추출물의 세포 독성을 확인하기 위한, 세포 생존율(cell viability, %)를 확인한 그래프로, 상기 그래프의 가로축은 추출물의 함량(μg/mL)을 의미하고, 상기 그래프의 세로축은 세포 생존율을 의미하며, 상기 각 그래프에서, G\는 열수 추출액을 의미하고, GM은 메탄올 추출액을 의미하며, GE7 은 70% 에탄올 수용액 추출액을 의미하고, GE는 95% 에탄올 수용액 추출액을 의미하며, GH 는 n-헥산 추출액을 의미하고, GEt 는 에틸아세테이트 추출액을 의미한다.3 is a graph confirming the cell viability (%) for confirming the cytotoxicity of the extract persimmon according to an embodiment of the present invention, the horizontal axis of the graph means the content of the extract (μg / mL) In addition, the vertical axis of the graph means cell viability, in each graph, G \ means hydrothermal extract, GM means methanol extract, GE7 means 70% ethanol aqueous extract, GE 95% Mean ethanol extract, GH means n-hexane extract, GEt means ethyl acetate extract.
도 4는 본 발명의 일 실시예에 따른 감송향 추출물의 NO 생성능을 대조군 대비 활성으로 나타낸 그래프(% of control)로, 상기 그래프의 가로축은 추출물의 종류를 의미하고, 상기 그래프의 세로축은 NO 생성능을 의미하며, 상기 각 그래프에서, GE는 95% 에탄올 수용액 추출액을 의미하고, GE7 은 70% 에탄올 수용액 추출액을 의미하며, GEt 는 에틸아세테이트 추출액을 의미하고, GH 는 n-헥산 추출액을 의미하며, G\는 열수 추출액을 의미하고, GM은 메탄올 추출액을 의미한다.Figure 4 is a graph (% of control) showing the NO generating ability of the extract according to an embodiment of the present invention as an activity compared to the control, the horizontal axis of the graph means the type of extract, the vertical axis of the graph is NO generating ability In each graph, GE means 95% ethanol aqueous extract, GE7 means 70% ethanol aqueous extract, GEt means ethyl acetate extract, GH means n-hexane extract, G \ stands for hot water extract and GM stands for methanol extract.
도 5는 본 발명의 일 실시예에 따른 감송향 추출물의 수용성 에폭사이드 가수분해효소를 나타낸 그래프(IC50)로, 상기 그래프의 가로축은 추출물의 종류를 의미하고, 상기 그래프의 세로축은 수용성 에폭사이드 가수분해효소를 50% 억제하기 위한 사용량(IC50,μg/mL)을 의미하며, 상기 각 그래프에서, GM은 메탄올 추출액을 의미하며, GE는 95% 에탄올 수용액 추출액을 의미하고, GE7 은 70% 에탄올 수용액 추출액을 의미하며, GEt 는 에틸아세테이트 추출액을 의미하고, GH 는 n-헥산 추출액을 의미하며, G\는 열수 추출액을 의미한다.Figure 5 is a graph showing the water-soluble epoxide hydrolase of the extract persimmon extract according to an embodiment of the present invention (IC 50 ), the horizontal axis of the graph means the type of extract, the vertical axis of the graph is a water-soluble epoxide Mean amount (IC 50 , μg / mL) to inhibit 50% hydrolase, in each graph, GM means methanol extract, GE means 95% ethanol aqueous solution, GE7 70% Mean ethanol extract, GEt means ethyl acetate extract, GH means n-hexane extract, G \ means hot water extract.
도 6은 본 발명의 일 실시예에 따른 감송향 추출물의 분획물의 NO 생성능을 대조군 대비 활성으로 나타낸 그래프(% of control)로, 상기 그래프의 가로축은 추출물의 종류를 의미하고, 상기 그래프의 세로축은 NO 생성능을 의미하며, 상기 각 그래프에서, n-hexane fr.은 헥산 분획을 의미하고, chloroform fr.은 클로로포름 분획을 의미하며, ethyl acetate fr.은 에틸 아세테이트 분획을 의미하고, butanol fr.은 부탄올 분획을 의미하며, water fr.은 물 분획을 의미한다.6 is a graph (% of control) showing the NO generation ability of the fraction of the persimmon extract according to an embodiment of the present invention as an activity compared to the control, the horizontal axis of the graph means the type of extract, the vertical axis of the graph In the graph, n-hexane fr. Means hexane fraction, chloroform fr. Means chloroform fraction, ethyl acetate fr. Means ethyl acetate fraction, butanol fr. Means butanol Fractions, water fr. Means water fraction.
도 7은 본 발명의 일 실시예에 따른 감송향 추출물의 헥산 분획물의 TLC 결과를 나타낸 사진으로, 상기 그래프의 가로축의 1은 헥산분획물을 의미하며, 상기 그래프의 가로축의 2는 헥산분획물의 서브-분획물 1을 의미하고, 3은 헥산분획물의 서브-분획물 2를 의미하며, 4는 헥산분획물의 서브-분획물 3을 의미하고, 4는 헥산분획물의 서브-분획물 5를 의미한다.Figure 7 is a photograph showing the TLC results of the hexane fraction of the extract of the persimmon flavor according to an embodiment of the present invention, 1 of the horizontal axis of the graph means hexane fraction, 2 of the horizontal axis of the graph sub- Means fraction 1, 3 means sub-fraction 2 of the hexane fraction, 4 means sub-fraction 3 of the hexane fraction, and 4 means sub-fraction 5 of the hexane fraction.
도 8은 본 발명의 일 실시예에 따른 감송향 추출물의 헥산 분획물의 서브-분획물 2의 컬럼 분획 결과물의 에폭사이드 가수분해효소 저해 활성을 나타낸 것으로, 상기 그래프의 가로축은 컬럼의 용매 용출량에 따른 분획 결과물의 종류를 의미하고, 상기 그래프의 세로축은 수용성 에폭사이드 가수분해효소를 50% 억제하기 위한 사용량(IC50,μg/mL)을 의미한다.Figure 8 shows the epoxide hydrolase inhibitory activity of the column fraction of the sub-fraction 2 of the hexane fraction of the extract of the persimmon extract according to an embodiment of the present invention, the horizontal axis of the graph is fraction according to the solvent elution amount of the column It means the type of the result, the vertical axis of the graph means the amount of use (IC 50 , μg / mL) for inhibiting 50% of the water-soluble epoxide hydrolase.
도 9는 본 발명의 일 실시예에 따른 감송향 추출물의 헥산 분획물의 서브-분획물 2의 No. 16 용출 분획 결과물의 Sephadex LH-20 크로마토그래피 결과물을 나타낸 사진이다.Figure 9 is a sub-fraction of No. 2 of the hexane fraction of the extract persimmon extract according to an embodiment of the present invention. Sephadex LH-20 Chromatography of the Elution Fraction Result.
도 10은 본 발명의 일 실시예에 따른 감송향 추출물의 헥산 분획물의 서브-분획물 3의 Sephadex LH-20 크로마토그래피 결과물을 나타낸 사진이다.10 is a photograph showing the Sephadex LH-20 chromatography results of the sub-fraction 3 of the hexane fraction of the extract of Persimmon flavor according to one embodiment of the present invention.
도 11은 본 발명의 일 실시예에 따른 감송향 추출물의 헥산 분획물의 서브-분획물 2의 No. 16 용출 분획 결과물로부터 분리된 compound 1의 GC-MS 스펙트럼 결과물을 나타낸 사진이다.11 is a No. 2 fraction of the hexane fraction of the hexane fraction of extract persimmon according to one embodiment of the present invention. Figure 16 shows the GC-MS spectrum of Compound 1 separated from the eluted fraction.
도 12 및 도 13은 본 발명의 일 실시예에 따른 감송향 추출물의 헥산 분획물의 서브-분획물 3으로부터 분리된 compound 2의 NMR 스펙트럼 결과물을 나타낸 사진으로, 도 12는 1H-NMR 스펙트럼 결과물을 나타낸 사진이고, 도 13은 13C-NMR 스펙트럼 결과물을 나타낸 사진이다.12 and 13 are photographs showing the NMR spectral output of Compound 2 separated from sub-fraction 3 of the hexane fraction of the extract of Persimmon Fragrance according to one embodiment of the present invention, and FIG. 12 shows the 1 H-NMR spectral output. It is a photograph, Figure 13 is a photograph showing the result of the 13 C-NMR spectrum.
도 14 내지 도 16은 본 발명의 일 실시예에 따른 감송향 추출물의 헥산 분획물의 서브-분획물 2의 No. 16 용출 분획 결과물로부터 분리된 compound 1의 NMR 스펙트럼 결과물을 나타낸 사진으로, 도 14는 1H-NMR 스펙트럼 결과물을 나타낸 사진이고, 도 15는 13C-NMR 스펙트럼 결과물을 나타낸 사진이며, 도 16은 EI-MS 스펙트럼 결과물을 나타낸 사진이다.14 to 16 are sub-fraction 2 of No. 2 of the hexane fraction of the persimmon extract according to an embodiment of the present invention. NMR spectrum product of Compound 1 separated from the eluted fraction product, Figure 14 is a picture showing the 1 H-NMR spectrum results, Figure 15 is a picture showing the 13 C-NMR spectrum results, Figure 16 is the EI -MS spectral photograph.
본 발명은 감송향 추출물을 유효성분으로 포함하는 심혈관계 질환 개선 또는 예방용 식품조성물에 관한 것이다. 상기 심혈관계 질환은 바람직하게는 고혈압일 수 있다.The present invention relates to a food composition for improving or preventing cardiovascular diseases, including the extract as an active ingredient. The cardiovascular disease may preferably be hypertension.
상기 감송향 추출물은 감송향 메탄올 추출물일 수 있고, 상기 감송향 추출물은 감송향 메탄올 추출물의 헥산 분획물일 수 있다. 또한, 상기 식품조성물은 기능성 식품 조성물일 수 있다.The persimmon extract may be a persimmon methanol extract, the persimmon extract may be a hexane fraction of the persimmon methanol extract. In addition, the food composition may be a functional food composition.
상기 감송향(Nardostachyschinensis)은 마타리과에 속하는 다년생 초본으로서 인도나 네팔의 산악지대가 원산지로, 그 맛이 달기 때문에 감송향이라고도 불리우며, 감송, 고미치, 향송이라고도 한다.The sense songhyang (Nardostachyschinensis) bulriwoomyeo is also known as a perennial plant belonging to the mountains to exchange valerianaceae originating in India or Nepal, fragrant spikenard Reply to this because its taste, spikenard, and Mitch, also known as pine fragrance.
상기 감송향은 외형이 활모양으로 구부러진 원주모양이고, 바깥면은 황갈색 내지 갈색으로 광택이 있으며, 자른면은 황색이 도는 갈색이다. 상기 바깥면은 5개 내지 6개의 세로 주름과 그 사이에 불규칙한 주림으 있으며, 단단하고, 원뿌리가 길고 두터우며, 수염뿌리가 달려 있고 질은 부드럽고 꺾이기 쉽다. 피층은 짙은 갈색으로 갈라져서 조각을 이루고 목부는 황백색이다.The persimmon fragrance is cylindrical in shape with a bow shape, and the outer surface is glossy to yellowish brown to brown, and the cut surface is yellowish brown. The outer surface has 5 to 6 longitudinal folds and irregular folds therebetween, which is hard, long and thick with roots, with beard roots and soft and easy to bend. Cortex divided into dark brown to form pieces, and neck is yellowish white.
상기 감송향은 청량감을 주고, 쓴맛이 돌며, 맵고 달고, 민간에서는 충치와 치질에 효과가 있다고 전해지고, 한방에서는 방향성 건위, 복부팽만 해소, 진통 또는 진정의 목적으로 처방되고 있다. 고대부터 진통제의 재료로 사용되기도 하였고, 유태인들은 결혼식에 사용하는 향유 중 하나인 나드향으로 알려져 있다.The sweet scent is said to give a refreshing feeling, bitter taste, spicy and sweet, and in the private sector, it is said to be effective for tooth decay and hemorrhoids. It has been used as an analgesic material since ancient times, and Jews are known as nad fragrance, one of the balms used for weddings.
상기 감송향은 감송향의 지상부 또는 지하부일 수 있고, 잎, 줄기, 뿌리, 꽃, 열매 및 이들의 혼합물로 이루어진 군 중에서 선택된 어느 하나일 수 있으며, 바람직하게는 감송향의 뿌리일 수 있다.The persimmon incense may be above ground or underground, and may be any one selected from the group consisting of leaves, stems, roots, flowers, fruits, and mixtures thereof.
본 발명의 추출물은 통상의 육상 식물, 일 예로 뿌리, 잎, 줄기, 꽃, 열매 또는 이들의 혼합물을 추출대상으로 한 추출물의 제조방법에 따라 제조된 것일 수 있으며, 용매 추출법, 초임계 추출법, 아임계 추출법, 속실렛 추출법, SDE 추출법 등 식물 추출물의 제조와 관련하여 사용되는 통상의 추출법으로 제조될 수 있다.Extract of the present invention may be prepared according to a conventional land plant, for example, root, leaf, stem, flower, fruit or a method for preparing an extract to extract a mixture thereof, solvent extraction method, supercritical extraction method, sub It can be prepared by conventional extraction methods used in connection with the preparation of plant extracts, such as critical extraction, soxhlet extraction, SDE extraction.
상기 용매 추출법(Solvent extraction method)은 추출대상인 식물로부터 특정 물질을 추출하기 위하여 용매를 써서 고체 또는 액체시료 중에서 성분물질의 1종 또는 2종 이상을 추출하는 방법을 의미한다. 상기 용매 추출법은 냉침추출법, 온침추출법 또는 열 추출법 등일 수 있으며, 통상의 추출기기, 초음파분쇄 추출기 또는 분획기를 이용하여 수행할 수 있다. 상기 초임계 추출법(supercritical extraction)은 초임계 유체를 이용한 추출법으로, 초임계 유체 추출법(supercritical fluid extraction, SFE)이라고도 한다. 상기 초임계 추출법은 임계온도 및 경계압력을 초과하는 상태의 유체인 초임계 유체를 이용한 것으로, 상기 초임계 유체는 높은 밀도가 있으면서 낮은 점성과 액체 보다 현저하게 높은 확산능력이 있기 때문에 기존 용매 추출법에 비하여 효율이 높은 장점이 있으며, 상기 초임계 유체로는 주로 이산화탄소 등이 사용된다. 상기 아임계 추출법(Subcritical Extraction)은 초임계 조건과 유사한 조건 즉, 임계온도 또는 임계압력보다 낮은 조건의 유체를 이용한 추출법으로, 주로 수용매를 이용한 아임계수 추출법(Subcritical Water Extraction, SWE)이 사용된다. 상기 SDE(Steam distillation extraction) 추출법은 동시증류추출장치를 이용한 추출법으로, 증기증류법이라고 불리우며, 정유를 추출하기 위해 가장 흔하게 사용되는 추출법이다. 뜨거운 증기를 추출대상물에 통과시킨 후, 다시 그 증기를 식히는 방법을 통해, 식물의 정유를 추출해내는 방법이다. 상기 속실렛 추출법은 속실렛 추출장치를 이용하여 추출하는 추출법으로, 주로 유지를 추출하기 위해 사용되는 방법이다.The solvent extraction method (Solvent extraction method) means a method of extracting one or two or more of the components in a solid or liquid sample using a solvent in order to extract a specific substance from the plant to be extracted. The solvent extraction may be cold extraction, hot extraction or thermal extraction, and may be performed using a conventional extraction device, an ultrasonic mill extractor or a fractionator. The supercritical extraction is an extraction method using a supercritical fluid, also called a supercritical fluid extraction (SFE). The supercritical extraction method uses a supercritical fluid that is a fluid in a state exceeding a critical temperature and a boundary pressure. The supercritical fluid has a high density and has a low viscosity and a remarkably higher diffusion ability than a liquid. Compared to the above, there is an advantage of high efficiency, and carbon dioxide is mainly used as the supercritical fluid. The subcritical extraction method is an extraction method using a fluid similar to a supercritical condition, that is, a critical temperature or a pressure lower than the critical pressure. Subcritical Water Extraction (SWE) using a solvent is mainly used. . The steam distillation extraction (SDE) extraction method is an extraction method using a simultaneous distillation extraction apparatus, called a steam distillation method, and is the extraction method most commonly used to extract essential oils. It is a method of extracting essential oils from plants by passing hot steam through the extract and cooling the steam again. The soxhlet extraction method is an extraction method using an soxhlet extraction device, which is mainly used for extracting oils and fats.
상기 감송향 추출물은 바람직하게는 용매 추출법, 초임계 추출법, 속실렛 추출법 또는 SDE 추출법으로 제조될 수 있다.The persimmon extract is preferably prepared by solvent extraction, supercritical extraction, Soxhlet extraction or SDE extraction.
상기 용매 추출법은 추출용매로 추출하거나 추출용매로 추출하여 제조한 조추출물에 분획용매를 가하여 분획하여 제조하는 방법으로 수행할 수 있다.The solvent extraction method may be performed by extracting with an extraction solvent or by adding a fractional solvent to the crude extract prepared by extraction with an extraction solvent.
상기 추출용매는 물 및 유기용매로 이루어진 군에서 선택된 1종 이상일 수 있다. 상기 유기용매는 메탄올, 에탄올 등의 탄소수 1 내지 5의 알코올, 에틸아세테이트 또는 아세톤 등의 극성용매와 에테르, 클로로포름, 벤젠, 헥산 또는 디클로로메탄의 비극성용매 또는 이들의 혼합용매일 수 있다.The extraction solvent may be at least one selected from the group consisting of water and an organic solvent. The organic solvent may be a polar solvent such as alcohol having 1 to 5 carbon atoms such as methanol or ethanol, ethyl acetate or acetone, and a nonpolar solvent of ether, chloroform, benzene, hexane or dichloromethane, or a mixed solvent thereof.
본 발명의 추출용매는 바람직하게는 물 및 탄소수 1 내지 4의 알코올로 이루어진 군에서 선택된 1종 이상의 용매일 수 있고, 더욱 바람직하게는 탄소수 1 내지 4의 알코올 및 헥산으로 이루어진 군에서 선택된 1종 이상일 수 있으며, 더더욱 바람직하게는 에탄올 또는 메탄올일 수 있고, 가장 바람직하게는 메탄올일 수 있다.The extraction solvent of the present invention may preferably be at least one solvent selected from the group consisting of water and alcohols having 1 to 4 carbon atoms, and more preferably at least one selected from the group consisting of alcohols having 1 to 4 carbon atoms and hexane. More preferably, ethanol or methanol, most preferably methanol.
또한, 본 발명의 추출물은 상기 용매로 추출한 조추출물에 대하여 분획용매를 가한 후, 분획과정을 더욱 실시한 분획물일 수 있다. 상기 분획용매는 에틸아세테이트, 에테르, 클로로포름, 벤젠, 헥산, 메틸렌클로라이드, 부탄올, 물 및 이들의 혼합용매로 이루어진 군에서 선택된 용매, 바람직하게는 헥산, 클로로포름 및 이들의 혼합물로 이루어진 군 중에서 선택된 용매, 더욱 바람직하게는 n-헥산(n-hexane)일 수 있다.In addition, the extract of the present invention may be a fraction further subjected to the fractionation process after adding a fractional solvent to the crude extract extracted with the solvent. The fractional solvent is a solvent selected from the group consisting of ethyl acetate, ether, chloroform, benzene, hexane, methylene chloride, butanol, water and mixed solvents thereof, preferably hexane, chloroform and a mixture thereof. More preferably, it may be n-hexane.
상기 제조된 추출물 또는 상기 분획과정을 수행하여 수득한 분획물은 이후 여과하거나 농축 또는 건조과정을 수행하여 용매를 제거할 수 있으며, 여과, 농축 및 건조를 모두 수행할 수 있다. 구체적으로 상기 여과는 여과지를 이용하거나 감압여과기를 이용할 수 있으며, 상기 농축은 감압 농축기, 일예로 회전 증발기를 이용하여 감압 농축할 수 있으며, 상기 건조는 일예로 동결건조법으로 수행할 수 있다.The prepared extract or the fraction obtained by performing the fractionation process can be filtered or concentrated or dried to remove the solvent, it can be carried out both filtration, concentration and drying. Specifically, the filtration may be performed using a filter paper or a reduced pressure filter, the concentration may be concentrated under reduced pressure using a reduced pressure concentrator, for example, a rotary evaporator, and the drying may be performed by, for example, a lyophilization method.
본 발명의 일예로, 상기 감송향 추출물은 하기 방법에 의해 수득될 수 있다.In one embodiment of the present invention, the extract may be obtained by the following method.
구체적으로, 상기 감송향 추출물은 감송향 뿌리에 메탄올을 가하여 추출하고, 여과 및 감압농축한 후, 동결건조시키는 방법으로 제조할 수 있다. 또한, 상기 감송향 분획물은 상기 감송향 추출물에 헥산, 클로로포름, 에틸 아세테이트, 부탄올 및 물의 분획용매을 순차적으로 첨가한 후, 각 용매 분획물을 분리하는 방법으로 제조할 수 있다.Specifically, the extract may be prepared by adding methanol to the root of the extract persimmon, filtered and concentrated under reduced pressure, and then lyophilized. In addition, the saenghyang fragrance fraction may be prepared by sequentially adding the fraction solvent of hexane, chloroform, ethyl acetate, butanol and water to the saenghyang extract, and then separating each solvent fraction.
상기 감송향 메탄올 추출물은 수용성 에폭사이드 가수분해효소의 저해능이 우수할 뿐만 아니라, 천연물 유래의 물질로 부작용이나 내성 등의 문제점이 발생될 가능성이 적고, 안전성이 우수하므로, 상기 감송향 메탄올 추출물은 고혈압을 비롯한 심혈관계 질환의 개선 또는 예방을 위한 식품 조성물의 유효성분으로 사용될 수 있다.The extract of Methanol fragrance extract is not only excellent in inhibiting ability of the water-soluble epoxide hydrolase, but also less likely to cause side effects or resistance as a substance derived from natural products, and has excellent safety. It can be used as an active ingredient of a food composition for the improvement or prevention of cardiovascular diseases, including.
이러한 측면에서, 본 발명은 감송향 추출물을 유효성분으로 포함하는 심혈관계 질환 개선 또는 예방용 조성물에 관한 것이다.In this aspect, the present invention relates to a cardiovascular disease improvement or prevention composition comprising the extract as an active ingredient.
상기 심혈관계 질환이란 심장질환과 혈관질환을 포함한 질환으로, 고혈압, 고혈압성 심장질환, 심장병, 부정맥, 뇌졸중, 혈전증, 협심증, 심부전, 심근경색, 죽상경화증 및 동맥경화로 이루어진 군중에서 선택된 어느 하나일 수 있으며, 바람직하게는 고혈압 또는 고혈압성 심장질환일 수 있다.The cardiovascular disease is a disease including heart disease and vascular disease, any one selected from the group consisting of hypertension, hypertensive heart disease, heart disease, arrhythmia, stroke, thrombosis, angina pectoris, heart failure, myocardial infarction, atherosclerosis and arteriosclerosis And preferably hypertension or hypertensive heart disease.
상기 고혈압은 혈관관련 질병으로, 주로 동맥의 혈압이 높은 동맥성 고혈압일 수 있다. 상기 고혈압은 동맥경화나 고혈압심장병(hypertensive heart disease) 등을 유발할 수 있다.The hypertension is a blood vessel-related disease, and may be arterial hypertension with high arterial blood pressure. The hypertension may cause arteriosclerosis or hypertensive heart disease.
상기 감송향 추출물은 수용성 에폭사이드 가수분해효소를 유효하게 저해할 수 있다는 결과로부터, 상기 감송향 추출물의 혈압강하능이 확인되었으므로, 상기 감송향 추출물은 고혈압 및 심장병, 동맥경화 또는 뇌출혈 등의 심혈관계 질환에 치료, 예방 또는 개선 효과가 있는 것으로 평가된다.From the result that the extract of S. extract is able to effectively inhibit the water-soluble epoxide hydrolase, the blood pressure lowering ability of the extract was confirmed, the extract of S. extract is a cardiovascular disease such as hypertension and heart disease, arteriosclerosis or cerebral hemorrhage. It is evaluated to have a therapeutic, prophylactic or ameliorating effect.
본 발명의 감송향 추출물을 유효성분으로 포함하는 심혈관계 질환 개선 또는 예방용 조성물은 조성물 총 중량에 대하여 상기 감송향 추출물을 0.001 내지 99.99중량%, 바람직하게는 0.1 내지 99 중량%로 포함할 수 있다.The cardiovascular disease improvement or prevention composition comprising the extract of Persimmon as an active ingredient of the present invention may include the extract of 0.001 to 99.99% by weight, preferably 0.1 to 99% by weight based on the total weight of the composition. .
상기 감송향 추출물을 유효성분으로 포함하는 심혈관계 질환 개선 또는 예방용 조성물은 천연물질의 추출물이라는 점에서 부작용의 문제가 발생되지 아니하고, 심혈관계 질환의 개선 또는 예방 효과가 있다는 점에서 우수한 효과를 가진다.The cardiovascular disease improvement or prevention composition comprising the extract as an active ingredient has an excellent effect in that there is no problem of side effects in that it is an extract of a natural substance, and there is an improvement or prevention effect of the cardiovascular disease. .
본 발명의 감송향 추출물을 유효성분으로 포함하는 심혈관계 질환 개선 또는 예방용 조성물은 인간에 직접 적용될 수 있다.The composition for improving or preventing cardiovascular diseases comprising the extract of Persimmon Fragrance of the present invention as an active ingredient may be directly applied to humans.
상기 감송향 추출물을 유효성분으로 포함하는 심혈관계 질환 개선 또는 예방용 조성물은 상기 감송향 추출물을 유효성분으로 단독으로 포함할 수 있고, 이외 제형, 사용방법 및 사용목적에 따라 추가성분 즉, 약제학적으로 허용되거나 영양학적으로 허용되는 담체, 부형제, 희석제 또는 부성분을 추가로 포함할 수 있다.The cardiovascular disease improvement or prevention composition comprising the extract as an active ingredient may include the extract as an active ingredient alone, and according to the other formulation, method and purpose of use additional ingredients, that is, pharmaceutical Or further nutritionally acceptable carriers, excipients, diluents or accessory ingredients.
본 명세서에서 식품이란 함은 영양소를 한 가지 또는 그 이상 함유하고 있는 천연물 또는 가공품을 의미하며, 바람직하게는 어느 정도의 가공 공정을 거쳐 직접 먹을 수 있는 상태가 된 것을 의미하며, 통상적인 의미로서, 건강기능식품, 음료, 식품 첨가제 및 음료 첨가제 등을 모두 포함하는 의도이다.In the present specification, the term "food" means a natural product or processed product containing one or more nutrients, and preferably means a state in which it can be directly eaten through some processing process. It is intended to include all dietary supplements, beverages, food additives and beverage additives.
본 발명의 식품은 예를 들어, 각종 식품류, 음료, 껌, 차, 비타민 복합제, 건강기능식품 등이 있다.Examples of the food of the present invention include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.
추가로, 본 발명에서 식품에는 특수영양식품(예, 조제유류, 영,유아식 등), 식육가공품, 어육제품, 두부류, 묵류, 면류(예, 라면류, 국수류 등), 건강보조식품, 조미식품(예, 간장, 된장, 고추장, 혼합장 등), 소스류, 과자류(예, 스넥류), 유가공품(예, 발효유, 치즈 등), 기타 가공식품, 김치, 절임식품(각종 김치류, 장아찌 등), 음료(예, 과실,채소류 음료, 두유류, 발효음료류 등), 천연조미료(예, 라면스프 등)을 포함하나 이에 한정되지 않는다. 상기 식품, 건강기능식품, 음료, 식품 첨가제 및 음료 첨가제는 통상의 제조방법으로 제조될 수 있다.In addition, the food in the present invention includes special nutritional products (e.g., prepared oils, infants, baby food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g. ramen, noodles, etc.), health supplements, seasoned foods ( For example, soy sauce, miso, red pepper paste, mixed soy sauce), sauces, confectionery (e.g. snacks), dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, pickles (various kimchi, pickles, etc.), beverages ( Examples include, but are not limited to, fruits, vegetable drinks, soy milk, fermented beverages, and the like, and natural seasonings (eg, ramen soup). The food, health functional food, beverages, food additives and beverage additives may be prepared by conventional manufacturing methods.
본 발명에서 건강기능식품이란 식품에 물리적, 생화학적, 생물공학적 수법 등을 이용하여 해당 식품의 기능을 특정 목적에 작용, 발현하도록 부가가치를 부여한 식품군이나 식품 조성이 갖는 생체방어리듬조절, 질병방지와 회복 등에 관한 체조절기능을 생체에 대하여 충분히 발현하도록 설계하여 가공한 식품을 의미한다.In the present invention, the health functional food is a biological defense rhythm control, disease prevention and the like having a food group or a food composition that has added value to the food by using physical, biochemical, biotechnological techniques, etc. It means a food that is designed and processed to fully express the gymnastics function on recovery.
상기 건강기능식품에는 식품학적으로 허용 가능한 식품 보조 첨가제를 포함할 수 있으며, 건강기능식품의 제조에 통상적으로 사용되는 적절한 담체, 부형제 및 희석제를 더욱 포함할 수 있다.The dietary supplement may include food acceptable food additives, and may further include appropriate carriers, excipients and diluents commonly used in the manufacture of dietary supplements.
본 발명에서 음료란 갈증을 해소하거나 맛을 즐기기 위하여 마시는 것의 총칭을 의미하며 건강기능음료를 포함하는 의도이다. 상기 음료는 지시된 비율로 필수 성분으로서 상기 감송향 추출물을 유효성분으로 포함하는 것 외에 다른 성분에는 특별한 제한이 없으며, 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다.In the present invention, the drink refers to a generic term for drinking to quench thirst or to enjoy the taste and is intended to include a functional beverage. The beverage is not particularly limited to other ingredients other than including the sweet persimmon extract as an active ingredient in the indicated ratio as an active ingredient, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. have.
상기의 천연 탄수화물의 예는 모노사카라이드, 예를 들어 포도당, 과당 등 디사카라이드, 예를 들어 말토스, 수크로스 등 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알코올이다. 상기한 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진 등) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다.Examples of such natural carbohydrates include monosaccharides such as glucose, fructose and other disaccharides such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like, and Sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. .
상기 천연 탄수화물의 비율은 본 발명의 식품 조성물 100㎖ 당 일반적으로 약 1 내지 20g, 바람직하게는 5 내지 12g일 수 있다. 그밖에 본 발명의 조성물은 천연 과일 주스, 과일 쥬스 음료, 야채 음료의 제조를 위한 과육을 추가로 함유할 수 있다.The ratio of the natural carbohydrate may be generally about 1 to 20 g, preferably 5 to 12 g per 100 ml of the food composition of the present invention. In addition, the composition of the present invention may further contain a pulp for the production of natural fruit juices, fruit juice drinks, vegetable drinks.
상기 외에 본 발명의 식품 조성물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있다. 이러한 성분을 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 그렇게 중요하지 않지만, 본 발명의 감송향 추출물 100 중량부 당 0 내지 2,000 중량부 범위에서 선택될 수 있다.In addition to the above, the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and Salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. These components can be used independently or in combination. The proportion of such additives is not so critical, but may be selected in the range of 0 to 2,000 parts by weight per 100 parts by weight of the persimmon extract of the present invention.
본 발명에서 건강기능음료란 음료에 물리적, 생화학적, 생물공학적 수법 등을 이용하여 해당 음료의 기능을 특정 목적에 작용, 발현하도록 부가가치를 부여한 음료 군이나 음료 조성이 갖는 생체방어리듬조절, 질병방지와 회복 등에 관한 체조절기능을 생체에 대하여 충분히 발현하도록 설계하여 가공한 음료를 의미한다.In the present invention, the health functional beverage is a biological defense rhythm control or disease prevention of a beverage group or beverage composition which has added value to the beverage by using physical, biochemical and biotechnological techniques to act and express the function of the beverage for a specific purpose. Means a beverage that is designed and processed to fully express the gymnastic function related to recovery and the like.
상기 건강기능음료는 지시된 비율로 필수 성분으로서 본 발명의 감송향 추출물을 함유하는 외에는 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다.The health functional beverage is not particularly limited to other ingredients except for containing the persimmon extract of the present invention as an essential ingredient in the indicated ratio, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. .
상기 천연 탄수화물의 예는 모노사카라이드, 예를 들어 포도당, 과당 등 디사카라이드, 예를 들어 말토스, 수크로스 등 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알코올이다. 상기한 것 이외의 향미제로서 천연 향미제(타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진 등) 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100㎖ 당 일반적으로 약 1 내지 20 g, 바람직하게는 5 내지 12 g이다.Examples of such natural carbohydrates include monosaccharides such as glucose, fructose and other disaccharides such as maltose, sucrose and the like and polysaccharides such as dextrin, cyclodextrin and the like, and xylitol Sugar alcohols such as sorbitol and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1 to 20 g, preferably 5 to 12 g per 100 ml of the composition of the present invention.
또한, 감송향 추출물을 유효성분으로 포함하는 심혈관계 질환 예방 또는 개선용 식품 조성물에 있어서, 상기 감송향 추출물의 양은 전체 식품 중량의 0.01 내지 15 중량%로 포함할 수 있으며, 음료 조성물은 100 ㎖를 기준으로 0.02 내지 5 g, 바람직하게는 0.3 내지 1g의 비율로 포함할 수 있다.In addition, in the food composition for preventing or improving cardiovascular disease, including the extract of Persimmon flavor as an active ingredient, the amount of the extract persimmon may be included in 0.01 to 15% by weight of the total food weight, the beverage composition is 100 ml It may be included in a ratio of 0.02 to 5 g, preferably 0.3 to 1 g as a reference.
본 발명의 감송향 추출물은 수용성 에폭사이드 가수분해효소를 저해하여, 심혈관계 질환, 구체적으로 고혈압 또는 고혈압성 심장질환을 갖는 환자 또는 이러한 위험성이 있는 경우에 유용하게 사용될 수 있을 것으로 판단된다.Persimmon extract of the present invention inhibits the water-soluble epoxide hydrolase, it is considered that it can be useful in patients with cardiovascular diseases, specifically hypertension or hypertensive heart disease or when there is such a risk.
또 다른 측면에 있어서, 본 발명은 감송향 추출물로부터 분리된 화합물 또는 그의 약학적으로 허용되는 염을 유효성분으로 포함하는 심혈관계 질환 치료 또는 예방용 의약조성물을 제공한다. 상기 심혈관계 질환은 고혈압 또는 고혈압성 심장질환일 수 있다.In another aspect, the present invention provides a pharmaceutical composition for treating or preventing cardiovascular diseases comprising a compound or a pharmaceutically acceptable salt thereof isolated from the extract of Persimmon Scent. The cardiovascular disease may be hypertension or hypertensive heart disease.
또한, 본 발명은 감송향 추출물로부터 분리된 화합물 또는 그의 약학적으로 허용되는 염을 유효성분으로 포함하는 심혈관계 질환 개선 또는 예방용 식품조성물을 제공한다. 상기 심혈관계 질환은 고혈압 또는 고혈압성 심장질환일 수 있다. 또한, 상기 식품조성물은 건강기능성 식품조성물일 수 있다.In another aspect, the present invention provides a food composition for improving or preventing cardiovascular diseases comprising a compound or a pharmaceutically acceptable salt thereof isolated from the extract of Scent. The cardiovascular disease may be hypertension or hypertensive heart disease. In addition, the food composition may be a health functional food composition.
상기한 바와 같이, 에폭사이드 가수분해효소를 저해하는 경우, 항고혈압 작용이 뛰어나므로, 에폭사이드 가수분해효소를 저해하는 물질의 경우 심혈관계 질환의 치료, 개선 또는 예방을 위해 사용되거나 상기 목적의 조성물의 유효성분으로 사용될 수 있다.As described above, when the epoxide hydrolase is inhibited, the antihypertensive action is excellent, and therefore, the substance that inhibits the epoxide hydrolase is used for the treatment, improvement or prevention of cardiovascular diseases, or the composition for the purpose. It can be used as an active ingredient of.
이러한 측면에 있어서, 본 발명은 감송향 추출물로부터 분리된 화합물의 용도, 구체적으로 에폭사이드 가수분해효소 저해용 용도 또는 고혈압 치료, 개선 또는 예방용 용도일 수 있다.In this aspect, the present invention may be the use of the compound isolated from the extract of the persimmon flavor, specifically for the use of epoxide hydrolase inhibition or for the treatment, improvement or prevention of hypertension.
상기 에폭사이드 가수분해효소를 저해하는 경우, 항염 효과 또는 항고혈압을 포함한 심혈관계 질환의 예방 또는 치료 효과가 있다. 따라서, 상기 감송향 추출물로부터 분리된 화합물의 용도는 항염 용도 또는 항고혈압 치료용 용도를 포함한 심혈관계 질환의 치료, 예방 또는 개선용 용도일 수 있다.When inhibiting the epoxide hydrolase, there is an anti-inflammatory effect or a prophylactic or therapeutic effect of cardiovascular diseases including antihypertensive. Therefore, the use of the compound isolated from the extract of the sweet persimmon may be for the treatment, prevention or improvement of cardiovascular diseases, including anti-inflammatory use or antihypertensive use.
구체적으로, 본 발명은 하기 화학식 1의 구조를 갖는 화합물 및 하기 화학식 2의 구조를 갖는 화합물로 이루어진 군 중에서 선택된 어느 하나의 화합물 또는 그의 약학적으로 허용되는 염을 유효성분으로 포함하는 심혈관계 질환 치료 또는 예방용 의약조성물에 관한 것이다.Specifically, the present invention is a cardiovascular disease treatment comprising any one compound selected from the group consisting of a compound having a structure of formula (1) and a compound having a structure of formula (2) or a pharmaceutically acceptable salt thereof as an active ingredient Or to preventive pharmaceutical compositions.
[화학식 1][Formula 1]
[규칙 제91조에 의한 정정 05.03.2013]　
Figure WO-DOC-FIGURE-126
[Revision under Rule 91 05.03.2013]
Figure WO-DOC-FIGURE-126
[화학식 2][Formula 2]
[규칙 제91조에 의한 정정 05.03.2013]　
Figure WO-DOC-FIGURE-128
[Revision under Rule 91 05.03.2013]
Figure WO-DOC-FIGURE-128
상기 심혈관계 질환은 고혈압, 고혈압성 심장질환, 심장병, 뇌졸중, 혈전증, 협심증, 심부전, 심근경색, 죽상경화증 및 동맥경화로 이루어진 군 중에서 선택된 어느 하나일 수 있으며, 바람직하게는 고혈압 또는 고혈압성 심장질환, 더욱 바람직하게는 고혈압일 수 있다.The cardiovascular disease may be any one selected from the group consisting of hypertension, hypertensive heart disease, heart disease, stroke, thrombosis, angina pectoris, heart failure, myocardial infarction, atherosclerosis and arteriosclerosis, preferably hypertension or hypertensive heart disease More preferably hypertension.
상기 고혈압은 혈관관련 질병으로, 주로 동맥의 협압이 높은 동맥성 고혈압을 의미한다. 상기 고혈압은 동맥경화나 고혈압성 심장질환(hypertensive heart disease) 등을 유발할 수 있다.The hypertension is a blood vessel-related disease, and means arterial hypertension with high arterial pressure. The hypertension may cause arteriosclerosis or hypertensive heart disease.
상기 동맥경화는 동맥의 벽이 두터워지고 동맥의 탄력이 저하되는 질환을 의미한다. 상기 동맥경화가 발병되면 경화된 동맥으로부터 혈액을 공급받던 장기에 대한 혈액 공급이 감소하게 되어, 추가적인 질병이 발병될 수 있다. 이와 관련된 대표적인 질병의 예는 심장의 근육에 혈액을 공급하는 심장동맥에 동맥경화의 변화가 오는 심장동맥병 등이 있다.The atherosclerosis refers to a disease in which the walls of the arteries are thickened and the elasticity of the arteries is reduced. When the atherosclerosis develops, the blood supply to the organs that received the blood from the hardened arteries is reduced, so that additional diseases may develop. An example of a typical disease related to this is coronary artery disease in which atherosclerosis changes in the coronary arteries that supply blood to the muscles of the heart.
상기 화학식 1의 구조를 갖는 화합물 및 상기 화학식 2의 구조를 갖는 화합물로 이루어진 군 중에서 선택된 어느 하나의 화합물 또는 그의 약학적으로 허용되는 염을 유효성분으로 포함하는 심혈관계 질환 치료 또는 예방용 의약조성물은 염수, 완충 염수, 덱스트로스, 물, 글리세롤 및 에탄올에서 선택되는 약학적 희석제 중 1종 이상 포함할 수 있으며, 희석제는 이에 한정되는 것은 아니다.A pharmaceutical composition for treating or preventing cardiovascular diseases comprising any one compound selected from the group consisting of the compound having the structure of Formula 1 and the compound having the structure of Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient It may include one or more of the pharmaceutical diluents selected from saline, buffered saline, dextrose, water, glycerol and ethanol, diluents are not limited thereto.
상기 심혈관계 질환 치료 또는 예방용 의약조성물은 투여목적 및 질병에 따라 상이하게 적용될 수 있다. 실질적으로 투여되는 활성 성분의 양은 다양한 관련 요소, 즉 치료하고자 하는 질병, 환자상태의 정도, 다른 약제(예를 들어 주화성 약제)와 공동 투여여부, 환자의 나이 성별, 체중, 음식, 투여시간, 투여경로, 및 조성물의 투여비율(ratio)을 고려하여 결정하여야 한다. 상기 심혈관계 질환 치료 또는 예방용 의약조성물은 투여량 및 투여경로가 질병의 형태 및 심각성에 따라 조절될 수 있으며, 하루에 한번 또는 1 내지 3번 나누어 투여될 수 있으나, 이에 한정되는 것은 아니다.The pharmaceutical composition for treating or preventing the cardiovascular disease may be applied differently depending on the purpose of administration and the disease. The amount of active ingredient administered substantially depends on a variety of related factors, such as the disease to be treated, the extent of the patient's condition, whether it is co-administered with other drugs (eg chemotactic drugs), the patient's age, gender, weight, food, time of administration, It should be determined in consideration of the route of administration and the ratio of the composition. The pharmaceutical composition for treating or preventing the cardiovascular disease may be adjusted according to the dosage form and the severity of the disease, and may be administered once or once or three times a day, but is not limited thereto.
본 발명의 심혈관계 질환 치료 또는 예방용 의약조성물은 경구 또는 비경구로 투여될 수 있다. 비경구 투여는 경구이외의 투여경로, 즉 직장, 정맥, 복막 및 근육, 동맥, 경피, 비강(nasal), 흡입, 안구, 및 피하를 통한 약제 투여를 의미한다.The pharmaceutical composition for treating or preventing the cardiovascular disease of the present invention may be administered orally or parenterally. Parenteral administration refers to administration of medications other than oral, namely rectal, intravenous, peritoneal and muscular, arterial, transdermal, nasal, inhaled, ocular, and subcutaneous.
상기 심혈관계 질환 치료 또는 예방용 의약조성물은 경구 투여 형태, 주입 가능한 용액 또는 국소제제와 같은 어떠한 형태로도 제제화될 수 있다. 제제화는 경구 및 주입 가능한 투여(진용액(true solution), 현탁액 또는 에멀젼)에 적합하도록 제조되는 것이 바람직하며, 정제, 캡슐, 연질캡슐, 수성 약제, 환제, 과립 등과 같은 경구 형태로 제조되는 것이 가장 바람직하다.The pharmaceutical composition for treating or preventing cardiovascular diseases may be formulated in any form such as oral dosage form, injectable solution or topical preparation. Formulations are preferably prepared for oral and injectable administration (true solution, suspension or emulsion), most preferably in oral form such as tablets, capsules, soft capsules, aqueous pharmaceuticals, pills, granules and the like. desirable.
상기 제제화에서 본 발명의 펩타이드는 부형제(excipient)없이 연질 캡슐에 충전될 수 있고, 담지체와 혼합되거나 희석된 후에 적당한 제제로 만들어질 수도 있다. 적합한 담지체의 예로는 전분, 물, 염수, 링거액, 덱스트로스 등이 있다.In the above formulation, the peptide of the present invention may be filled into a soft capsule without an excipient, and may be made into a suitable formulation after being mixed or diluted with the carrier. Examples of suitable carriers include starch, water, saline, Ringer's solution, dextrose and the like.
상기 심혈관계 질환 치료 또는 예방용 의약조성물은 약학 조성물일 수 있다.The pharmaceutical composition for treating or preventing the cardiovascular disease may be a pharmaceutical composition.
상기 약학 조성물은 인간을 포함한 동물에 직접 적용될 수 있다. 상기 동물은 식물에 대응하는 생물군으로 주로 유기물을 영양분으로 섭취하며, 소화나 배설 및 호흡기관이 분화되어 있는 것을 말하고, 바람직하게는 척추동물, 더욱 바람직하게는 포유류일 수 있다. 상기 포유류는 인간, 돼지, 소 또는 염소 등일 수 있으며, 바람직하게는 인간일 수 있다.The pharmaceutical composition may be directly applied to animals including humans. The animal is a biological group corresponding to a plant, and mainly ingests organic substances as nutrients, and means that digestion, excretion, and respiratory organs are differentiated, and preferably, vertebrates, more preferably mammals. The mammal may be human, pig, cow or goat, etc., preferably human.
상기 약학 조성물은 상기 화학식 1의 구조를 갖는 화합물 및 상기 화학식 2의 구조를 갖는 화합물로 이루어진 군 중에서 선택된 어느 하나의 화합물 또는 그의 약학적으로 허용되는 염을 유효성분으로 단독으로 포함할 수 있고, 이외 제형, 사용방법 및 사용목적에 따라 추가성분 즉, 약제학적으로 허용되거나 영양학적으로 허용되는 담체, 부형제, 희석제 또는 부성분을 추가로 포함할 수 있다.The pharmaceutical composition may include any one compound selected from the group consisting of the compound having the structure of Formula 1 and the compound having the structure of Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient, and Depending on the formulation, the method of use and the purpose of use, it may further comprise additional ingredients, ie, pharmaceutically acceptable or nutritionally acceptable carriers, excipients, diluents or accessory ingredients.
보다 상세하게는 상기 약학 조성물은 상기 유효성분 외에 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, 탄산 음료에 사용되는 탄산화제 등을 추가로 함유할 수 있다.More specifically, the pharmaceutical composition may be added to the nutrients, vitamins, electrolytes, flavors, coloring agents, neutralizing agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners, pH regulators, and stabilizers in addition to the active ingredient. It may further contain an agent, a preservative, glycerin, alcohol, a carbonation agent used in the carbonated beverage, and the like.
또한, 상기 담체, 부형제 또는 희석제는 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 자이리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로오스, 메틸 셀룰로오스, 미정질 셀루로오스, 폴리비닐 피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유, 덱스트린, 칼슘카보네이드, 프로필렌글리콜, 리퀴드 파라핀, 생리식염수로 이루어진 군에서 선택된 1이상 일 수 있으나, 이에 한정되는 것은 아니며 통상의 담체, 부형제 또는 희석제 모두 사용가능하다.In addition, the carrier, excipient or diluent may be lactose, dextrose, sucrose, sorbitol, mannitol, ziitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose Selected from the group consisting of loose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin, saline It may be one or more, but is not limited to any conventional carrier, excipient or diluent can be used.
상기 성분들은 상기 유효성분인 상기 상기 화학식 1의 구조를 갖는 화합물 및 상기 화학식 2의 구조를 갖는 화합물로 이루어진 군 중에서 선택된 어느 하나의 화합물 또는 그의 약학적으로 허용되는 염에 독립적으로 또는 조합하여 추가될 수 있다.The components may be added independently or in combination to any one compound selected from the group consisting of a compound having the structure of Formula 1 and a compound having the structure of Formula 2 or a pharmaceutically acceptable salt thereof as the active ingredient. Can be.
상기 약학 조성물은 조성물 총 중량에 대하여 상기 화학식 1의 구조를 갖는 화합물 및 상기 화학식 2의 구조를 갖는 화합물로 이루어진 군 중에서 선택된 어느 하나의 화합물 또는 그의 약학적으로 허용되는 염을 0.001 중량% 내지 99.9 중량%, 바람직하게는 0.1 중량% 내지 99 중량%, 더욱 바람직하게는 1중량% 내지 50 중량% 포함할 수 있다.The pharmaceutical composition is 0.001% to 99.9% by weight of any one compound selected from the group consisting of a compound having the structure of Formula 1 and a compound having the structure of Formula 2 or a pharmaceutically acceptable salt thereof based on the total weight of the composition %, Preferably 0.1% to 99% by weight, more preferably 1% to 50% by weight.
또한, 상기 약학 조성물은 약제화하는 경우, 통상의 충진제, 증량제, 결합제, 붕해제, 계면활성제, 항응집제, 윤활제, 습윤제, 향료, 유화제 또는 방부제 등을 더욱 포함할 수 있으며, 경구 또는 비경구 모두 사용 할 수 있다.In addition, the pharmaceutical composition may further include conventional fillers, extenders, binders, disintegrants, surfactants, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers or preservatives when formulated, and oral or parenteral. Can be used
구체적으로 경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 상기 유효성분에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(calcium carbonate), 수크로스(sucrose) 또는 락토오스(lactose), 젤라틴 등을 섞어 조제될 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제들도 사용된다. 경구를 위한 액상 제제로는 현탁제, 내용액제, 유제, 시럽제 등이 해당되는데, 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제 예를 들면, 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다.Specifically, solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations may include at least one excipient such as starch, calcium carbonate, water, or the like in the active ingredient. It may be prepared by mixing cross or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, solvents, emulsions, and syrups.In addition to the commonly used simple diluents, water and liquid paraffin, various excipients may include, for example, wetting agents, sweeteners, fragrances, and preservatives. have.
또한, 본 발명의 약학 조성물의 제형은 사용방법에 따라 바람직한 형태일 수 있으며, 특히 포유동물에 투여된 후 활성 성분의 신속, 지속 또는 지연된 방출을 제공할 수 있도록 당업계에 공지된 방법을 채택하여 제형화할 수 있다. 구체적인 제형의 예로는 경고제, 과립제, 로션제, 리니멘트제, 리모나데제, 산제, 시럽제, 안연고제, 액제, 에어로솔제, 엑스제(EXTRACTS), 엘릭실제, 연고제, 유동엑스제, 유제, 현탁제, 전제, 침제, 점안제, 정제, 좌제, 주사제, 주정제, 캅셀제, 크림제, 환제, 연질 또는 경질 젤라틴 캅셀 등이 있다.In addition, the formulation of the pharmaceutical composition of the present invention may be in a preferred form depending on the method of use, in particular by adopting methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to a mammal. It can be formulated. Examples of specific formulations include warnings, granules, lotions, linings, limonades, powders, syrups, ointments, liquids, aerosols, EXTRACTS, elixirs, ointments, liquid extracts, emulsions, Suspensions, premises, acupuncture, eye drops, tablets, suppositories, injections, spirits, capsules, creams, pills, soft or hard gelatin capsules.
더 나아가 본 발명의 약학 조성물은 당해 기술 분야의 공지된 적절한 방법을 사용하여 또는 레밍턴의 문헌(Remington's Pharmaceutical Science(최근판), Mack Publishing Company, Easton PA)에 개시되어 있는 방법을 이용하여 바람직하게 제형화될 수 있다.Furthermore, the pharmaceutical compositions of the present invention are preferably formulated using appropriate methods known in the art or using methods disclosed in Remington's Pharmaceutical Science (Recent Edition), Mack Publishing Company, Easton PA. Can be converted.
본 발명에 따른 약학 조성물의 투여량은, 투여방법, 복용자의 연령, 성별 및 체중, 및 질환의 중증도 등을 고려하여 당업자에 의해 적절하게 선택될 수 있다. 일예로, 본 발명의 약학 조성물은 상기 화학식 1의 구조를 갖는 화합물 및 상기 화학식 2의 구조를 갖는 화합물로 이루어진 군 중에서 선택된 어느 하나의 화합물을 기준으로 할 때, 0.000001 mg/kg/day 내지 1000 mg/kg/day로 투여할 수 있다. 투여는 하루에 한번 투여할 수도 있고, 수회 나누어 투여할 수도 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.The dosage of the pharmaceutical composition according to the present invention may be appropriately selected by those skilled in the art in consideration of the method of administration, the age, sex and weight of the recipient, the severity of the disease, and the like. In one embodiment, the pharmaceutical composition of the present invention is 0.000001 mg / kg / day to 1000 mg based on any one compound selected from the group consisting of a compound having a structure of Formula 1 and a compound having a structure of Formula 2 It can be administered as / kg / day. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.
또한, 본 발명의 약학 조성물은, 유효성분인 상기 화학식 1의 구조를 갖는 화합물 및 상기 화학식 2의 구조를 갖는 화합물로 이루어진 군 중에서 선택된 어느 하나의 화합물 또는 그의 약학적으로 허용되는 염 이외에 공지의 심혈관 질환 치료효과를 갖는 화합물을 더욱 포함할 수 있으며, 상기 유효성분 100 중량부에 대하여 각각 5 중량부 내지 100 중량부로 포함될 수 있다.In addition, the pharmaceutical composition of the present invention, in addition to any one compound selected from the group consisting of a compound having a structure of the formula (1) and a compound having the structure of the formula (2) or a pharmaceutically acceptable salt thereof is a known cardiovascular system The compound may further include a compound having a therapeutic effect on the disease, and may be included in an amount of 5 parts by weight to 100 parts by weight based on 100 parts by weight of the active ingredient.
본 발명은 또한 상기 화학식 1의 구조를 갖는 화합물 및 상기 화학식 2의 구조를 갖는 화합물로 이루어진 군 중에서 선택된 어느 하나의 화합물 또는 그의 염을 유효성분으로 포함하는 심혈관계 질환 개선 또는 예방용 식품조성물을 제공한다. 본 발명의 식품 조성물의 예로는 식품, 식품첨가제, 음료 또는 음료첨가제를 들 수 있다.The present invention also provides a food composition for improving or preventing cardiovascular diseases comprising any one compound selected from the group consisting of a compound having the structure of Formula 1 and a compound having the structure of Formula 2 or a salt thereof as an active ingredient. do. Examples of the food composition of the present invention include food, food additives, beverages or beverage additives.
상기 화학식 1의 구조를 갖는 화합물 및 상기 화학식 2의 구조를 갖는 화합물로 이루어진 군 중에서 선택된 어느 하나의 화합물 또는 그의 염을 유효성분으로 포함하는 식품 조성물은 그 제조에 통상적으로 사용하는 적절한 담체, 부형제 및 희석제를 더욱 포함할 수 있다.A food composition comprising any one compound selected from the group consisting of a compound having the structure of Formula 1 and a compound having the structure of Formula 2 or a salt thereof as an active ingredient is an appropriate carrier, excipient, and the like commonly used in the preparation thereof. Diluents may be further included.
본 명세서에서 식품이란 함은 영양소를 한 가지 또는 그 이상 함유하고 있는 천연물 또는 가공품을 의미하며, 바람직하게는 어느 정도의 가공 공정을 거쳐 직접 먹을 수 있는 상태가 된 것을 의미하며, 통상적인 의미로서, 식품, 식품 첨가제, 건강 기능성 식품 및 음료를 모두 포함하는 의도이며, 바람직하게는 껌 또는 캔디일 수 있다.In the present specification, the term "food" means a natural product or processed product containing one or more nutrients, and preferably means a state in which it can be directly eaten through some processing process. It is intended to include all foods, food additives, health functional foods and beverages, preferably gum or candy.
본 발명의 상기 화학식 1의 구조를 갖는 화합물 및 상기 화학식 2의 구조를 갖는 화합물로 이루어진 군 중에서 선택된 어느 하나의 화합물 또는 그의 염을 첨가할 수 있는 식품으로는 예를 들어, 각종 식품류, 음료, 껌, 캔디, 차, 비타민 복합제, 기능성 식품 등이 있다. 추가로, 본 발명에서 식품에는 특수영양식품(예, 조제유류, 영, 유아식 등), 식육가공품, 어육제품, 두부류, 묵류, 면류(예, 라면류, 국수류 등), 건강보조식품, 조미식품(예, 간장, 된장, 고추장, 혼합장 등), 소스류, 과자류(예, 스넥류), 유가공품(예, 발효유, 치즈 등), 기타 가공식품, 김치, 절임식품(각종 김치류, 장아찌 등), 음료(예, 과실, 채소류 음료, 두유류, 발효음료류, 아이스크림류 등), 천연조미료(예, 라면 스프 등), 비타민 복합제, 알코올 음료, 주류 및 그 밖의 건강보조식품류를 포함하나 이에 한정되지 않는다. 상기 식품, 음료 또는 식품첨가제는 통상의 제조방법으로 제조될 수 있다.As a food to which any compound selected from the group consisting of a compound having the structure of Formula 1 and a compound having the structure of Formula 2 or a salt thereof of the present invention may be added, for example, various foods, drinks, gum , Candy, tea, vitamin complexes, and functional foods. In addition, the food in the present invention includes special nutritional products (e.g., crude oil, young, baby food, etc.), processed meat products, fish products, tofu, jelly, noodles (e.g. ramen, noodles, etc.), health supplements, seasonings ( For example, soy sauce, miso, red pepper paste, mixed soy sauce), sauces, confectionery (e.g. snacks), dairy products (e.g. fermented milk, cheese, etc.), other processed foods, kimchi, pickles (various kimchi, pickles, etc.), beverages ( Examples include, but are not limited to, fruits, vegetable drinks, soy milk, fermented beverages, ice cream, etc., natural seasonings (eg, ramen soup, etc.), vitamin complexes, alcoholic beverages, alcoholic beverages, and other dietary supplements. The food, beverage or food additives may be prepared by a conventional manufacturing method.
본 발명에서 기능성 식품이란 식품에 물리적, 생화학적, 생물공학적 수법 등을 이용하여 해당 식품의 기능을 특정 목적에 작용, 발현하도록 부가가치를 부여한 식품군이나 식품 조성이 갖는 생체방어리듬조절, 질병방지와 회복 등에 관한 체조절기능을 생체에 대하여 충분히 발현하도록 설계하여 가공한 식품을 의미하며, 바람직하게는 본 발명의 기능성 식품은 심혈관계 질환, 구체적으로 고혈압의 예방 또는 개선에 관한 체조절기능을 생체에 대하여 충분히 발현할 수 있는 식품을 의미한다. 상기 기능성 식품에는 식품학적으로 허용 가능한 식품 보조 첨가제를 포함할 수 있으며, 기능성 식품의 제조에 통상적으로 사용되는 적절한 담체, 부형제 및 희석제를 더욱 포함할 수 있다.Functional food in the present invention is the control of biological defense rhythm, disease prevention and recovery of food groups or food compositions that have added value to the food by using physical, biochemical, biotechnological techniques, etc. Means the food processed and designed to fully express the gymnastic function related to the living body, preferably the functional food of the present invention to the biological body for the prevention or improvement of cardiovascular disease, specifically hypertension It means the food which can fully express. The functional food may include food acceptable food additives, and may further include appropriate carriers, excipients and diluents commonly used in the manufacture of functional foods.
또한, 본 발명은 다른 측면에 있어서, 감송향 추출물로부터 분리된 화합물을 유효성분으로 포함하는 에폭사이드 가수분해효소(epoxide hydrolase, EH) 저해용 조성물을 제공한다. 상기 에폭사이드 가수분해효소는 가용성 에폭사이드 가수분해효소(soluble epoxide hydrolase, sEH)일 수 있다.In another aspect, the present invention provides a composition for inhibiting epoxide hydrolase (EH) comprising a compound isolated from the extract of Persimmon Scent as an active ingredient. The epoxide hydrolase may be a soluble epoxide hydrolase (sEH).
상기 저해용 조성물은 상기 에폭사이드 가수분해효소의 활성을 저해하거나 상기 효소의 활성을 억제할 수 있는 조성물을 의미한다.The inhibitory composition refers to a composition capable of inhibiting the activity of the epoxide hydrolase or inhibiting the activity of the enzyme.
보다 구체적으로, 본 발명은 하기 화학식 1의 구조를 갖는 화합물 및 하기 화학식 2의 구조를 갖는 화합물로 이루어진 군 중에서 선택된 어느 하나 또는 그의 약학적으로 허용되는 염을 유효성분으로 포함하는 에폭사이드 가수분해효소(epoxide hydrolase, EH) 저해용 조성물일 수 있다.More specifically, the present invention is an epoxide hydrolase comprising any one or a pharmaceutically acceptable salt thereof selected from the group consisting of a compound having a structure of Formula 1 and a compound having the structure of Formula 2 as an active ingredient It may be a composition for inhibiting (epoxide hydrolase, EH).
[화학식 1][Formula 1]
[규칙 제91조에 의한 정정 05.03.2013]　
Figure WO-DOC-FIGURE-159
[Revision under Rule 91 05.03.2013]
Figure WO-DOC-FIGURE-159
[화학식 2][Formula 2]
[규칙 제91조에 의한 정정 05.03.2013]　
Figure WO-DOC-FIGURE-161
[Revision under Rule 91 05.03.2013]
Figure WO-DOC-FIGURE-161
상기 에폭사이드 가수분해효소는 가용성 에폭사이드 가수분해효소(soluble epoxide hydrolase, sEH)일 수 있다.The epoxide hydrolase may be a soluble epoxide hydrolase (sEH).
상기 화학식 1의 구조를 갖는 화합물 또는 상기 화학식 2의 구조를 갖는 화합물은 에폭사이드 가수분해효소의 저해 효과를 갖는 것이므로, 이러한 측면에서 본 발명은 상기 화학식 1의 구조를 갖는 화합물 또는 상기 화학식 2의 구조를 갖는 화합물의 용도일 수 있다. 상기 용도는 에폭사이드 가수분해효소의 저해용 용도일 수 있다.Since the compound having the structure of Formula 1 or the compound having the structure of Formula 2 has an inhibitory effect of epoxide hydrolase, in this aspect, the present invention provides a compound having the structure of Formula 1 or structure of Formula 2 It may be the use of the compound having a. The use may be for the inhibition of epoxide hydrolase.
상기 에폭사이드 가수분해효소를 저해하는 경우, 항염 효과 또는 항고혈압을 포함한 심혈관계 질환의 예방 또는 치료 효과가 있다. 따라서, 상기 화학식 1의 구조를 갖는 화합물 또는 상기 화학식 2의 구조를 갖는 화합물의 용도는 항염 용도 또는 항고혈압 치료용 용도를 포함한 심혈관계 질환의 예방 또는 치료용 용도일 수 있다.When inhibiting the epoxide hydrolase, there is an anti-inflammatory effect or a prophylactic or therapeutic effect of cardiovascular diseases including antihypertensive. Therefore, the compound having the structure of Formula 1 or the compound having the structure of Formula 2 may be used for the prevention or treatment of cardiovascular diseases including anti-inflammatory use or anti-hypertensive use.
상기 화학식 1의 구조를 갖는 화합물 및 상기 화학식 2의 구조를 갖는 화합물로 이루어진 군 중에서 선택된 어느 하나의 화합물은 상기 에폭사이드 가수분해효소 저해용 조성물 내에 단독으로 사용될 수 있으며, 그 외 약리학적으로 허용가능한 담체, 부형제, 희석제 또는 부성분을 추가로 포함할 수 있다.Any one compound selected from the group consisting of a compound having the structure of Formula 1 and a compound having the structure of Formula 2 may be used alone in the composition for inhibiting epoxide hydrolase, and other pharmacologically acceptable Carriers, excipients, diluents or accessory ingredients may further be included.
보다 상세하게는, 상기 화학식 1의 구조를 갖는 화합물 및 상기 화학식 2의 구조를 갖는 화합물로 이루어진 군 중에서 선택된 어느 하나의 화합물을 포함하는 조성물이 약제로 사용되거나, 의약 또는 약학적 용도로 사용되는 경우, 상기 상기 화학식 1의 구조를 갖는 화합물 및 상기 화학식 2의 구조를 갖는 화합물로 이루어진 군 중에서 선택된 어느 하나의 화합물은 통상적인 방법에 따라 약학적으로 허용되는 담체 또는 부형제와 혼합하거나 희석제로 희석하여 사용될 수 있다.More specifically, when the composition comprising any one compound selected from the group consisting of a compound having a structure of Formula 1 and a compound having the structure of Formula 2 is used as a medicament, or used in medicine or pharmaceutical use , Any one compound selected from the group consisting of a compound having the structure of Formula 1 and a compound having the structure of Formula 2 may be mixed with a pharmaceutically acceptable carrier or excipient or diluted with a diluent according to a conventional method. Can be.
이 경우 상기 조성물 내 상기 상기 화학식 1의 구조를 갖는 화합물 및 상기 화학식 2의 구조를 갖는 화합물로 이루어진 군 중에서 선택된 어느 하나의 화합물의 함량은 0.001 중량 % 내지 99.9 중량 %, 0.1 중량% 내지 99 중량% 또는 1 중량% 내지 20 중량%일 수 있으나, 이에 한정되는 것은 아니며, 조성물의 사용태양 및 사용방법에 따라 상기 화합물의 함량은 바람직한 함량으로 적절히 조절하여 사용될 수 있다.In this case, the content of any one compound selected from the group consisting of the compound having the structure of Formula 1 and the compound of Formula 2 in the composition is 0.001% to 99.9%, 0.1% to 99% by weight Or it may be 1% to 20% by weight, but is not limited thereto, the content of the compound may be used to appropriately adjust to the desired content according to the mode of use and method of use of the composition.
상기 약학적으로 허용되는 담체, 부형제 또는 희석제의 예로는, 락토즈, 덱스트로즈, 수크로즈, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로즈, 폴리비닐 피롤리돈, 물, 메틸하이드록시벤조에이트, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유, 프로필하이드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유, 덱스트린, 칼슘카보네이트, 프로필렌글리콜, 리퀴드 파라핀 및 생리 식염수로 이루어진 군에서 선택된 1 이상을 들 수 있으나, 이에 한정되는 것은 아니며 통상의 담체, 부형제 또는 희석제 모두 사용가능하다.Examples of the pharmaceutically acceptable carrier, excipient or diluent 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 and mineral oil, propylhydroxybenzoate, talc, magnesium stearate and mineral oil , Dextrin, calcium carbonate, propylene glycol, liquid paraffin, and one or more selected from the group consisting of physiological saline, but is not limited thereto, and any conventional carrier, excipient or diluent may be used.
본 발명의 조성물은 당해 기술 분야의 공지된 적절한 방법을 사용하여 또는 레밍턴의 문헌(Remington's Pharmaceutical Science(최근판), Mack Publishing Company, Easton PA)에 개시되어 있는 방법을 이용하여 제형화될 수 있다.The compositions of the present invention can be formulated using any suitable method known in the art or using methods disclosed in Remington's Pharmaceutical Science (Recent Edition, Mack Publishing Company, Easton PA).
상기 조성물의 투여량은 투여방법, 복용자의 연령, 성별, 환자의 중증도, 상태, 체내에서 활성 성분의 흡수도, 불활성율 및 병용되는 약물을 고려하여 결정할 수 있으며, 일 예로 1일 유효성분을 기준으로 하였을 때 0.1 mg/kg(체중) 내지 500 mg/kg(체중), 0.1 mg/kg(체중) 내지 400 mg/kg(체중) 또는 1 mg/kg(체중) 내지 300 mg/kg(체중)으로 투여할 수 있으며, 1회 또는 수회로 나누어 투여할 수 있다. 상기 투여량은 어떠한 면으로든 본 발명의 범위를 한정하는 것은 아니다.The dosage of the composition may be determined in consideration of the method of administration, age, sex, severity, condition of the patient, absorption of the active ingredient in the body, inactivation rate and the drug used in combination, for example, based on the daily effective ingredient 0.1 mg / kg (body weight) to 500 mg / kg (body weight), 0.1 mg / kg (body weight) to 400 mg / kg body weight or 1 mg / kg body weight to 300 mg / kg body weight It can be administered, and can be administered once or divided into several times. The dosage does not limit the scope of the invention in any aspect.
이하, 본 발명을 더욱 구체적으로 설명하기 위하여 제조예 및 실시예를 제시한다. 그러나, 하기 제조예 및 실시예는 본 발명의 이해를 돕기 위하여 예시한 것을 뿐, 여러 가지 다른 형태로 변형될 수 있으며, 본 발명의 범위가 하기 예들에 한정되는 것은 아니다.Hereinafter, the preparation examples and examples are presented in order to explain the present invention in more detail. However, the following Preparation Examples and Examples are merely illustrated to aid the understanding of the present invention, and may be modified in various other forms, and the scope of the present invention is not limited to the following Examples.
[실시예]EXAMPLE
실시예 1: 감송향 추출물 및 분획물의 제조Example 1 Preparation of Persimmon Extract and Fractions
1-1. 추출물의 제조1-1. Preparation of Extract
감송향(Nardostachyschinensis)은 2009년도에 채취된 것으로 전남 목포시 상동 소재의 전원인삼당제약에서 한약재로 시판되는 감송향의 뿌리 건조물을 구입하여 4℃에서 냉장보관하며 사용하였다.Persimmon Scent ( Nardostachyschinensis ) was collected in 2009, and the root dried dried persimmon scent was purchased as a medicinal herb from Power Ginseng Dang Pharmaceutical Co., Ltd., Sangdong, Mokpo-si, Jeonnam, Korea.
상기 감송향 추출물의 제조를 위하여, 상기 냉장 보관 중인 감송향 뿌리 건조물에 물 도는 유기용매, 구체적으로 메탄올(GM), 95% 에탄올 수용액(GE), 70% 에탄올 수용액(GE7), 에틸아세테이트(GEt) 및 n-헥산(GH)을 각각 500 mL 첨가하고, homogenizer로 마쇄하여 20℃에서 24시간 동안 침지하여 추출하는 방법으로 추출물을 제조하였다. 상기 용매로 물을 이용하는 경우에는 상기 추출온도를 100℃에서 수행하였다.In order to prepare the extract of the persimmon extract, water or organic solvents in the dried persimmon root dried material in the cold storage, specifically methanol (GM), 95% ethanol aqueous solution (GE), 70% ethanol aqueous solution (GE7), ethyl acetate (GEt) ) And n-hexane (GH) were added 500 mL, and the extract was prepared by crushing with a homogenizer and immersing at 20 ° C. for 24 hours for extraction. When water was used as the solvent, the extraction temperature was performed at 100 ° C.
상기 제조된 추출액은 Whatman NO. 2(Whatman, England)로 감압 여과하여 여액과 잔사로 각각 구분하여 수득하였다. 상기 수득한 잔사는 상기와 같은 방법으로 2회 반복하여 추출 및 여과과정을 수행하였다. The prepared extract was Whatman NO. Filtration under reduced pressure with 2 (Whatman, England) yielded each of filtrate and residue. The obtained residue was repeated twice in the same manner as described above to perform the extraction and filtration process.
상기 수득한 여액 중 열수 추출액은 동결건조(Freezing dryer, Samwon, Korea)하여, 최종적으로 감송향 열수 추출물을 수득하였고, 나머지 유기용매 추출액은 cooling aspirator(CA-11, Eyela, Tokyo, Japan)가 장착된 vacuum evaporator(N-2N, Eyela, Tokyo, Japan)를 사용하여 35±2℃에서 감압 농축하는 방법으로 추출물을 제조하였다.The hot water extract in the obtained filtrate was freeze-dried (Freezing dryer, Samwon, Korea), and finally a saccharin hot water extract was obtained, and the remaining organic solvent extract was equipped with a cooling aspirator (CA-11, Eyela, Tokyo, Japan). Extract was prepared by vacuum concentration at 35 ± 2 ℃ using a vacuum evaporator (N-2N, Eyela, Tokyo, Japan).
상기 추출물의 추출 수율을 측정하여 도 2에 나타내었다. 상기 추출수율은 추출물 제조에 투여된 감송향 대비 수득한 추출물의 중량비(w/w, %)로 구하였다.The extraction yield of the extract was measured and shown in FIG. 2. The extraction yield was determined by the weight ratio (w / w,%) of the extract obtained relative to the sweet scent administered in the preparation of the extract.
상기 도 2에 나타낸 바와 같이, 감송향의 각 용매별 추출 수율은 메탄올 추출물(GM)의 경우, 14.1%로 가장 높았고, 상기 95% 에탄올 수용액 추출물(GE)의 경우, 12.3%이었으며, 상기 70% 에탄올 수용액 추출물(GE7)의 경우, 11.7%이었고, 상기 에틸아세테이트 추출물(GEt)의 경우, 7.1%이었으며, 상기 n-헥산 추출물(GH)의 경우, 7.2%이었다. 또한, 상기 열수 추출물 즉, 열수 환류 추출의 경우, 열수 추출물의 수율은 8.7%이었다.As shown in FIG. 2, the extraction yield for each solvent of the persimmon aroma was 14.1% for methanol extract (GM), 12.3% for the 95% ethanol aqueous solution extract (GE), and 70% for the extract. 11.7% of the ethanol aqueous solution extract (GE7), 7.1% of the ethyl acetate extract (GEt), 7.2% of the n-hexane extract (GH). In addition, in the case of the hydrothermal extract, that is, hydrothermal reflux extraction, the yield of the hydrothermal extract was 8.7%.
1-2. 분획물의 제조1-2. Preparation of Fractions
감송향(Nardostachyschinensis)분획물을 제조하기 위하여, 우선 감송향 메탄올 추출물을 제조하였다.In order to prepare a Nardostachyschinensis fraction, first, a methanol extract was prepared.
상기 실시예 1-1의 냉장 보관 중인 감송향 뿌리 3.0 kg에 메탄올 20 L를 넣고 homogenizer(BM-2 Nissei bio-mixer, Nihonseiki Kaiseiki LTD, Japan)로 마쇄하면서 상기 실시예 1-1과 같이, 3회 반복 추출하여 총 60 L의 감송향 뿌리 메탄올 추출액을 얻었다.20 L of methanol was added to 3.0 kg of the persimmon incense roots in the cold storage of Example 1-1, and ground with a homogenizer (BM-2 Nissei bio-mixer, Nihonseiki Kaiseiki LTD, Japan), as in Example 1-1, 3 Extraction was repeated several times to obtain a total of 60 L of extract of persimmon root methanol.
상기 수득한 메탄올 추출액은 Whatman NO. 2로 감압 여과한 후 여액은 cooling aspirator가 장착된 vacuum evaporator 사용하여 35±2℃에서 감압 농축하는 방법으로 추출물을 제조하여, 추출물 425 g을 수득였다. 상기 추출물은 -20℃ 보관하였다.The methanol extract obtained above was Whatman NO. After filtering under reduced pressure to 2, the filtrate was prepared by extracting the filtrate under a reduced pressure at 35 ± 2 ° C. using a vacuum evaporator equipped with a cooling aspirator to obtain 425 g of extract. The extract was stored at -20 ° C.
상기 추출물의 용매 분획은 우선, 상기 메탄올 추출물 20.0g(141.2 g dry wt. eq.)을 증류수에 녹인 후, n-헥산(5.9 g), 클로로포름(10.3 g), 에틸아세테이트(0.3 g), 부탄올(1.7 g) 및 물(0.8 g)의 순서대로 순차적으로 분획용매를 첨가하고, 용매분획하는 방법으로 수행하였다. 상기 얻어진 분획액은 유기용매를 이용한 분획층의 경우 감압 농축하여 분획물을 수득하였고, 물 분획은 동결건조하여 분획물을 수득하였다.The solvent fraction of the extract was first dissolved in distilled water 20.0 g (141.2 g dry wt. Eq.) Of methanol extract, then n-hexane (5.9 g), chloroform (10.3 g), ethyl acetate (0.3 g), butanol (1.7 g) and water (0.8 g) were sequentially added to the fractional solvent, followed by solvent fractionation. The obtained fraction was concentrated under reduced pressure in the case of a fraction layer using an organic solvent to obtain a fraction, and the water fraction was lyophilized to obtain a fraction.
상기 분획물의 분획 수율은 추출 단계에서 투여된 감송향 대비 수득한 분획물의 중량비(w/w, %)로 구하였다. 감송향의 각 용매별 분획물은 n-헥산의 경우, 5.9 g을 수득하였고, 클로로포름의 경우, 10.3 g을 수득하였으며, 에틸아세테이트의 경우, 0.3 g을 수득하였고, 부탄올의 경우, 1.7 g을 수득하였으며, 물의 경우, 0.8 g을 수득하였다.Fraction yield of the fraction was determined by the weight ratio (w / w,%) of the fraction obtained relative to the sweet smell administered in the extraction step. For each solvent, the fraction of persimmon was obtained 5.9 g for n-hexane, 10.3 g for chloroform, 0.3 g for ethyl acetate, and 1.7 g for butanol. For water, 0.8 g was obtained.
실시예 2: 감송향 추출물 및 분획물의 추출용매 별 기능성 확인Example 2: Verification of functionalities of extracts of persimmon extract and fractions
2-1. 감송향 추출물의 추출용매 별 안전성(세포독성) 확인2-1. Confirmation of Safety (Cytotoxicity) by Extraction Solvent of Persimmon Extract
상기 실시예 1에서 제조된 감송향 추출물의 추출용매별 nitric oxide(NO) 생성 저해능을 측정하기 위하여, 세포주로 RAW 264.7 macropharge(mouse) cell line을 ATCC에서 구입하여 배양하였다.In order to measure the inhibitory ability of nitric oxide (NO) production for each extract solvent of the extract of Persimmon extract prepared in Example 1, a RAW 264.7 macropharge (mouse) cell line as a cell line was purchased and cultured at ATCC.
상기 RAW 264.7 macropharge (mouse) cell line는 5% CO2,95%습도 조건 및 37℃의 배양기에서 10% FBS(GIBCO, 16000-044)와 1% penicillin-streptomycin(GIBCO, 15140-122)가 함유된 DMEM medium(GIBCO, 12100-046)이 담긴 75 cm2T-flask를 이용하여 배양하였다.The RAW 264.7 macropharge (mouse) cell line contains 10% FBS (GIBCO, 16000-044) and 1% penicillin-streptomycin (GIBCO, 15140-122) in an incubator at 5% CO 2 , 95% humidity and 37 ° C. Cultured using a 75 cm 2 T-flask containing the DMEM medium (GIBCO, 12100-046).
상기 배양 세포는 2일 또는 3일에 한번씩 계대 배양을 하면서 실험에 사용하였다. 상기 RAW 264.7의 세포수 측정은, single cell로 만든 다음 hemocytometer로 이용하여 수행하였다.The cultured cells were used for the experiment while subcultured every two or three days. The cell number of the RAW 264.7 was measured using a hemocytometer made of a single cell.
상기 실시예 1에서 제조된 감송향 추출물의 세포독성은 상기 RAW 264.7 cell을 이용한 세포 생존율(cell viability, %)을 MTT assay를 통한 방법으로 확인하였다.Cytotoxicity of the extract of the persimmon extract prepared in Example 1 was confirmed by the method of cell viability (%) using the RAW 264.7 cells through the MTT assay.
상기 MTT assay는 상기 RAW 264.7 cell을 0.25% trypsin-EDTA를 사용해 monolayer로 부유시키고, 10% FBS MEM medium이 96well plate의 1 well당 1 x 105/200μL가 되도록 분주하여 전 배양하였다. 24시간 후 새로운 배지로 교환한 후 시료 최종 농도를 10 μg/mL, 20 μg/mL, 50 μg/mL 및 100 μg/mL이 되도록 하였고, 전체 부피(total volume)를 200 μL로 하여 72시간 동안 배양하였다.In the MTT assay, the RAW 264.7 cells were suspended in a monolayer using 0.25% trypsin-EDTA, and precultured by dispensing 10% FBS MEM medium to 1 x 10 5 / 200μL per well of a 96well plate. After 24 hours, the sample was replaced with fresh medium, and the final sample concentration was 10 μg / mL, 20 μg / mL, 50 μg / mL and 100 μg / mL, and the total volume was 200 μL for 72 hours. Incubated.
MTT formazan의 형성은 2 mg/mL 의 MTT(2,5-diphenyl-2H-tetrazoliumbromide, Sigma, USA) 용액(in PBS) 20 μL를 첨가하여 3시간 내지 4 시간 동안 배양하는 방법으로 수행하였다. 상기 보라색의 MTT formazan을 DMSO용매 1 mL으로 정확히 녹여, 540 nm에서 ELISA microplate reader(680, BIO-RAD, JAPAN)로 흡광도(absorbance)를 측정하였다. 상기 측정한 결과는 도 3에 나타내었다.Formation of MTT formazan was performed by adding 20 μL of 2 mg / mL MTT (2,5-diphenyl-2H-tetrazoliumbromide, Sigma, USA) solution (in PBS) and incubating for 3 to 4 hours. The purple MTT formazan was correctly dissolved in 1 mL of DMSO solvent, and absorbance was measured at 540 nm with an ELISA microplate reader (680, BIO-RAD, JAPAN). The measurement results are shown in FIG. 3.
상기 도 3에 나타낸 바와 같이, 감송향의 열수 추출물(GW), 95% 에탄올 수용액 추출물(GE) 및 70% 에탄올 수용액 추출물(GE7)은 100 μg/mL의 농도까지 세포에 대한 독성이 나타나지 않았고, 메탄올 추출물(GM)의 경우, 50 μg/mL의 농도에 96.8%의 세포 생존율을 보여, 50 μg/mL의 농도까지는 안전한 것으로 확인하였으며, n-헥산 추출물(GH) 및 에틸아세테이트 추출물(GEt)의 경우 100 μg/mL의 농도에 83%의 세포 생존율을 보여 낮은 독성을 나타내었다.As shown in FIG. 3, the hydrothermal extract (GW), 95% ethanol aqueous solution extract (GE), and 70% ethanol aqueous solution extract (GE7) of the extracts did not show toxicity to cells up to a concentration of 100 μg / mL, In the case of methanol extract (GM), it showed 96.8% cell viability at a concentration of 50 μg / mL, and it was confirmed that it was safe up to a concentration of 50 μg / mL, and that of n-hexane extract (GH) and ethyl acetate extract (GEt) The case showed low virulence with 83% cell viability at a concentration of 100 μg / mL.
상기 세포독성과 관련된 결과에 따라, 각 추출용매에 따른 추출물의 NO 생성저해능 및 수용성 에폭사이드 가수분해효소의 저해능을 확인하기 위한 실험에서 각 추출용매 별 추출물의 시료 처리 농도는 50 μg/mL로 결정하였다.According to the results related to the cytotoxicity, the sample treatment concentration of the extract for each extract solvent was determined to be 50 μg / mL in the experiment to confirm the NO production inhibitory ability and the inhibitory ability of the water-soluble epoxide hydrolase of the extract according to each extract solvent. It was.
2-2. 감송향 추출물의 추출용매 별 nitric oxide(NO)생성 저해능 확인2-2. Inhibition of nitric oxide (NO) production by extractant
상기 실시예 1에서 제조된 감송향 추출물의 추출용매별 nitric oxide(NO) 생성 저해능을 측정하기 위하여, 24 well plate에 2×105cells/well의 세포를 주입한 후 24시간 동안 배양하여 세포를 well에 부착시켰다.In order to measure the inhibitory ability of nitric oxide (NO) production for each extract solvent of the extract of Persimmon extract prepared in Example 1, 2 × 10 5 cells / well of cells were injected into a 24 well plate and cultured for 24 hours. Attached to the well.
상기 실시예 1에서 제조된 감송향 추출물을 상기 RAW 264.7 cell에 농도별(25 ㎍/mL, 50 ㎍/Ml 및 100 ㎍/mL)로 처리한 후, LPS(50 ㎍/mL)와 IFN-γ(0.005 U/mL)을 주입하고, 24시간 배양하였다.LPS (50 μg / mL) and IFN-γ after treatment of the extracts prepared in Example 1 by concentration (25 μg / mL, 50 μg / mL and 100 μg / mL) to the RAW 264.7 cells (0.005 U / mL) was injected and incubated for 24 hours.
상기 배양액의 상등액 450 μL와 Griess reagent인 1% sulfanilamide 가 함유된 5% H3PO4및 0.1% N-(1-naphthyl)-ethylendiamide dihydro chloride를 각 well 상의 상등액과 동량 섞어 실온에서 10분간 반응시킨 후, UV-visible spectrophotometer를 이용하여 543 nm에서 흡광도를 측정하였다. 상기 결과로부터 NO(nitric oxide) 발현량을 측정한 결과를 도 4에 나타내었다.450 μL of the supernatant of the culture solution and 5% H 3 PO 4 and 0.1% N- (1-naphthyl) -ethylendiamide dihydro chloride containing 1% sulfanilamide as Griess reagent were mixed with the supernatant of each well and reacted at room temperature for 10 minutes. After, the absorbance was measured at 543 nm using a UV-visible spectrophotometer. The result of measuring the amount of expression of nitric oxide (NO) is shown in FIG. 4.
상기 도 4에 나타낸 바와 같이, 시료 무처리군(control)에 대하여, 추출물 전반적으로 NO 생성 저해능을 보였으며, 비교적 극성 용매 추출물인 열수 추출물(GW)과 70% 에탄올 수용액 추출물(GE7)의 NO 생성 저해능이 80.2% 및 78.3%으로 다른 추출물에 비해 낮은 저해 활성을 나타내었다. 반면, 비극성 용매 추출물인 n-헥산 추출물(GH), 에틸 아세테이트 추출물(GEt), 메탄올 추출물(GM)의 NO 생성 저해능은 각각 61.3%, 62.4% 및 56.3%로 나타나, 높은 저해 활성을 나타내었다.As shown in FIG. 4, for the sample untreated group (control), the extract showed overall NO production inhibiting ability, and the NO production of the relatively polar solvent extract of the hydrothermal extract (GW) and the 70% ethanol aqueous solution extract (GE7) The inhibitory activity was 80.2% and 78.3%, showing a lower inhibitory activity than the other extracts. On the other hand, the non-polar solvent extracts of n-hexane extract (GH), ethyl acetate extract (GEt), methanol extract (GM) NO production was 61.3%, 62.4% and 56.3%, respectively, indicating a high inhibitory activity.
상기 결과로부터 감송향의 메탄올 추출물은 NO 생성 저해능이 가장 우수한 것으로 확인되었다.From the results, it was confirmed that the methanol extract of the persimmon aroma was the most excellent in NO production inhibitory ability.
2-3. 감송향 추출물의 추출용매 별 수용성 에폭사이드 가수분해효소 저해능 확인2-3. Determination of Water Soluble Epoxide Hydrolase Inhibition by Extract Solvents
상기 실시예 1에서 제조된 감송향 추출물의 추출용매별 수용성 에폭사이드 가수분해효소(sEH) 저해능을 측정하기 위하여, 상기 감송향 추출물을 5 μg/mL 내지 500 μg/mL의 농도로 DMSO에 녹인 후 형광기질 CMNPC[Cyano-(2-methoxynaphthalen-6-yl) methyl(3-phenyloxiran-2-yl) methyl carbonate] 및 재조합 hsEH(human soluble epoxide hydrolase produced by baculovirus expression system from insect cell)와 반응하여 수용성 에폭사이드 가수분해효소(sEH)의 저해활성을 측정하였다.In order to measure the water-soluble epoxide hydrolase (sEH) inhibitory ability of each extract of the extract prepared in Example 1, the persimmon extract was dissolved in DMSO at a concentration of 5 μg / mL to 500 μg / mL. Water-soluble epoxides react with fluorescent substrate CMNPC [Cyano- (2-methoxynaphthalen-6-yl) methyl (3-phenyloxiran-2-yl) methyl carbonate] and recombinant human soluble epoxide hydrolase produced by baculovirus expression system from insect cell Inhibitory activity of side hydrolase (sEH) was measured.
상기 측정방법은 구체적으로 다음과 같다. 우선, 96 well plate에 25 mM bis/TrisHCl buffer(pH 7.0, BSA 0.1 mg/mL 포함) 용액 150 μL을 넣고, blank well에 DMSO 2 μL와 상기 실시예 1에서 제조된 감송향 추출물 DMSO 용액을 농도 별로 2 μL씩 분주하였다.The measuring method is specifically as follows. First, 150 μL of 25 mM bis / TrisHCl buffer (pH 7.0, containing 0.1 mg / mL of BSA) solution was added to a 96 well plate, and 2 μL of DMSO in the blank well and the DMSO solution prepared in Example 1 were prepared. Each 2 μL was dispensed.
Microplate reader를 이용하여 30℃에서 5분간 warm-up한 다음, blank well에 25 mM bis/Tris HCl buffer 20 μL을 넣고, activity well에는 baculovirus expression system에 의해 얻어진 재조합 hsEH(0.6 mg/L in 25 mM bis/Tris HCl buffer) 20 μL씩 넣었다. 이 black 96-well plate를 microplate reader에 넣고 10초 동안 혼합(mix)한 후 30℃에서 5분간 warm-up하였다.After warming up at 30 ° C for 5 minutes using a microplate reader, 20 μL of 25 mM bis / Tris HCl buffer was added to a blank well, and the recombinant hsEH (0.6 mg / L in 25 mM) obtained by a baculovirus expression system was added to the activity well. bis / Tris HCl buffer) 20 μL each. The black 96-well plate was placed in a microplate reader, mixed for 10 seconds, and warmed up at 30 ° C. for 5 minutes.
이어서 기질인 CMNPC 용액 30 μL를 넣어 최종 농도가 5 μM이 되게 조절한 다음, 30℃에서 10분 동안 형광 microplate reader(excitation 340 nm, emission 460 nm)를 이용하여 측정하였다. 상기 측정결과를 도 5에 나타내었다.Subsequently, 30 μL of a CMNPC solution, which is a substrate, was adjusted to a final concentration of 5 μM, and then measured using a fluorescent microplate reader (excitation 340 nm, emission 460 nm) at 30 ° C. for 10 minutes. The measurement results are shown in FIG. 5.
상기 도 5에 나타낸 바와 같이, 감송향 추출물 중 메탄올 추출물(GM)의 IC50값은 4.5 μg/mL로 가장 좋은 저해활성을 나타내었고, 95% 에탄올 수용액 추출물(GE) 및 n-헥산 추출물(GH)의 IC50값은 각각 7.0 μg/ mL 또는 7.5 μg/mL 로 나타났으며, 열수 추출물(GW)의 IC50값은 각각 123.5 μg/ mL로 가장 낮은 저해활성을 나타내었다.As shown in FIG. 5, the IC 50 value of the methanol extract (GM) among the extracts of Saengsonghyang showed the best inhibitory activity at 4.5 μg / mL, 95% ethanol aqueous solution extract (GE) and n-hexane extract (GH). The IC 50 value of) was 7.0 μg / mL or 7.5 μg / mL, respectively, and the IC 50 value of the hydrothermal extract (GW) was 123.5 μg / mL, respectively, showing the lowest inhibitory activity.
실시예3: 감송향 분획물의 분획용매 별 기능성 확인Example 3 Functional Confirmation of Fractionant Solvents
3-1. 감송향 메탄올 추출물의 분획물의 분획용매 별 nitric oxide(NO)생성 저해능 확인3-1. Inhibition of nitric oxide (NO) production by fractional solvents
상기 실시예 2에서 확인된 최적의 추출용매인 메탄올을 이용한 감송향 메탄올 추출물의 분획용매별 nitric oxide(NO) 생성 저해능을 측정하기 위하여, 상기 실시예 2-2와 같은 방법으로 수행하였다. 상기 측정한 결과를 도 6에 나타내었다.In order to measure the inhibitory ability of nitric oxide (NO) generation by fractional solvent of the extract of the saenghyanghyang methanol using the optimum extraction solvent identified in Example 2, it was performed in the same manner as in Example 2-2. The measurement results are shown in FIG. 6.
상기 도 6에 나타낸 바와 같이, 시료 무처리군(control)에 대해서 NO 생성 저해능은 n-헥산 분획물이 49.7%로 가장 높았고, 클로로포름 분획이 38.7%, 에틸 아세테이트 분획이 21.1%, 부탄올 분획이 11.6% 순으로 나타났으며, 물 분획은 1.9%로 NO 생성 저해능이 가장 약한 것으로 확인되었다.As shown in FIG. 6, the NO production inhibitory activity was highest in the n-hexane fraction, 49.7%, 38.7% in the chloroform fraction, 21.1% in the ethyl acetate fraction, and 11.6% in the butanol fraction. It was found that the water fraction was 1.9%, which was the weakest inhibitor of NO production.
3-2. 감송향 메탄올 추출물의 분획물의 분획용매 별 수용성 에폭사이드 가수분해효소 저해능 확인3-2. Identification of Water Soluble Epoxide Hydrolase Inhibition by Fraction Solvents
상기 실시예 2에서 확인된 최적의 추출용매인 메탄올을 이용한 감송향 메탄올 추출물의 분획용매별 수용성 에폭사이드 가수분해효소(sEH) 저해능을 측정하기 위하여, 상기 감송향 메탄올 추출물의 분획물을 0.5 μg/mL 내지 200 μg/mL의 농도로 DMSO에 녹인 후 형광기질 CMNPC 및 재조합 hsEH와 반응하여 수용성 에폭사이드 가수분해효소(sEH)의 저해활성을 상기 실시예 2-3과 같은 방법으로 측정하였다.In order to measure the ability of the water-soluble epoxide hydrolase (sEH) to inhibit the fractional solvent of the extract of the saenghyang methanol using methanol which is the optimum extraction solvent identified in Example 2, 0.5 μg / mL of the fraction of the saenghyang methanol extract The inhibitory activity of the water-soluble epoxide hydrolase (sEH) was measured in the same manner as in Example 2-3 by dissolving in DMSO at a concentration of 200 μg / mL and reacting with fluorescent substrate CMNPC and recombinant hsEH.
상기 실시예 2에서 확인된 최적의 추출용매인 메탄올을 이용한 감송향 메탄올 추출물의 분획용매별 수용성 에폭사이드 가수분해효소(sEH)의 저해활성을 측정한 결과를 하기 표 1에 나타내었다.Table 1 shows the results of measuring the inhibitory activity of the water-soluble epoxide hydrolase (sEH) by fractional solvents of the extracts of saenghyang-methanol using methanol as the optimal extraction solvent identified in Example 2.
표 1
분획용매 IC50값(μg/mL)
메탄올 추출물 4.5 ± 0.12
n-헥산 분획물 3.5 ± 0.09
클로로포름 분획물 7.0 ± 1.33
에틸 아세테이트 분획물 21.7 ± 1.92
부탄올 분획물 50.3 ± 1.08
물 분획물 220.1± 8.4
Table 1
Fractional solvent IC50 value (μg / mL)
Methanol extract 4.5 ± 0.12
n-hexane fraction 3.5 ± 0.09
Chloroform fraction 7.0 ± 1.33
Ethyl acetate fraction 21.7 ± 1.92
Butanol fraction 50.3 ± 1.08
Water fraction 220.1 ± 8.4
상기 표 1에 나타낸 바와 같이, n-헥산 분획의 IC50값이 3.5 μg/mL로 가장 강한 저해 활성을 나타내었고, 다음은 클로로포름 분획으로 IC50값이 7.0 μg/mL으로 나타난 반면, 에틸아세테이트 분획과 부탄올 분획에서는 IC50값이 각각 21.7 μg/Ml 및 50.3 μg/mL으로 나타났다.As shown in Table 1, the IC 50 value of the n-hexane fraction showed the strongest inhibitory activity as 3.5 μg / mL, the next is the chloroform fraction IC 50 value of 7.0 μg / mL, while the ethyl acetate fraction The IC 50 values were 21.7 μg / Ml and 50.3 μg / mL in the and butanol fractions, respectively.
상기 결과는 감송향 추출물, 바람직하게는 감송향 메탄올 추출물이 우수한 가용성 에폭사이드 가수분해효소의 저해 효과를 가지고 있고, NO 생성 저해능이 우수함을 나타내며, 상기 결과로부터 혈압 저하능과 항고혈압능을 비롯한 심혈관계 질환 치료, 예방 또는 개선 효과를 가지고 있음을 시사하는 것으로 사료된다. 또한, 감송향 메탄올 추출물의 분획물 중, 헥산 분획물이 가장 우수한 가용성 에폭사이드 가수분해효소의 저해 활성을 가지므로, 상기 결과로부터 가용성 에폭사이드 가수분해효소 저해능과 관련된 혈압 저하능과 항고혈압능을 비롯한 심혈관계 질환 치료 효과를 가지고 있음을 확인할 수 있다.The results indicate that the extract of saenghyanghyang, preferably the extract of saenghyanghyang, has an excellent inhibitory effect of soluble epoxide hydrolase, and is excellent in inhibiting NO production, and from the above results, cardiovascular system including blood pressure lowering ability and antihypertensive activity It is suggested that it has a treatment, prevention, or amelioration effect on a related disease. In addition, since the hexane fraction has the highest inhibitory activity of the soluble epoxide hydrolase among the fractions of the extract of Gamsong-hyang, the cardiovascular system including the blood pressure lowering ability and the antihypertensive activity related to the soluble epoxide hydrolase inhibition activity from the above results. It can be confirmed that the disease has a therapeutic effect.
실시예 4: 헥산 분획층으로부터 유효성분의 분리Example 4 Separation of Active Ingredient from Hexane Fractionated Layer
4-1. 헥산 분획층으로부터의 유효성분의 분리4-1. Separation of Active Ingredients from Hexane Fractionated Beds
감송향 메탄올 추출물의 용매분획 획분 중 가장 활성이 높게 나타난 n-hexane 획분에 함유된 활성 본체의 구명하기 위해 활성물질의 단리를 시도하였다. 먼저 n-hexane 획분(600 mg, 14.2 g dry wt. eq.)을 n-hexane/ethyl acetate 용매계(99:1, 80:20 v/v)로 한 silica gel adsorption column chromatography에 의해 용출 및 분획하였다.Isolation of the active substance was attempted to identify the active body contained in the n-hexane fraction, which was the most active among the solvent fractions of the extract of Gamsong-hyang. First eluted and fractionated by silica gel adsorption column chromatography using n-hexane fraction (600 mg, 14.2 g dry wt.eq.) in n-hexane / ethyl acetate solvent system (99: 1, 80:20 v / v). It was.
구체적으로, silica gel adsorption column은 시료의 200배량에 상당하는 silica gel(40-63㎛ 60, SILICYCLE, Canada)을 n-hexane : ethyl acetate 99:1(v/v)와 혼합하여 slurry를 만든 후 column(35 mm × 700 mm)에 충진하여 제조하였다. 이어서 시료를 n-hexane : ethyl acetate 99:1(v/v) 혼합용매에 녹여 column에 충진한 후 n-hexane : ethyl acetate 99:1(v/v) 혼합용매계로 1 L로 용출한 다음 n-hexane : ethyl acetate 80:20(v/v) 혼합용매로 용출하였다. 용출한 각 sub-fraction은 30 mL test tube에 20 mL씩 분취한 다음 TLC(thin-layer chromatography, 250 ㎛, SILICYCLE, Canada)을 이용(전개용매 n-hexane : ethyl acetate 99:1, 발색제 0.2% naphthoresorcinol), 물질의 spot을 확인하고 비슷한 Rf 값을 갖는 spot의 분획은 서로 합쳐 감압 농축하였다.Specifically, the silica gel adsorption column is made of slurry by mixing silica gel (40-63㎛ 60, SILICYCLE, Canada) equivalent to 200 times of the sample with n-hexane: ethyl acetate 99: 1 (v / v). It was prepared by filling in a column (35 mm × 700 mm). Subsequently, the sample was dissolved in n-hexane: ethyl acetate 99: 1 (v / v) mixed solvent and filled in a column, and then eluted with 1 L of n-hexane: ethyl acetate 99: 1 (v / v) mixed solvent. -hexane: eluted with ethyl acetate 80:20 (v / v) mixed solvent. Each eluted sub-fraction was aliquoted into 30 mL test tube, 20 mL, and then using TLC (thin-layer chromatography, 250 μm, SILICYCLE, Canada) (developing solvent n-hexane: ethyl acetate 99: 1, color developer 0.2% naphthoresorcinol), spots of the material were identified and fractions of spots with similar Rf values were combined and concentrated under reduced pressure.
각 용출 획분들을 각각 TLC에 전개, 확인하여 TLC상에서 나타난 동일한 Rf를 갖는 용출 획분을 합친 결과 4 활성 획분으로 나눌 수 있었다. 상기 결과는 도 7에 나타내었다.Each of the eluted fractions was developed and identified in TLC, and the eluted fractions having the same Rf as shown on the TLC were combined and divided into four active fractions. The results are shown in FIG.
상기 구분된 각각 활성 획분의 hsEH 저해 활성을 상기 실시예 2-3과 같은 방법으로 검정한 결과를 하기 표 2에 나타내었다.As a result of assaying the hsEH inhibitory activity of each of the separated active fractions in the same manner as in Example 2-3, it is shown in Table 2 below.
표 2
분획종류 IC50값(μg/mL)
n-헥산 분획물 3.5 ± 0.09
서브분획물 1(sub-fr. 1) 14.6 ± 0.20
서브분획물 2(sub-fr. 2) 2.5 ± 0.11
서브분획물 3(sub-fr. 3) 3.3 ± 0.02
서브분획물 4(sub-fr. 4) 20.5 ± 1.8
TABLE 2
Type of fraction IC50 value (μg / mL)
n-hexane fraction 3.5 ± 0.09
Sub-fragment 1 14.6 ± 0.20
Sub-fr. 2 2.5 ± 0.11
Sub-fr. 3 3.3 ± 0.02
Sub-fragment 4 20.5 ± 1.8
상기 표 2에 나타낸 바와 같이, 서브-분획물 2 및 서브-분획물 3이 가장 좋은 활성을 보였다.As shown in Table 2 above, sub-fraction 2 and sub-fraction 3 showed the best activity.
상기 한 바와 같이, 감송향 메탄올 추출물의 헥산 분획의 silica gel absorption chromatography에 의해 얻진 서브-분획물 중에서 hsEH 저해 활성 용출 분획이 서브-분획물 2와 서브-분획물 3인 것으로 나타났다. 특히, TLC 양상에 나타났듯 서브-분획물 2의 단일 물질이 아닌 2종의 물질이 혼입되어 있는 것으로 나타나 Sephadex LH-20 column chromatography를 이용하여 순차 정제를 행하였다.As described above, it was shown that the hsEH inhibitory activity eluted fractions were sub-fraction 2 and sub-fraction 3 in the sub-fraction obtained by silica gel absorption chromatography of the hexane fraction of the extract of the persimmon methanol. In particular, as shown in the TLC pattern, it was found that two kinds of substances were mixed instead of a single substance of sub-fraction 2. Thus, purification was performed sequentially using Sephadex LH-20 column chromatography.
구체적으로, Sephadex LH-20 column은 Sephadex LH-20(17-0090-01, GE Healthcare, Sweden)을 chloroform : methanol 1:1(v/v)와 혼합하여 slurry를 만들고, 24 hr swelling한 후 column(30mm × 700mm)에 500 mm까지 충진하여 제조하였다. 시료 분획물을 loading하고 chloroform : methanol 1:1(v/v)의 혼합용매계로 700 mL 용출하였다. 용출한 각 sub-fraction은 25 mL test tube에 15 mL씩 분취한 다음 0.6 mg/dry weight 상당량을 취해 감압 농축한 후 hsEH 저해활성을 측정하였다. 상기 측정 결과를 도 8에 나타내었다.Specifically, the Sephadex LH-20 column is prepared by mixing Sephadex LH-20 (17-0090-01, GE Healthcare, Sweden) with chloroform: methanol 1: 1 (v / v) to make slurry, and after 24 hr swelling (30 mm × 700 mm) was prepared by filling up to 500 mm. The sample fractions were loaded and eluted with 700 mL with a mixed solvent system of chloroform: methanol 1: 1 (v / v). Each eluted sub-fraction was aliquoted into 25 mL test tubes, 15 mL, concentrated to 0.6 mg / dry weight, and concentrated under reduced pressure to measure hsEH inhibitory activity. The measurement results are shown in FIG. 8.
상기 도 8에 나타낸 바와 같이, 그 결과 bed volumn에 대한 elution volumn의 비(Ve/Vt)가 0.60∼0.68의 elution 범위인 No. 14 내지 No. 16이 각각 34.6%, 51.1%, 80.8%의 hsEH 저해활성을 보였다. No. 14 내지 No. 16 용출 분획층을 TLC로 확인한 결과, No. 16 용출 분획이 단일 spot으로 확인되어 상기 No. 16 용출 분획을 HPLC(high performance liquid chromatography)에 의해 정제를 위한 분획으로 선택하였다.As shown in FIG. 8, as a result, the ratio (Ve / Vt) of the elution volumn to the bed volumn is in the range of 0.60 to 0.68. 14 to No. 16 showed 34.6%, 51.1%, and 80.8% of hsEH inhibitory activity, respectively. No. 14 to No. 16 eluted fractionation layer was confirmed by TLC. 16 elution fractions were identified as a single spot. The 16 eluting fractions were selected as fractions for purification by high performance liquid chromatography (HPLC).
상기 HPLC에 의한 정제는 구체적으로 다음과 같은 방법으로 수행하였다. 우선, 상기 HPL에 사용된 컬럼은 C18 column(10×250 mm, 5C18-AR-Ⅱ, Nacalai, JAPAN)을 이용하였고, 4.72 mL/min(Model 980E, JASCO, Japan)의 유속으로 210 nm의 검출파장(MD-2010 Multiwavelength detector, JASCO, Japan)에서 수행하였다.The purification by HPLC was specifically carried out in the following manner. First, the column used for HPL was C18 column (10 × 250 mm, 5C18-AR-II, Nacalai, JAPAN), and 210 nm detection at a flow rate of 4.72 mL / min (Model 980E, JASCO, Japan). It was performed at a wavelength (MD-2010 Multiwavelength detector, JASCO, Japan).
상기 서브-분획물 2의 No. 16 용출 분획 결과물과 서브-분획물 3을 대상으로 C18 column을 이용한 HPLC 분석을 수행한 결과, main peak를 확인할 수 있었다. 상기 확인 결과를 도 9 및 도 10에 나타내었다.No. of the sub-fraction 2 above. As a result of HPLC analysis using the C18 column, 16 main eluted fractions and sub-fraction 3 were identified. The confirmation results are shown in FIGS. 9 and 10.
상기 도 9에 나타낸 바와 같이, 상기 서브-분획물 2의 No. 16 용출 분획 결과물을 50% acetonitrile을 이동상으로 한 ODS-HPLC에 의해 분리한 후 t R 25.86분의 용출 획분에서 main peak를 반복 분취하여 활성 물질인 compound 1을 얻었다.As shown in FIG. 9, the No. The resulting eluted fraction was separated by ODS-HPLC using 50% acetonitrile as a mobile phase, and the main peak was repeatedly extracted from the elution fraction of t R 25.86 minutes to obtain the active compound 1.
또한, 상기 도 10에 나타낸 바와 같이, 서브-분획물 3을 50% acetonitrile을 이동상으로 한 ODS-HPLC에 의해 t R 12.85분에서 용출 획분에서 main peak를 반복 분취하여 활성 물질인 compound 2를 얻었다.In addition, as shown in FIG. 10, sub-fraction 3 was repeatedly fractionated from the elution fraction at t R 12.85 minutes by ODS-HPLC using 50% acetonitrile as a mobile phase to obtain compound 2 as an active substance.
상기 단리된 compound 1과 compound 2의 hsEH저해 활성을 상기 실시예 2-3과 같은 방법으로 측정한 결과, 하기 표 3과 같이 각각 IC50값이 1.60 μg/mL, 2.05 μg/mL의 저해활성을 나타냈다. As a result of measuring the hsEH inhibitory activity of the isolated compound 1 and compound 2 in the same manner as in Example 2-3, the IC 50 value of 1.60 μg / mL and 2.05 μg / mL, respectively, as shown in Table 3 below. Indicated.
표 3
Compound IC50값(μg/mL)
1 1.60 ± 0.02
2 2.05 ± 0.01
TABLE 3
Compound IC50 value (μg / mL)
One 1.60 ± 0.02
2 2.05 ± 0.01
4-2. 유효성분의 확인4-2. Confirmation of active ingredient
상기 실시예 4-1에서 확인된 2가지 유효성분(compound)의 구조를 확인하기 위하여, GC-MS 분석 및 LC-MS 분석과 FT-NMR 분석을 수행하였다.In order to confirm the structures of the two effective compounds identified in Example 4-1, GC-MS analysis, LC-MS analysis, and FT-NMR analysis were performed.
우선, GC-MS 분석은 GC-MS(5975C, Agilent Tech., USA)법으로 분석하였다. 구체적으로, 분석조건은 초기온도를 60℃로 하여 3분간 유지한 다음, 1분에 5℃씩 상승시켜 230℃까지 올린 다음, 이 온도에서 20분간 유지시켰다. 질량분석은 GC에서 분리되어 나온 성분을 70 eV에서 이온화하여 측정하였으며, 이 때의 fragmentation pattern을 Wiely/NIST library를 이용하여 성분을 정성분석하였다. LC-MS 분석, 구체적으로 LC-ESI-MS 분석은 MS(ESI Ion Trap LC-MS System, Bruker, Germany)가 장착된 LC(Agilent 1100 HPLC)를 이용하여 분석을 실시하였다.First, GC-MS analysis was analyzed by GC-MS (5975C, Agilent Tech., USA) method. Specifically, the analysis conditions were maintained for 3 minutes at an initial temperature of 60 ℃, and then raised to 230 ℃ by 5 ℃ by 1 minute, and then maintained at this temperature for 20 minutes. Mass spectrometry was performed by ionizing the components separated from GC at 70 eV. The fragmentation patterns were qualitatively analyzed using the Wiely / NIST library. LC-MS analysis, specifically LC-ESI-MS analysis was performed using LC (Agilent 1100 HPLC) equipped with MS (ESI Ion Trap LC-MS System, Bruker, Germany).
또한, FT-NMR 분석, 구체적으로, 1H-nuclearmagneticresonance(1H-NMR),13C-nuclearmagneticresonance(13C-NMR)및 2D-NMR 등의 기기 분석은 Fourier transform-nuclear magnetic resonance 기기(400 MHz, Avance 400, Bruker, Germany)를 사용하였고, 용매는 deuterium chloroform(CD3Cl),그리고 내부 표준 물질은 tetramethylsilane(TMS, δ=0)을 각각 사용하였다. 1H-NMR과 13C-NMR 분석외에 2D-NMR[shift correlated spectroscopy(COSY), heteronuclear multiple quantum coherence(HMQC), heteronuclear multiple bond correlation(HMBC)]을 분석하여 구조해석하였다.In addition, FT-NMR analysis, specifically, 1 H-nuclearmagnetic resonance ( 1 H-NMR), 13 C-nuclearmagnetic resonance ( 13 C-NMR) and 2D-NMR instrument analysis, such as Fourier transform-nuclear magnetic resonance instrument (400 MHz , Avance 400, Bruker, Germany), deuterium chloroform (CD 3 Cl) as a solvent, and tetramethylsilane (TMS, δ = 0) as an internal standard. In addition to 1 H-NMR and 13 C-NMR analysis, 2D-NMR (shift correlated spectroscopy (COSY), heteronuclear multiple quantum coherence (HMQC), heteronuclear multiple bond correlation (HMBC)) was analyzed.
상기 compound 1에 대한 분석한 결과를 도 11 내지 도 13 및 하기 표 4에 나타내었다.The analysis results of the compound 1 are shown in FIGS. 11 to 13 and Table 4 below.
상기 도 11에 나타낸 바와 같이, compound 1의 구조해석을 위해 GC-MS 분석을 실시한 결과, t R 21.86분에서 222.2 m/z, 207.2 m/z, 189.2 m/z, 179.2 m/z, 161.1 m/z, 151.1 m/z, 138.1 m/z, 125.1 m/z, 109.1 m/z, 98.1 m/z, 83.1 m/z, 69.1 m/z, 55.1 m/z의 Mass spectrum을 보였으며, Wiely/NIST library 이용fragmentation pattern을 비교분석한 결과 patchouli alcohol과 99%로 매우 유사한 경향를 보였다.As shown in FIG. 11, GC-MS analysis for the structural analysis of compound 1 showed 222.2 m / z , 207.2 m / z , 189.2 m / z , 179.2 m / z , 161.1 m at 21.86 min at t R. Mass spectrum of / z , 151.1 m / z , 138.1 m / z , 125.1 m / z , 109.1 m / z , 98.1 m / z , 83.1 m / z , 69.1 m / z , 55.1 m / z Comparative analysis of fragmentation pattern using / NIST library showed very similar trend with patchouli alcohol (99%).
또한, 상기 도 12에 나타낸 바와 같이, 1H-NMR(400MHz,CD3Cl)분석 결과, δ 0.790(3H, d, J=6.68Hz, CH3),δ 0.850(3H, s, CH3),δ 1.072(3H, s, CH3),δ 1.083(3H, s, CH3)의 methyl proton signal를 나타내어 sesquiterpene인 patchouli alcohol의 특징과 유사한 것으로 확인되었다.In addition, as shown in FIG. 12, 1 H-NMR (400 MHz, CD 3 Cl) analysis result showed δ 0.790 (3H, d, J = 6.68 Hz, CH 3 ), δ 0.850 (3H, s, CH 3 ). It was confirmed that the methyl proton signal of δ 1.072 (3H, s, CH 3 ) and δ 1.083 (3H, s, CH 3 ) was similar to that of patchouli alcohol, a sesquiterpene.
또한, 상기 도 13에 나타낸 바와 같이, 단리 화합물의 13C-NMR data는 patchouli alcohol의 특징과 유사한 것으로 확인되었다. 이를 확인하기 위하여, Nishiya 등의 patchouli alcohol data와 비교한 결과를 하기 표 4에 나타내었다.In addition, as shown in FIG. 13, 13 C-NMR data of the isolated compound was confirmed to be similar to the characteristics of patchouli alcohol. To confirm this, the results compared with patchouli alcohol data of Nishiya et al. Are shown in Table 4 below.
표 4
Position δC
Compound 1(CD3Cl) Patchouli alcohol(CD3Cl)
1 76.0 75.6
2 33.0 32.8
3 28.9 28.7
4 28.5 28.2
5 44.0 44.0
6 24.7 24.7
7 39.4 39.6
8 24.9 24.9
9 29.2 29.1
10 38.0 37.8
11 40.5 40.4
12 27.2 27.2
13 24.7 24.4
14 21.0 20.7
15 18.9 18.7
Table 4
Position δC
Compound 1 (CD 3 Cl) Patchouli alcohol (CD 3 Cl)
One 76.0 75.6
2 33.0 32.8
3 28.9 28.7
4 28.5 28.2
5 44.0 44.0
6 24.7 24.7
7 39.4 39.6
8 24.9 24.9
9 29.2 29.1
10 38.0 37.8
11 40.5 40.4
12 27.2 27.2
13 24.7 24.4
14 21.0 20.7
15 18.9 18.7
상기 표 4에 나타낸 바와 같이, compound 1에서 확인된 15개의 signal이 기존의 patchouli alcohol의 signal과 일치하여 이 화합물이 patchouli alcohol임을 확인하였다.As shown in Table 4, the 15 signals identified in compound 1 is consistent with the signal of the existing patchouli alcohol, it was confirmed that the compound is patchouli alcohol.
또한, 상기 compound 2에 대한 분석한 결과를 도 14 내지 도 16 에 나타내었다.In addition, the analysis results for the compound 2 is shown in Figure 14 to 16.
상기 도 14에 나타낸 바와 같이, compound 2의 구조해석을 위해 1H-NMR(400 MHz, CD3Cl) 분석을 실시한 결과, δ 1.025(3H, d, J=6.50Hz, CH3),δ 1.136(3H, s, CH3),δ 1.1.186(3H, s, CH3),δ 1.386(3H, s, CH3)의 methyl proton signal를 나타냈다. 상기 도 15에 나타낸 바와 같이, 13C-NMR(100MHz,CD3Cl)분석 결과, 15개의 carbon signal spectrum을 보였고, 상기 도 16에 나타낸 바와 같이, Direct-positive-EI-MS의 분석결과, M+ion이 250.9 m/z에서 나타났으며, 이물질의 fragment 이온의 패턴으로부터 compound 2와 유사한 sesquiterpene임을 시사하였다.As shown in FIG. 14, 1 H-NMR (400 MHz, CD3Cl) analysis was performed for structural analysis of compound 2, δ 1.025 (3H, d, J = 6.50 Hz, CH 3 ), δ 1.136 (3H , s, CH 3 ), δ 1.1.186 (3H, s, CH 3 ), δ 1.386 (3H, s, CH 3 ) showed a methyl proton signal. As shown in FIG. 15, 13 C-NMR (100 MHz, CD3Cl) analysis showed 15 carbon signal spectrums, and as shown in FIG. 16, the analysis result of Direct-positive-EI-MS, M + ion Was found at 250.9 m / z, suggesting that compound 2 is a sesquiterpene similar to that of fragment ions.
상기 compound 2의 13C-NMR data를 Masuyama 등이 분석한 nardosinone의 13C-NMR data와 비교한 결과를 하기 표 5에 나타내었다.The results are a comparison of the 13 C-NMR data of the compound 2 and 13 C-NMR data of this analysis, a nardosinone Masuyama shown in Table 5.
표 5
Position δC
Compound 2CD3Cl) nardosinone(CD3Cl)
1 137.7 137.6
2 25.7 25.8
3 25.7 25.8
4 32.9 33.0
5 39.8 39.9
6 59.5 59.7
7 77.9 78.0
8 38.3 38.5
9 196.4 196.4
10 139.9 140.1
11 85.0 85.0
12 23.7 23.8
13 26.7 26.9
14 22.0 22.1
15 16.0 16.1
Table 5
Position δC
Compound 2CD 3 Cl) nardosinone (CD 3 Cl)
One 137.7 137.6
2 25.7 25.8
3 25.7 25.8
4 32.9 33.0
5 39.8 39.9
6 59.5 59.7
7 77.9 78.0
8 38.3 38.5
9 196.4 196.4
10 139.9 140.1
11 85.0 85.0
12 23.7 23.8
13 26.7 26.9
14 22.0 22.1
15 16.0 16.1
상기 표 5에 나타낸 바와 같이, compound 2에서 확인된 15개의 signal이 기존의 nardosinone를 분석한 signal과 일치하여 compound 2가 nardosinone임을 확인하였다.As shown in Table 5, it was confirmed that compound 2 is nardosinone by matching 15 signals identified in compound 2 with the conventional signal analysis of nardosinone.

Claims (10)

  1. 감송향(Nardostachyschinensis)추출물을 유효성분으로 포함하는 심혈관계 질환 개선 또는 예방용 식품 조성물.Food composition for improving or preventing cardiovascular diseases, including the extract of Nardostachyschinensis as an active ingredient.
  2. 제1항에 있어서,The method of claim 1,
    상기 심혈관계 질환은 고혈압 또는 고혈압성 심장질환인 심혈관계 질환 개선 또는 예방용 식품 조성물.The cardiovascular disease is a food composition for improving or preventing cardiovascular disease, which is hypertension or hypertensive heart disease.
  3. 제1항에 있어서,The method of claim 1,
    상기 감송향 추출물은 물, 50% 내지 100%의 탄소수 1 내지 4의 알콜, 에틸아세테이트, 헥산, 클로로포름, 메틸렌 클로라이드 및 디클로로메탄으로 이루어진 군 중에서 1종 이상 선택된 추출용매로 추출한 것인 심혈관계 질환 개선 또는 예방용 식품 조성물.The extract of persimmon is improved cardiovascular disease that is extracted with at least one extraction solvent selected from the group consisting of water, alcohol having 1 to 4 carbon atoms of 50% to 100%, ethyl acetate, hexane, chloroform, methylene chloride and dichloromethane Or prophylactic food composition.
  4. 제3항에 있어서,The method of claim 3,
    상기 감송향 추출물은 메탄올로 추출한 것인 심혈관계 질환 개선 또는 예방용 식품 조성물.Wherein the extract is a food composition for improving or preventing cardiovascular disease that is extracted with methanol.
  5. 제4항에 있어서,The method of claim 4, wherein
    상기 감송향 추출물은 상기 메탄올로 추출한 것에 물, 탄소수 1 내지 4의 알콜, 에틸아세테이트, 헥산 및 디클로로메탄으로 이루어진 군 중에서 1종 이상 선택된 분획용매로 분획한 것인 심혈관계 질환 개선 또는 예방용 식품 조성물.The extract of the extract of the sense of flavor is a food composition for improving or preventing cardiovascular disease that is fractionated with one or more fractions selected from the group consisting of water, alcohol having 1 to 4 carbon atoms, ethyl acetate, hexane and dichloromethane extracted with methanol. .
  6. 제5항에 있어서,The method of claim 5,
    상기 감송향 추출물은 상기 메탄올로 추출한 것에 헥산을 분획용매로 분획한 것인 심혈관계 질환 개선 또는 예방용 식품 조성물.Wherein the extract persimmon extract is a food composition for improving or preventing cardiovascular disease that is extracted with hexane as a fraction solvent extracted with the methanol.
  7. [규칙 제91조에 의한 정정 05.03.2013]
    하기 화학식 1의 구조를 갖는 화합물 및 하기 화학식 2의 구조를 갖는 화합물로 이루어진 군 중에서 선택된 어느 하나의 화합물 또는 그의 약학적으로 허용되는 염을 유효성분으로 포함하는 심혈관계 질환 치료 또는 예방용 의약조성물.
    [화학식 1]
    Figure WO-DOC-FIGURE-165a
    [화학식 2]
    Figure WO-DOC-FIGURE-165b
    [Revision under Rule 91 05.03.2013]
    A pharmaceutical composition for treating or preventing cardiovascular diseases comprising any one compound selected from the group consisting of a compound having the structure of Formula 1 and a compound having the structure of Formula 2 or a pharmaceutically acceptable salt thereof as an active ingredient.
    [Formula 1]
    Figure WO-DOC-FIGURE-165a
    [Formula 2]
    Figure WO-DOC-FIGURE-165b
  8. 제7항에 있어서, 상기 심혈관계 질환은 고혈압 또는 고혈압성 심장질환인 심혈관계 질환 치료 또는 예방용 의약조성물.8. The pharmaceutical composition for treating or preventing cardiovascular diseases according to claim 7, wherein the cardiovascular disease is hypertension or hypertensive heart disease.
  9. [규칙 제91조에 의한 정정 05.03.2013]
    하기 화학식 1의 구조를 갖는 화합물 및 하기 화학식 2의 구조를 갖는 화합물로 이루어진 군 중에서 선택된 어느 하나의 화합물 또는 그의 약학적으로 허용되는 염을 유효성분으로 포함하는 에폭사이드 가수분해효소 저해용 조성물.
    [화학식 1]
    Figure WO-DOC-FIGURE-c9a
    [화학식 2]
    Figure WO-DOC-FIGURE-c9b
    [Revision under Rule 91 05.03.2013]
    Epoxide hydrolase inhibition composition comprising any one compound selected from the group consisting of a compound having a structure of formula (1) and a compound having a structure of formula (2) or a pharmaceutically acceptable salt thereof as an active ingredient.
    [Formula 1]
    Figure WO-DOC-FIGURE-c9a
    [Formula 2]
    Figure WO-DOC-FIGURE-c9b
  10. 제9항에 있어서,The method of claim 9,
    상기 에폭사이드 가수분해효소는 가용성 에폭사이드 가수분해효소인 에폭사이드 가수분해효소 저해용 조성물.The epoxide hydrolase is a soluble epoxide hydrolase composition for inhibiting epoxide hydrolase.
PCT/KR2012/011835 2012-02-29 2012-12-31 Composition for treating or preventing hypertension WO2013129769A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR1020120021519A KR101819606B1 (en) 2012-02-29 2012-02-29 Food composition for preventing or improving the hypertension containing nardostachys chinensis extract
KR10-2012-0021519 2012-02-29
KR10-2012-0031444 2012-03-28
KR1020120031444A KR101400735B1 (en) 2012-03-28 2012-03-28 A composition for preventing or improving hypertension

Publications (1)

Publication Number Publication Date
WO2013129769A1 true WO2013129769A1 (en) 2013-09-06

Family

ID=49082929

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2012/011835 WO2013129769A1 (en) 2012-02-29 2012-12-31 Composition for treating or preventing hypertension

Country Status (1)

Country Link
WO (1) WO2013129769A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108721257A (en) * 2017-04-25 2018-11-02 成都中医药大学 Purposes of the patchouli alcohol in preparing the non-dependent vasodilator of endothelium

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101023780B1 (en) * 2009-02-23 2011-03-21 주식회사한국전통의학연구소 Composition for preventing and treating acute pancreatitis comprising Nardostachys jatamansi extract as an active ingredient

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101023780B1 (en) * 2009-02-23 2011-03-21 주식회사한국전통의학연구소 Composition for preventing and treating acute pancreatitis comprising Nardostachys jatamansi extract as an active ingredient

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
REVERMANN, MARC ET AL., J HYPERTENS., vol. 27, no. 2, February 2009 (2009-02-01), pages 322 - 331 *
SONG, MI-YOUNG ET AL., WORLD J GASTROENTEROL, vol. 16, no. 26, 14 July 2010 (2010-07-14), pages 3249 - 3257 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108721257A (en) * 2017-04-25 2018-11-02 成都中医药大学 Purposes of the patchouli alcohol in preparing the non-dependent vasodilator of endothelium
CN108721257B (en) * 2017-04-25 2021-09-10 成都中医药大学 Application of patchouli alcohol in preparation of endothelium-independent vasodilator

Similar Documents

Publication Publication Date Title
WO2018124508A1 (en) Composition for prevention and treatment of muscular diseases or for improvement of muscle function containing 3,5-dicaffeoylquinic acid or chrysanthemum extract
WO2012033378A2 (en) Composition comprising cudrania tricuspidata and coix lachryma-jobi having an obesity-suppressing activity and blood-sugar lowering effect, and a use therefor
WO2010087577A2 (en) Use of thymus capitatus extract, satureja hortensis extract, or carvacrol for treating metabolic diseases
WO2020218720A1 (en) Composition for preventing or treating muscular disorders or improving muscular functions, containing leonurus japonicus extract or leonurine
WO2015037778A1 (en) Composition containing lignan compound as active ingredient for preventing or treating cancer
WO2012067316A1 (en) Composition for prevention or treatment of metabolic diseases or complications thereof containing pterocarpan-based compounds or pharmaceutically acceptable salts thereof as active ingredient, or composition for antioxidation
WO2021080297A1 (en) Composition containing evening primrose flower extract as active ingredient for preventing or treating obesity or metabolic syndromes induced thereby
WO2014003232A1 (en) Composition comprising dendropanax morbifera léveille extract as active ingredient for promoting hair growth
WO2010090498A2 (en) Pharmaceutical composition and health food composition containing youngia denticulata extract, fraction thereof, or compound isolated therefrom as active ingredient for improving liver function
WO2010027221A9 (en) Skin-whitening composition comprising an extract, fraction or compound derived from lindera erythrocarpa
WO2020085826A1 (en) Composition for alleviation of skin irritation induced by environmental pollution factors or for skin protection, containing nutmeg extract or macelignan as active ingredient
WO2018221922A1 (en) Composition for preventing and treating muscle-related diseases, containing coptidis rhizoma extract, and use thereof
WO2013012117A1 (en) Pharmaceutical compositions for preventing or treating inflammatory diseases, comprising phytosterol compound
WO2013129769A1 (en) Composition for treating or preventing hypertension
WO2010041908A2 (en) Novel use of panduratin derivative or boesenbergia pandurata extract
WO2009151236A2 (en) The composition comprising extracts or fractions of magnolia obovata thunb for treating and preventing inflammation disease
WO2020145619A1 (en) Composition for allergy prevention, atopic dermatitis alleviation or skin regeneration, containing, as active ingredient, undecane or undecanal
WO2021256858A1 (en) Composition for improving, preventing or treating non-alcoholic fatty liver disease and preparation method therefor
WO2016204493A1 (en) A novel compound (ks 513) isolated from pseudolysimachion rotundum var. subintegrum, the composition comprising the same as an active ingredient for preventing or treating allergy disease, inflammatory disease, asthma or chronic obstructive pulmonary disease and the use thereof
WO2020130478A1 (en) Anti-allergy, atopic dermatitis alleviation or skin regeneration composition containing jasmone as active ingredient
WO2017030419A1 (en) Composition including euphorbia supina raf. extract or fraction as active ingredient for preventing or treating obesity
WO2016190689A2 (en) Composition for preventing, alleviating or treating muscle diseases or improving muscular function
WO2020189911A1 (en) Composition including noranhydroicaritin, for preventing or treating cardiovascular metabolic diseases
WO2009142458A2 (en) Functional health food composition for preventing carcinogenesis containing aster koraiensis extract,aster koraiensis fractions, gymnasterkoreaynes derivatives separated therefrom or pharmaceutically acceptable salts thereof as effective ingredients
WO2019172566A1 (en) Anti-stress agent, anti-depressant or anti-anxiety agent composition containing ionone as active ingredient

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 12870070

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12870070

Country of ref document: EP

Kind code of ref document: A1