KR101400735B1 - A composition for preventing or improving hypertension - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the treatment or prevention of cardiovascular diseases, which comprises a compound isolated from ginseng root extract as an active ingredient. The cardiovascular disease may be hypertension or hypertensive heart disease. Compounds isolated from the ginseng root extract, which is an active ingredient of the composition of the present invention, have excellent inhibitory effects on NO production, and inhibit epoxide hydrolase, such as epoxyeicosatrienoic acids (EETs) It can inhibit the hydrolysis of an epoxy fatty acid intermediate substance and thus can exert a therapeutic or preventive action effect of a cardiovascular disease including an antihypertensive action.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing or improving hypertension,

The present invention relates to a composition for preventing or improving hypertension.

Epoxide hydrolase (EH, EC 3.3.2.3.) Is a naturally occurring enzyme group and is a hydrolytic enzyme detected in both animals and plants. Epoxide hydrolase (E ₂ H ₂ O) It is a hydrolytic enzyme capable of hydrolyzing an oxide to a diol.

The epoxide hydrolase plays an important role in the metabolism of harmful foreign substances such as hormones, chemotherapeutic drugs, calcinoin, environmental pollutants and fungal poisons.

The epoxide hydrolase is divided into microsomal epoxide hydrolase (mEH) and soluble epoxide hydrolase (sEH) according to the intracellular location and substrate selectivity, and the plant natural product Lt; RTI ID = 0.0 > decomposition. ≪ / RTI >

The soluble epoxide hydrolase mainly participates in the metabolism of lipid oxides such as arachidonic acid and linoleic acid. The hydrolysis of epoxyeicosatrienoic acid (EET) of arachidonic acid shows that they can regulate endothelial function by soluble epoxide hydrolase (sEH) by controlling their fusion to tubular endothelial phospholipids.

Recently, spontaneous hypertensive rats (SHRs) were treated with selective soluble epoxide hydrolase inhibitors, and blood pressure was significantly lowered. In addition, male knockout sEH rats show significantly lower blood pressure than wild-type mice, indicating that soluble epoxide hydrolase can regulate blood pressure.

In addition, the epoxide of arachidonic acid exhibits an anti-inflammatory effect in endothelial cells. On the other hand, epoxynololates, i.e., diols derived from leukotoxin, disturb membrane permeability and calcium homeostasis, leading to inflammation regulated by nitric oxide synthase and endothelin-1.

The concentration of the leukotoxin micromolar level is associated with anti-inflammatory and hypoxic conditions, and it is known to lower mitochondrial respiration in vitro and induce cardiopulmonary resuscitation of mammals in vivo. In addition, toxicity of the leukotoxin causes a number of symptoms of organ dysfunction and acute respiratory distress syndrome (ARDS). In both cellular and individual models, leukotoxin mediated toxicity is dependent on epoxide hydrolysis, so soluble epoxide hydrolase is known to modulate vascular permeability, in particular.

Therefore, it has been reported that the epoxide hydrolase inhibitor (sEH inhibitor) can be a pharmaceutical target for the treatment or prevention of cardiovascular diseases such as hypertension, heart disease, arteriosclerosis or cerebral hemorrhage. . Therefore, it is urgently required to develop an epoxide hydrolase inhibitor derived from a natural substance, in which side effects are not a problem and safety is secured.

KR2008-0109846A KR2009-0064480A KR2010-0110532A KR2011-0005536A

 Hammock, B. D., et al., COMPERIENSIVE TOXICOLOGY Oxford: Pergamon Press, 283-305 (1977)  Fretland, A. J., et al., Chem. Bio. Interact, 129, 41-59 (2000)  Zeldin, D. C., et al., J. Bio. Chem., 268, 6402-6407 (1993)  Node, K., et al., Science, 285, 1276-1279 (1999)  Campbell, W. B., Trends Pharmarcol. Sci., 21, 125-127 (2000)  Moghaddam, M. F., et al., Nat. Med., 3, 562-567 (1997)  Ishizaki, T., et al., Am. J. Physiol., 269, 65-70 (1995)

It is an object of the present invention to provide a composition for treating or preventing cardiovascular diseases, which is less likely to cause side effects due to edible natural products and is safe.

The present invention provides a pharmaceutical composition for the treatment or prevention of cardiovascular diseases comprising as an active ingredient a compound or a pharmaceutically acceptable salt thereof isolated from the ginseng root extract. The cardiovascular disease may be hypertension or hypertensive heart disease.

In addition, the present invention provides a food composition for improving or preventing cardiovascular diseases, which comprises a compound isolated from ginseng root extract or a pharmaceutically acceptable salt thereof as an active ingredient. The cardiovascular disease may be hypertension or hypertensive heart disease. In addition, the food composition may be a health functional food composition.

In the case of inhibiting the above epoxide hydrolase, the antihypertensive effect is excellent. Therefore, in the case of a substance inhibiting epoxide hydrolase, it may be used for the treatment, improvement or prevention of cardiovascular diseases or as an active ingredient of the above- Can be used.

In the present invention, the compound is not limited to the whole of the specified molecule, but also to a pharmaceutically acceptable or pharmacologically acceptable salt thereof, including salts, prodrug conjugates, such as esters and amides, metabolites, hydrates and solvates, ≪ / RTI >

In the present invention, the composition is intended to include any product that contains the specified ingredients in the specified amounts, as well as any product that results, directly or indirectly, from the combination of the specified ingredients in the specified amounts.

In the present invention, the term pharmaceutically acceptable means that the carrier, diluent or excipient should be compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

In the present invention, soluble epoxide hydrolase (sEH) can be used as a therapeutic agent for epoxyeicosatrienoic acids (EETs) in endothelial cells, smooth muscle and other cell types by hydroxyeicosatrienoic acid DHET "). ≪ / RTI > The soluble epoxide hydrolase may be, for example, a human soluble epoxide hydrolase.

As a result of research on plant-derived substances having an effect of treating, ameliorating or preventing cardiovascular diseases, the present inventors have found that the compounds of the present invention isolated from ginseng root extract can inhibit NO production, As a result of inhibiting the enzymes, it is possible to inhibit the hydrolysis of epoxy fatty acid intermediates such as epoxyeicosatrienoic acids (EETs), which are associated with hypertension, Or prevention of cardiovascular diseases including cardiovascular diseases.

Hereinafter, the present invention will be described in more detail.

The present invention relates to a pharmaceutical composition for the treatment or prevention of cardiovascular diseases comprising as an active ingredient any one compound selected from the group consisting of a compound having a structure represented by the following formula (1) and a compound having a structure represented by the following formula (2) or a pharmaceutically acceptable salt thereof To a pharmaceutical composition.

[Chemical Formula 1]

(2)

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 , And more preferably hypertension.

The hypertension is a vascular-related disease, which means arterial hypertension mainly with arterial narrowing. The hypertension may cause arteriosclerosis or hypertensive heart disease.

The atherosclerosis means a disease in which the wall of the artery is thickened and the elasticity of the artery is lowered. When the arteriosclerosis is caused, blood supply to organs that have been supplied with blood from the hardened artery is decreased, and further diseases may be caused. An example of a related disease is coronary artery disease, where atherosclerosis changes in the heart arteries supplying blood to the heart muscle.

A pharmaceutical composition for treating or preventing cardiovascular diseases comprising, as an active ingredient, 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, Saline, buffered saline, dextrose, water, glycerol, and ethanol, and the diluent is not limited thereto.

The pharmaceutical composition for treating or preventing cardiovascular diseases may be applied differently depending on the purpose of administration and disease. The amount of the active ingredient to be substantially administered depends on a variety of relevant factors, such as the disease to be treated, the severity of the patient's condition, whether or not to co-administer with other medicaments (e.g., a chemotherapeutic agent), the patient's age, The route of administration, and the ratio of administration of the composition. The pharmaceutical composition for the treatment or prevention of cardiovascular diseases may be regulated depending on the type and severity of the disease, and may be administered once a day or one to three times, but is not limited thereto.

The pharmaceutical composition for treating or preventing cardiovascular diseases of the present invention may be administered orally or parenterally. Parenteral administration refers to administration of the drug through a route other than oral, that is, rectal, intravenous, peritoneal and muscular, arterial, percutaneous, nasal, inhalation, ocular, and subcutaneous.

The pharmaceutical composition for the treatment or prevention of cardiovascular diseases may be formulated in any form such as an oral administration form, an injectable solution or a topical preparation. The formulations are preferably prepared to be suitable for oral and injectable administration (true solutions, suspensions or emulsions) and are preferably prepared in oral form such as tablets, capsules, soft capsules, aqueous medicaments, pills, desirable.

In the formulation, the peptides of the present invention may be filled into soft capsules without an excipient, and may be made into a suitable formulation after mixing or diluting with the carrier. Examples of suitable carriers include starch, water, saline, Ringer's solution, dextrose, and the like.

The pharmaceutical composition for treating or preventing cardiovascular diseases may be a pharmaceutical composition.

The pharmaceutical compositions can be applied directly to animals, including humans. The animal is a biological group corresponding to a plant. It mainly consumes organic matter as nutrients, and has digestion or excretion and differentiation of the respiratory organs. Preferably, it may be a vertebrate, more preferably a mammal. The mammal may be a human, a pig, a cow, or a goat, and preferably a human.

The pharmaceutical composition may contain, as an active ingredient, 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, Depending on the formulation, the method of use and the intended use, it may further comprise additional components, that is, pharmaceutically acceptable or nutritionally acceptable carriers, excipients, diluents or subcomponents.

More specifically, the pharmaceutical composition may further contain, in addition to the active ingredient, a nutritional agent, a vitamin, an electrolyte, a flavoring agent, a coloring agent, a stabilizer, a pectic acid and a salt thereof, an alginic acid and a salt thereof, an organic acid, a protective colloid thickening agent, A preservative, a glycerin, an alcohol, a carbonating agent used in a carbonated drink, and the like.

The carrier, excipient or diluent may be selected from the group consisting of lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, Selected from the group consisting of water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, dextrin, calcium carbonate, propylene glycol, liquid paraffin, physiological saline But it is not limited thereto, and any conventional carrier, excipient or diluent may be used.

These components may be added to the compound or a pharmaceutically acceptable salt thereof, independently or in combination, with 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 .

The pharmaceutical composition may contain 0.001 wt% to 99.9 wt% of at least 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, %, Preferably 0.1 wt% to 99 wt%, and more preferably 1 wt% to 50 wt%.

The pharmaceutical composition may further comprise conventional fillers, extenders, binders, disintegrants, surfactants, anti-coagulants, lubricants, wetting agents, flavoring agents, emulsifiers or preservatives, Can be used.

Particularly, solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like. These solid preparations may contain at least one excipient such as starch, calcium carbonate, Sucrose, lactose, gelatin, and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used. Liquid preparations for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweetening agents, have.

In addition, the formulation of the pharmaceutical composition of the present invention may be in a desired form depending on the method of use, and employing methods known in the art to provide rapid, sustained or delayed release of the active ingredient after administration to the mammal Can be formulated. Exemplary formulations include, but are not limited to, excipients, granules, lotions, liniments, rimonadense, powders, syrups, ointments, liquids, aerosols, EXTRACTS, elixirs, ointments, Suspensions, premixes, infusions, eye drops, tablets, suppositories, injections, main tablets, capsules, creams, pills, soft or hard gelatin capsules.

Further, the pharmaceutical compositions of the present invention may be formulated using any of the known methods known in the art or by methods disclosed in Remington ' s Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA) .

The dosage of the pharmaceutical composition according to the present invention can be suitably selected by those skilled in the art in view of the administration method, the age, sex, and weight of the recipient, severity of the disease, and the like. For example, the pharmaceutical composition of the present invention may contain 0.000001 mg / kg / day to 1000 mg / day of a compound selected from the group consisting of the compound having the structure of Formula 1 and the compound having the structure of Formula 2 / kg / day. < / RTI > The administration may be carried out once a day or divided into several times. The dose is not intended to limit the scope of the invention in any way.

In addition, the pharmaceutical composition of the present invention may contain, in addition to any 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, A compound having a disease-treating effect, and may be contained in an amount of 5 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, as an active ingredient, 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 do. Examples of the food composition of the present invention include foods, food additives, beverages or beverage additives.

The food composition comprising the compound having the structure of Formula 1 and the compound having the structure of Formula 2 or a salt thereof as an active ingredient may be prepared by mixing a suitable carrier, May further comprise a diluent.

As used herein, the term " food " means a natural product or a processed product containing one or more nutrients. Preferably, it means that the food can be directly eaten through a certain degree of processing. Food, food additive, health functional food and beverage, preferably gum or candy.

Examples of the food to which the compound having the structure of the formula 1 of the present invention and the compound having the structure of the formula 2 or the salt thereof can be added include various foods, , Candy, tea, vitamin complex, and functional foods. In addition, in the present invention, the food may contain special nutritional foods (e.g., crude oil, spirits, infant food, etc.), meat products, fish products, tofu, jelly, noodles (Such as soy sauce, soybean paste, hot pepper paste, mixed sauce), sauces, confectionery (eg snacks), dairy products (eg fermented milk, cheese), other processed foods, kimchi, pickled foods But are not limited to, natural flavors (eg, ramen soup, etc.), vitamin complexes, alcoholic beverages, alcoholic beverages and other health supplement foods. The food, beverage or food additive may be prepared by a conventional production method.

In the present invention, the functional food refers to a food group which is imparted with added value to function and express the function of the food by using physical, biochemical, biotechnological techniques and the like, the regulation of the biological defense rhythm of the food composition, The functional food of the present invention is preferably a food which is processed by designing the body so as to sufficiently express the body control function with respect to the living body. Preferably, the functional food of the present invention is a food having a body control function for prevention or improvement of cardiovascular diseases, Means foods that can be fully expressed. The functional food may include a food-acceptable food-aid additive, and may further comprise suitable carriers, excipients and diluents conventionally used in the production of functional foods.

According to another aspect of the present invention, there is provided a composition for inhibiting epoxide hydrolase (EH) comprising as an active ingredient a compound isolated from a ginseng extract. The epoxide hydrolase may be a soluble epoxide hydrolase (sEH).

The inhibitory composition means a composition capable of inhibiting the activity of the epoxide hydrolase or inhibiting the activity of the enzyme.

More particularly, the present invention relates to a pharmaceutical composition comprising an epoxide hydrolase (hereinafter referred to as " epoxide hydrolase ") comprising, as an active ingredient, any one selected from the group consisting of a compound having a structure represented by the following formula and epoxide hydrolase (EH).

[Chemical Formula 1]

(2)

The epoxide hydrolase may be a soluble epoxide hydrolase (sEH).

The compound having the structure of Formula 1 or the compound having the structure of Formula 2 has an inhibitory effect on the epoxide hydrolase. Thus, the present invention provides a compound having the structure of Formula 1 or a structure of Formula 2 ≪ / RTI > The use may be for the inhibition of epoxide hydrolase.

When the epoxide hydrolase is inhibited, it has 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 prevention or treatment of cardiovascular diseases including anti-inflammatory use or antihypertensive treatment.

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 can be used alone in the epoxide hydrolase activating composition and the other pharmacologically acceptable carrier , An excipient, a diluent or a subcomponent.

More specifically, when a composition 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 is used as a pharmaceutical agent or used for medicinal or pharmaceutical use , The compound having the structure of Formula 1 and the compound having the structure of Formula 2 may be mixed with a pharmaceutically acceptable carrier or excipient according to a conventional method, or may be diluted with a diluent to be used .

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 having the structure of Formula 2 in the composition is 0.001 to 99.9 wt%, 0.1 to 99 wt% Or 1% by weight to 20% by weight. However, the present invention is not limited thereto, and the content of the compound may be appropriately adjusted depending on the manner of use of the composition and the method of use.

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, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, propylhydroxybenzoate, talc, magnesium stearate and mineral oil , Dextrin, calcium carbonate, propylene glycol, liquid paraffin, and physiological saline. However, the present invention is not limited thereto, and any conventional carrier, excipient or diluent may be used.

The compositions of the present invention may 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 can be determined in consideration of the administration method, the age, sex, the severity of the patient, the condition of the patient, the degree of absorption of the active ingredient in the body, the inactivation rate and the drug to be used together. 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, And may be administered once or several times in divided doses. The dose is not intended to limit the scope of the invention in any way.

The compound derived from ginseng root extract of the present invention and the pharmaceutical composition containing the same inhibit the production of NO and can inhibit epoxide hydrolase so that the preventive or therapeutic effect of cardiovascular diseases is recognized and safe plant extract- It can be used for the purpose of treatment, improvement or prevention of cardiovascular diseases, especially hypertension.

FIG. 1 is a schematic view showing a process for preparing an extract and a fraction of persimmon extract according to an embodiment of the present invention.
FIG. 2 is a graph showing a yield according to an extraction solvent of ginseng extract according to an embodiment of the present invention, wherein the horizontal axis represents the extraction solvent and the vertical axis represents the extraction yield.
FIG. 3 is a graph showing the cell viability (%) for confirming the cytotoxicity of the ginseng root extract according to an embodiment of the present invention. The abscissa of the graph indicates the content (μg / mL) of the extract And the vertical axis of the graph represents cell viability. In the graphs, G represents hot water extract, GM represents methanol extract, GE7 represents 70% ethanol aqueous solution extract, GE represents 95% Ethanol aqueous solution, GH means n-hexane extract, and GEt means ethyl acetate extract.
FIG. 4 is a graph showing the NO production ability of the ginseng root extract according to an embodiment of the present invention as the activity of the control group (% of control), wherein the abscissa of the graph indicates the kind of the extract, GE represents an extract of aqueous ethanol solution of 95%, GE7 represents an extract of 70% ethanol aqueous solution, GEt represents an ethyl acetate extract, GH represents an n-hexane extract, G \ means hot water extract, and GM means methanol extract.
FIG. 5 is a graph (IC ₅₀ ) showing the water-soluble epoxide hydrolase of ginseng root extract according to an embodiment of the present invention, wherein the abscissa in the graph indicates the kind of the extract and the ordinate axis in the graph indicates the water- a hydrolase refers to a 50% utilization (IC _50, μg / mL) for inhibiting, and wherein in each graph, GM means the methanol extract, and GE means a 95% ethanol aqueous solution extract and GE7 70% Ethanol solution, GEt means an ethyl acetate extract, GH means an n-hexane extract, and G \ means a hot-water extract.
FIG. 6 is a graph showing the NO production ability of the fractions of the ginseng root extract according to an embodiment of the present invention as the activity of the control group (% of control), wherein the abscissa of the graph indicates the kind of the extract, N-hexane fr. Means hexane fraction, chloroform fr. Means chloroform fraction, ethyl acetate fr means ethyl acetate fraction, butanol fr. Means butanol fr. Means water fraction, and water fr. Means water fraction.
FIG. 7 is a photograph showing the TLC results of the hexane fraction of the ginseng root extract according to an embodiment of the present invention. In the graph, 1 in the horizontal axis represents hexane fraction, and 2 in the graph represents the sub- Means fraction 1, 3 means sub-fraction 2 of hexane fraction, 4 means sub-fraction 3 of hexane fraction, and 4 means sub-fraction 5 of hexane fraction.
FIG. 8 shows the activity of inhibiting the epidosidase activity of the column fraction of the sub-fraction 2 of the hexane fraction of the ginseng root extract according to an embodiment of the present invention. The abscissa of the graph shows the fraction according to the elution amount of the solvent in the column , And the vertical axis of the graph means the amount (IC ₅₀ , μg / mL) for inhibiting the water-soluble epoxide hydrolase by 50%.
FIG. 9 is a graph showing the results of the sub-fraction 2 of the hexane fraction of ginseng root extract according to an embodiment of the present invention. 16 eluted fraction was subjected to Sephadex LH-20 chromatography.
10 is a photograph showing the result of Sephadex LH-20 chromatography of sub-fraction 3 of hexane fraction of ginseng root extract according to an embodiment of the present invention.
FIG. 11 is a graph showing the percentage of the fraction of hexane in sub-fraction 2 of the ginseng root extract according to an embodiment of the present invention. 16 < / RTI > shows the result of GC-MS spectroscopy of compound 1 isolated from the eluted fraction.
FIG. 12 is a photograph showing the result of NMR spectroscopy of compound 2 separated from sub-fraction 3 of the hexane fraction of ginseng extract according to an embodiment of the present invention. FIG. 13A is a photograph showing the result of ¹ H-NMR spectroscopy, 13B is a photograph showing the result of ¹³ C-NMR spectroscopy.
FIG. 13 is a graph showing the results of the sub-fraction 2 of the hexane fraction of ginseng root extract according to one embodiment of the present invention. FIG. 13A is a photograph showing the result of ¹ H-NMR spectroscopy, FIG. 13B is a photograph showing the result of ¹³ C-NMR spectroscopy, FIG. 13C is a photograph showing the result of NMR spectroscopy of EI -MS spectral product.

Hereinafter, the present invention will be described in more detail with reference to production examples and examples. However, the following Preparation Examples and Examples are merely illustrative examples for the purpose of facilitating understanding of the present invention, and can be modified into various other forms, and the scope of the present invention is not limited to the following Examples.

[Example]

Example  One: Gam-cho  Preparation of extract

1-1. Preparation of extract

Nardostachys chinensis was collected in 2009, and purchased from the ginseng herbal medicine market in Mokpo city, Jeonnam, Korea, and stored at 4 ℃ in cold storage.

For the preparation of the ginseng extract, the ginseng root dried in the refrigerator is dissolved in an organic solvent such as methanol (GM), 95% ethanol aqueous solution (GE), 70% ethanol aqueous solution (GE7), ethyl acetate ) And n-hexane (GH) were each added, and the mixture was ground with a homogenizer and immersed at 20 ° C for 24 hours for extraction. In the case of using water as the solvent, the extraction temperature was carried out at 100 占 폚.

 The extract solution prepared was dissolved in Whatman NO. 2 (Whatman, England) to obtain filtrate and residue, respectively. The residue thus obtained was subjected to extraction and filtration by repeating this process twice.

The hot water extract solution from the filtrate thus obtained was freeze-dried (Samwon, Korea), and finally, a hot-water extract was obtained. The remaining organic solvent extract was loaded with a cooling aspirator (CA-11, Eyela, Tokyo, Japan) (N-2N, Eyela, Tokyo, Japan) at a temperature of 35 ± 2 ° C.

The extraction yield of the extract was measured and shown in FIG. The extraction yield was determined as the weight ratio (w / w,%) of the extract obtained relative to the sour taste of the extract.

As shown in FIG. 2, the extraction yield of each solvent of ginseng extract was highest at 14.1% for methanol extract (GM), 12.3% for aqueous extract of 95% ethanol (GE) (GE7), 7.1% in the case of the ethyl acetate extract (GEt) and 7.2% in the case of the n-hexane extract (GH). In addition, in the case of the hot-water extraction, that is, the hot-water reflux extraction, the yield of the hot-water extract was 8.7%.

1-2. Fraction  Produce

Nardostachys chinensis ) fractions were first prepared.

20 kg of methanol was added to 3.0 kg of the ginseng root in the refrigerated storage of Example 1-1 and the mixture was pulverized with a homogenizer (BM-2 Nissei bio-mixer, Nihonseiki Kaiseiki LTD, Japan) The total amount of 60 L of ginseng root methanol extract was obtained.

The obtained methanol extract solution was dissolved in Whatman NO. 2, and the filtrate was concentrated under reduced pressure at 35 ± 2 ° C using a vacuum evaporator equipped with a cooling aspirator to prepare an extract, and 425 g of an extract was obtained. The extract was stored at -20 < 0 > C.

The solvent fraction of the extract was prepared by dissolving 20.0 g (141.2 g dry wt. Eq.) Of the methanol extract in distilled water and then adding n-hexane (5.9 g), chloroform (10.3 g), ethyl acetate (1.7 g) and water (0.8 g) in that order, followed by solvent fractionation. The obtained fraction solution was concentrated under reduced pressure in the case of a fraction layer using an organic solvent, and a water fraction was lyophilized to obtain a fraction.

The fraction yield of the fractions was determined as the weight ratio (w / w,%) of the fractions obtained relative to the syrup fractions administered in the extraction step. 5.9 g of each solvent fraction of gherkin was obtained in the case of n-hexane, 10.3 g in the case of chloroform, 0.3 g in the case of ethyl acetate and 1.7 g in the case of butanol , And in the case of water, 0.8 g.

Example  2: Gam-cho  Identification of Extract Solvent Functionality

2-1. Gam-cho  Identification of the safety (cytotoxicity) of extracts by extraction solvent

To determine the nitric oxide (NO) production inhibitory activity of the extracts of the ginseng root extract prepared in Example 1, RAW 264.7 macropharge (mouse) cell line was purchased from ATCC and cultured.

The RAW 264.7 macropharge (mouse) cell line contained 10% FBS (GIBCO, 16000-044) and 1% penicillin-streptomycin (GIBCO, 15140-122) in a 5% CO ₂ , 95% And cultured in a 75 cm ² T-flask containing DMEM medium (GIBCO, 12100-046).

The cultured cells were used for the experiment while subculturing every 2 or 3 days. The RAW 264.7 cell count was performed using a hemocytometer after being made into a single cell.

The cytotoxicity of the ginseng root extract prepared in Example 1 was confirmed by the MTT assay using cell viability (%) using the RAW 264.7 cell.

The MTT assay is incubated before the frequency division is the RAW 264.7 cell and using a 0.25% trypsin-EDTA in the floating monolayer, 10% FBS MEM medium so that 1 1 x 10 ^5/200 μL per well of a 96well plate. After 24 hours, the final concentration of the sample was changed to 10 μg / mL, 20 μg / mL, 50 μg / mL and 100 μg / mL, and the total volume was changed to 200 μL for 72 hours Lt; / RTI >

The 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) for 3 to 4 hours. The purple MTT formazan was accurately dissolved in 1 mL of DMSO solvent and the absorbance was measured at 540 nm with an ELISA microplate reader (680, BIO-RAD, JAPAN). The measurement results are shown in Fig.

(GW), 95% ethanol aqueous solution extract (GE) and 70% ethanol aqueous solution extract (GE7) showed no toxicity to the cells up to a concentration of 100 μg / mL, In the case of methanol extract (GM), cell viability was found to be 96.8% at a concentration of 50 μg / mL, and it was found to be safe up to a concentration of 50 μg / mL and that of n-hexane extract (GH) and ethyl acetate extract In the case of 100 μg / mL, the cell viability was 83%, indicating low toxicity.

Based on the results related to the above cytotoxicity, in order to examine the inhibitory effect of the extract on NO production and the inhibitory effect of the water-soluble epoxide hydrolase according to the respective extracting solvents, the sample treatment concentration of each extracting solvent was determined to be 50 μg / mL Respectively.

2-2. Gam-cho  Extraction solvent extraction nitric oxide ( NO )produce Low performance  Confirm

To measure nitric oxide (NO) production inhibitory activity of the ginseng extract prepared in Example 1, 2 × 10 ⁵ cells / well were injected into a 24-well plate and cultured for 24 hours. well.

LPS (50 μg / mL) and IFN-γ (50 μg / mL) were treated with RAW 264.7 cells (25 μg / mL, 50 μg / mL and 100 μg / mL) (0.005 U / mL), and cultured for 24 hours.

450 μL of the supernatant of the culture and 5% H ₃ PO ₄ and 0.1% N- (1-naphthyl) -ethylendiamide dihydrochloride containing 1% sulfanilamide as a griess reagent were mixed with the supernatant of each well and incubated at room temperature for 10 minutes The absorbance was measured at 543 nm using a UV-visible spectrophotometer. The results of measurement of NO (nitric oxide) expression level from the above results are shown in FIG.

As shown in FIG. 4, the NO production inhibitory effect of the extract on the overall control of the sample was shown, and the NO production of the relatively polar solvent extract (GW) and the 70% ethanol aqueous solution extract (GE7) The inhibitory activity was 80.2% and 78.3%, respectively. On the other hand, NO production inhibitory activities of n - hexane extract (GH), ethyl acetate extract (GEt) and methanol extract (GM) were 61.3%, 62.4% and 56.3%, respectively.

From the above results, it was confirmed that the methanol extract of ginseng extract had the best inhibitory effect on NO production.

2-3. Gam-cho  Solubility of extracts by solvent Epoxide  Hydrolase Low performance  Confirm

In order to measure the water-soluble epoxide hydrolase (sEH) inhibitory activity of the extract of the ginseng root extract prepared in Example 1, the ginseng root extract was dissolved in DMSO at a concentration of 5 μg / mL to 500 μg / mL Reacted with fluorescent substrate CMNPC [Cyano- (2-methoxynaphthalen-6-yl) methyl (3-phenyloxiran-2- yl) methyl carbonate] and recombinant hsEH (human soluble epoxide hydrolase produced by baculovirus expression system from insect cell) The inhibitory activity of the side hydrolase (sEH) was measured.

The measurement method is as follows. First, 150 μL of 25 mM bis / Tris-HCl buffer (pH 7.0, containing 0.1 mg / mL of BSA) was added to a 96-well plate, and 2 μL of DMSO and DMSO solution Were dispensed at 2 μL per concentration.

After incubation for 5 min at 30 ° C using a microplate reader, 20 μl of 25 mM bis / Tris · HCl buffer was added to the blank well. The activity wells contained recombinant hsEH (0.6 mg / L in 25 mM bis / Tris · HCl buffer). The black 96-well plate was placed in a microplate reader, mixed for 10 seconds, and warmed up at 30 ° C for 5 minutes.

Subsequently, 30 μL of the substrate CMNPC solution was added to adjust the final concentration to 5 μM, followed by measurement at 30 ° C. for 10 minutes using a fluorescent microplate reader (excitation 340 nm, emission 460 nm). The measurement results are shown in Fig.

As shown in FIG. 5, the IC ₅₀ value of the methanol extract (GM) of the ginseng extract was the best inhibitory activity of 4.5 μg / mL, and the 95% ethanol aqueous solution extract (GE) and the n-hexane extract ) Showed IC ₅₀ values of 7.0 μg / mL or 7.5 μg / mL, respectively, and the IC ₅₀ values of hot water extract (GW) were 123.5 μg / mL, respectively.

Example  3: Gam-cho Fraction  Confirmation of functional properties by fraction solvent

3-1. Gam-cho  Methanol extract Fraction  By fraction solvent nitric oxide ( NO )produce Low performance  Confirm

In order to measure the nitric oxide (NO) production inhibitory activity of each of the fractionated methanol extracts of methanol extracts using the optimal extraction solvent identified in Example 2, the same procedure as in Example 2-2 was carried out. The measurement results are shown in Fig.

As shown in FIG. 6, the n-hexane fraction had the highest NO production inhibitory effect (49.7%), the chloroform fraction was 38.7%, the ethyl acetate fraction was 21.1%, the butanol fraction was 11.6% , And water fraction was 1.9%, indicating that NO production inhibition was the weakest.

3-2. Gam-cho  Methanol extract Fraction  Water soluble fractional solvent Epoxide  Hydrolase Low performance  Confirm

In order to determine the water-soluble epoxide hydrolase (sEH) inhibitory activity of each of the fractionated methanol extracts of methanol of the ginseng extract using methanol as the optimal extraction solvent as identified in Example 2, the fraction of the methanol extract of Ginseng extract was added at a concentration of 0.5 μg / mL (200 μg / mL), and then reacted with fluorescent substrate CMNPC and recombinant hsEH to measure the inhibitory activity of the water-soluble epoxide hydrolase (sEH) in the same manner as in Example 2-3.

The inhibitory activity of water-soluble epoxide hydrolase (sEH) was determined by the fraction solvent of methanol extract of Ganoderma lucidum using methanol as an optimal extraction solvent as shown in Example 2, as shown in Table 1 below.

Fraction solvent IC ₅₀ 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

As shown in Table 1, the IC ₅₀ value of the n-hexane fraction showed the strongest inhibitory activity at 3.5 μg / mL, and the IC ₅₀ value was 7.0 μg / mL in the chloroform fraction, while the ethyl acetate fraction And butanol fractions showed IC ₅₀ values of 21.7 μg / Ml and 50.3 μg / mL, respectively

Example  4: Hexane From the fraction layer  Isolation of active ingredient

4-1. Hexane From the fraction layer  Isolation of active ingredient

The active substance contained in the n-hexane fraction, which is the most active fraction of the solvent fraction of methanol extract, was isolated in order to investigate the active substance. First, elution and fractionation were performed by silica gel adsorption column chromatography using n-hexane fraction (600 mg, 14.2 g dry wt. Eq.) With n-hexane / ethyl acetate solvent (99: 1, 80:20 v / v) Respectively.

Specifically, silica gel adsorption column was prepared by mixing silica gel (40 ㎛ -63 ㎛, 60 Å, SILICYCLE, Canada) corresponding to 200 times of the sample with n-hexane: ethyl acetate 99: 1 (v / v) And then packed in a column (35 mm × 700 mm). Then, the sample was dissolved in a mixed solvent of n-hexane and ethyl acetate (99: 1, v / v) and packed in a column. The sample was eluted with 1 L of n-hexane: ethyl acetate mixed solvent system -hexane: ethyl acetate 80:20 (v / v) mixed solvent. The eluted sub-fractions were collected by 20 mL into a 30 mL test tube and then eluted with TLC (thin-layer chromatography, 250 μm, SILICYCLE, Canada) (developing solvent n-hexane: ethyl acetate 99: naphthoresorcinol), the spot of the substance was confirmed, and the fractions of the spot having similar Rf values were combined and concentrated under reduced pressure.

Each elution fraction was elucidated on TLC, and the elution fractions with the same Rf as shown on TLC were combined and divided into 4 active fractions. The results are shown in Fig.

The hsEH inhibitory activity of each of the above-identified active fractions was assayed in the same manner as in Example 2-3, and the results are shown in Table 2 below.

Fraction type IC ₅₀ value (μg / mL) n-hexane fraction 3.5 ± 0.09 Sub-fraction 1 (sub-fr. 1) 14.6 ± 0.20 Sub-fraction 1 (sub-fr. 1) 2.5 ± 0.11 Sub-fraction 1 (sub-fr. 1) 3.3 ± 0.02 Sub-fraction 1 (sub-fr. 1) 20.5 ± 1.8

As shown in Table 2 above, sub-fraction 2 and sub-fraction 3 showed the best activity.

As described above, among the sub-fractions obtained by the silica gel absorption chromatography of the hexane fraction of the methanol extract of persimmon extract, the fraction eluting with hsEH inhibitory activity was sub-fraction 2 and sub-fraction 3. Particularly, as shown in the TLC aspect, it was confirmed that two substances other than the single substance of sub-fraction 2 were incorporated, and sequential purification was carried out using Sephadex LH-20 column chromatography.

Specifically, Sephadex LH-20 column was prepared by mixing Sephadex LH-20 (17-0090-01, GE Healthcare, Sweden) with chloroform: methanol 1: 1 (v / v) (30 mm x 700 mm) to fill up to 500 mm. The sample fractions were loaded and eluted with 700 mL of chloroform: methanol 1: 1 (v / v) mixed solvent system. Each sub-fraction eluted was fractionated into 15 mL of a 25 mL test tube and then concentrated to a concentration of 0.6 mg / dry weight. The hsEH inhibitory activity was measured. The measurement results are shown in Fig.

As shown in FIG. 8, as a result, the ratio of elution volumn to bed volum (Ve / Vt) was in the elution range of 0.60 to 0.68. 14 to No. 16 showed hsEH inhibitory activity of 34.6%, 51.1% and 80.8%, respectively. No. 14 to No. 16 eluted fraction layer was confirmed by TLC. 16 eluted fractions were identified as a single spot, 16 eluted fraction was selected as the fraction for purification by HPLC (high performance liquid chromatography).

Purification by HPLC was carried out in the following manner. First, the column used for the HPL was a C18 column (10 x 250 mm, 5C18-AR-II, Nacalai, JAPAN) Wavelength (MD-2010 Multiwavelength detector, JASCO, Japan).

No. of the sub-fraction 2. 16 elution fraction and sub - fraction 3 were analyzed by HPLC using C18 column. As a result, the main peak was confirmed. The results of the above confirmation are shown in FIG. 9 and FIG.

As shown in Fig. 9, the No. 1 of the sub- 16 eluted fractions were separated by ODS-HPLC using 50% acetonitrile as a mobile phase, and the main peak was repeatedly obtained from elution fractions of t _R 25.86 minutes to obtain compound 1 as an active ingredient.

In addition, the 10, the sub-fractions by the three repeated obtain a main peak in the elution fraction at t _R 12.85 minutes by an ODS-HPLC to 50% acetonitrile as the mobile phase to obtain a compound 2 active material.

The hsEH inhibitory activities of the isolated compound 1 and compound 2 were measured by the same method as in Example 2-3. As a result, IC ₅₀ values were 1.60 μg / mL and 2.05 μg / mL, respectively, .

Compound IC ₅₀ value (μg / mL) One 1.60 + 0.02 2 2.05 ± 0.01

4-2. Identification of active ingredients

GC-MS analysis, LC-MS analysis and FT-NMR analysis were performed to confirm the structures of the two active compounds identified in Example 4-1.

First, GC-MS analysis was analyzed by GC-MS (5975C, Agilent Tech., USA) method. Specifically, the analysis conditions were as follows: the initial temperature was kept at 60 DEG C for 3 minutes, then increased by 5 DEG C per minute to 230 DEG C, and then maintained at this temperature for 20 minutes. Mass spectrometry was performed by ionization at 70 eV. The fragmentation pattern was analyzed by 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).

5) FT-NMR analysis, specifically, ¹ H-nuclear magnetic resonance ( ¹ H-NMR), ¹³ C-nuclear magnetic resonance ( ¹³ C- magnetic resonance instrument (400 MHz, Avance 400, Bruker, Germany) was used, deuterium chloroform (CD ₃ Cl) was used as solvent and tetramethylsilane (TMS, δ = 0) as internal standard. ^{In addition to 1} H-NMR and ¹³ C-NMR analysis, 2D-NMR (shift-correlated spectroscopy (COZY), heteronuclear multiple quantum coherence (HMQC) and heteronuclear multiple bond correlation (HMBC)

The results of the analysis for compound 1 are shown in Figs. 11 to 12 and Table 4 below.

As shown in FIG. 11, when GC-MS analysis was performed for the structural analysis of compound 1, t _R At 21.86 minutes 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 and 55.1 m / z , respectively. The fragmentation pattern of the Wiely / NIST library was comparatively analyzed and showed a similar tendency to patchouli alcohol 99%.

In addition, as shown in FIG. ^{12a, 1 H-NMR (400} MHz, CD 3 Cl) analysis, δ 0.790 (3H, d, J = 6.68Hz, CH 3), δ 0.850 (3H, s, CH 3 ), δ 1.072 (3H, s, CH ₃ ), and δ 1.083 (3H, s, CH ₃ ), which were similar to those of patchouli alcohol, sesquiterpene.

Further, as shown in FIG. The ¹³ C-NMR data of the isolated compound was found to be similar to that of phosphorus patchouli alcohol. To confirm this, the results of comparison with patchouli alcohol data of Nishiya et al. Are shown in Table 4 below.

Position δ _C Compound 1 (CD ₃ Cl) Reference compound ^{1 )} (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

As shown in Table 4 above, the 15 signals identified in compound 1 were consistent with the existing patchouli alcohol, confirming that the compound was a patchouli alcohol.

FIG. 13 shows the result of the analysis on the compound 2. FIG.

As shown in FIG. 13A, ¹ H-NMR (400 MHz, CD 3 Cl) analysis was performed to analyze the structure of compound 2. As a result, δ 1.025 (3H, d, J = 6.50 Hz, CH ₃ ), δ 1.136 , s, CH ₃ ), δ 1.1.186 (3H, s, CH ₃ ), and δ 1.386 (3H, s, CH ₃ ). As shown in Fig. ^{13b, 13 C-NMR (100} MHz, CD3Cl) analysis showed 15 carbon showed the signal spectrum, as shown in FIG 13c, the Direct-positive-EI-MS analysis, M ⁺ ion at 250.9 m / z, suggesting that it is a similar sesquiterpene to compound 1 from the fragment ion pattern of foreign matter.

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.

Position δ _C Compound 2 (CD ₃ Cl) Reference compound ^{1 )} (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

As shown in Table 5, 15 signals identified in compound 2 were consistent with the conventional nardosinone and compound 2 was found to be nardosinone.

Claims (4)

A pharmaceutical composition for treating or preventing a hypertensive disease, comprising a compound having a structure represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
[Chemical Formula 1]

1. A food composition for improving or preventing hypertension comprising a compound having a structure of the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.
[Chemical Formula 1]

3. The method of claim 2,
Wherein the food composition is a health functional food composition. delete

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