KR20130104103A - Composition comprising extract of myristica fragrans or lignan compounds isolated therefrom for treating or preventing vascular diseases - Google Patents

Abstract

PURPOSE: A pharmaceutical composition for preventing or treating vascular diseases is provided to suppress NO generation and neointimal formation, thereby effectively preventing or suppressing vascular diseases. CONSTITUTION: A pharmaceutical composition for preventing or treating vascular diseases contains 0.5 wt% or less of myristicin and 10-30 wt % of an ethanol solution extract of Myristica fragrans. The ethanol solution contains one or more compounds selected among nectandrin A, fragransin C1, verrucosin, saucernetindiol, tetrahydrofuroguaiacin, and nectandrin B of chemical formula 1. The pharmaceutical composition suppresses NO generation and neointimal formation caused by phosphorylation of endothelial nitric oxide synthase (eNOS).

Description

Composition comprising extract of Myristica fragrans or lignan compounds isolated there from for treating or preventing vascular diseases}

The present invention relates to a nutritional supplement ( Myristica fragrans ) extract or a composition for the prevention or treatment of vascular diseases containing a lignan-based compound isolated therefrom, and more specifically, 10-30% aqueous ethanol extract or 2,5-bis-aryl separated therefrom It relates to a composition for the prevention or treatment of vascular diseases containing -3,4-dimethyltetrahydrofuran lignan compounds (2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan compounds) as an active ingredient.

In modern society, vascular disease is becoming a serious social problem. Examples of the vascular diseases include arteriosclerosis, hypertension, angina pectoris, myocardial infarction, ischemic heart disease, heart failure, complications arising after pericardial artery arteriography, cerebral infarction, cerebral hemorrhage, and stroke. According to the statistics released by the National Statistical Office in 2010, vascular diseases including hypertensive diseases, ischemic heart diseases and cerebrovascular diseases are the second leading cause of death in Korea, followed by malignant tumors, Or more, the mortality rate of vascular disease is greatly increased in women over 65 years of age. In particular, risk factors associated with atherosclerosis are age, sex, hypertension, hyperlipidemia, diabetes, smoking, lack of exercise and obesity. Patients with vascular disease receive continuous medication to treat it, but it is not easy to cure it in modern medicine. If you take the medicine for a long time, the side effect is big.

Among the vascular diseases, atherosclerosis is mainly due to genetic variation, hypercholesterolemia, homocystinemia, metabolic damage such as diabetes, physical damage such as vortex, hypertension, or immunological damage after heart and kidney transplantation. It is known that vascular endothelial cells are caused by dysfunctional states that do not maintain normal homeostasis due to constant or repeated stress. In addition, the expression of adhesion molecules of vascular endothelial cells is significantly increased, and cell permeability is increased to increase adhesion of blood immune cells such as monocytes, platelets and lipids, and permeability to tissues. Atherosclerotic lesions are known to occur due to inflammatory reactions such as secretion of inflammation mediators and growth factors. For example, low density lipoprotein (LDL) in the blood is transformed into modified-LDL through oxidation and glycosylation through the endothelium, which stimulates and damages vascular endothelial cells and smooth muscle. In addition, the expression of adherents including VCAM-1 and ICAM-1 promotes the influx / accumulation of LDL into endothelial cells, and the process of oxidative LDL inducing macrophages and the like and activation of foam cells. Repeatedly promotes the inflammatory response.

In addition, one of the vascular diseases, the vascular wall thickening (intimal hyperplasia, neointima) phenomenon is known as the primary cause of vascular restenosis occurring after stent surgery and balloon surgery. This is due to overproliferation of vascular smooth muscle cells in the inner wall and increased smooth muscle cell migration in the medial wall. Vascular wall thickening may be a disorder of blood circulation because it has an effect of narrowing the flow path of blood, and is also known as the cause of atherosclerosis.

Meanwhile, endothelial nitric oxide synthase (eNOS) present in vascular endothelial cells produces NO (nitric oxide) based on L-arginine (L-arginine), thereby promoting vascular relaxation and inhibiting LDL oxidation. It inhibits the progression of atherosclerosis. Thus, deactivation of eNOS acts as a cause or representative pathologic marker of vascular endothelial dysfunction. Conversely, a substance that promotes eNOS activity can be used as a candidate drug to improve chronic vascular disease. Nebivolol, a third-generation beta-blocker, enhances vascular relaxation through eNOS activation. It is known to make.

Dysfunction of the vascular endothelium acts as a key cause of most vascular diseases including atherosclerosis, but as described above, various pathologies are involved and development of a therapeutic agent for improving this is insufficient. Therefore, the discovery of candidate substances that control the vascular endothelial dysfunction may suggest important clues in the development of therapeutic agents for vascular diseases.

On the other hand, the regulation of eNOS has been reported various pathways such as calcium accumulation in endothelial cells, the regulation of the coenzyme tetrahydrobiopterin (tetrahydrobiopterin), the phosphorylation of eNOS is known to be the most important regulatory mechanism recently. eNOS has various phosphorylation sites, but the phosphorylation of Ser-1177 is estrogen receptor (Circulation, 2002, 105 (11), 1368 ~ 1373), PI3-kinase / Akt (Nature, 1999, 399, 601 ~ 605), MAP kinase (J. Biol. Chem., 2001, 276 (50), 47642 ~ 47649) and AMP kinase (J. Biol. Chem., 2003, 278 (34), 31629 ~ 31639) Is regulated, and plays an important role in changing the activity of eNOS. In fact, in postmenopausal women whose female hormone estrogen secretion is weakened, the incidence of cardiovascular disease is significantly increased, which can be attributed to the decreased activity of eNOS. Therefore, the discovery of natural substances that enhance enzymatic activity by eNOS phosphorylation may be an important strategy for dysfunction of blood vessels and improvement of blood circulation.

Nutmeg ( Myristica fragrans ), a natural product of the present invention, is an evergreen tree belonging to the nutmeg family and is a herbaceous plant grown in Sumatra or Java. Fruits are egg-shaped flesh, and include seeds surrounded by a red scarlet seed coat in the center. Nutmeg has been widely used as a flavoring agent for foods such as sauces, and has been used in herbal medicine as an aromatic nutrient for diarrhea, bloating, vomiting, and loss of appetite.

However, in addition to the usefulness of these nutmeg, many literatures report that acute toxicity occurs when a large amount of nutmeg is used. The nutmeg extract is a representative substance which is toxic to humans, and is an alkylbenzene derivative, myristi, which is disclosed in the following figure. It is known that myristicin, elemicin, safrole and the like are extracted together.

These compounds are known to exhibit toxic effects similar to those of antipsychotic drugs when they are ingested by humans and are converted into amphetamine derivatives in the body. Traditionally, to remove the toxicity of nutmeg, the nutmeg is soaked in lime for one day, It was also used in medicinal materials. The most common toxic substance in nutmeg is myricitin (Yagaku Zasshi, 128 (1), 2008, 129 ~ 133), and the nutmeg extract has been reported to contain 2.1% of mysticin on average. Natural Toxins, 5 (5), 1997, 186-192).

Prior patents related to nutmeg extract include "composition for inhibiting cholesterol esterase containing herbal extract (Korean Patent No. 399529)" and "prevention or treatment of coronary heart disease or arteriosclerosis containing herbal extract as an active ingredient. Pharmaceutical composition (Korean Patent No. 793204) ”and the like, but they are only used as the nutmeg extract itself, whose active ingredient is not identified, and is less relevant to the present invention confirming the activity of the compound contained in the nutmeg extract.

The prior patents related to the compounds isolated from the extract of nutmeg include "inhibitor of toxicity caused by anticancer agent and anticancer composition containing it (Korean Patent No. 646574)", "treatment or prevention of acne containing lignan compound as active ingredient , A pharmaceutical composition for the treatment or prevention of an inflammatory disease containing a lignan compound (Korean Patent Registration No. 567431), a medicinal composition for treating liver diseases and liver diseases (Korean Patent No. 619498) Korean Patent No. 579752), "" Pharmaceutical composition for the treatment or prevention of type 2 diabetes (Korean Patent No. 627643) "," PPAR alpha containing the macelignan compound or its pharmaceutically acceptable salt as an active ingredient (Korean Patent No. 830192) ", "a composition for the treatment or prevention of cranial nerve diseases containing lignan compounds ", " Composition (Korean Patent No. 679306) ", etc., but these are disclosed as various activities using a macelignan compound, which may be a 2,5-bis-aryl-3,4-dimethyl of the present invention. Its structure and properties are different from those of tetrahydrofuran lignan-based compounds, and their application is also different.

Registered Korean Patent No. 399529 (Cholesterol esterase inhibition composition containing herbal extracts, 2003.09.26. Notification) Korean Registered Patent No. 793204 (Pharmaceutical composition for the prevention or treatment of coronary heart disease or atherosclerosis, containing herbal extract as an active ingredient, announcement 2008.01.10.) Korean Registered Patent No. 646574 (Inhibitors of Toxicity Induced by Anticancer Agents and Anticancer Compositions Containing the Same, 2006.11.23. Korean Registered Patent No. 567431 (A composition for treating or preventing acne containing a lignan-based compound as an active ingredient, announcement 2006.04.04.) Korean Registered Patent No. 619498 (Medicinal composition for liver protection and liver disease treatment, 2006.09.06. Notification) Korean Registered Patent No. 579,522 (Pharmaceutical composition for the treatment or prevention of inflammatory diseases containing lignan-based compounds, Announced May 15, 2006) Korean Registered Patent No. 626643 (Pharmaceutical composition for treating or preventing type 2 diabetes mellitus 2006.09.25. Notification) Korean Registered Patent No. 830192 (A composition for the prevention or treatment of diseases mediated by PPARα containing mayligignan compound or a pharmaceutically acceptable salt thereof as an active ingredient, notification of 2008.05.19.) Korean Registered Patent No. 679306 (Pharmaceutical composition for the treatment or prevention of cerebral neurological diseases containing lignan-based compounds, 2007.02.06. Notification)

Simoncini T., Genazzani A.R., Liao J.K., Nongenomic mechanisms of endothelial nitric oxide synthase activation by the selective estrogen receptor modulator raloxifene., Circulation, 2002, 105 (11), 1368-1373 Hisamoto K., Ohmichi M., Kanda Y., Adachi K., Nishio Y., Hayakawa J., Mabuchi S., Takahashi K., Tasaka K., Miyamoto Y., Taniguchi N., Murata Y., Induction of endothelial nitric-oxide synthase phosphorylation by the raloxifene analog LY117018 is differentially mediated by Akt and extracellular signal-regulated protein kinase in vascular endothelial cells., Nature, 1999, 399, 601--605 Hisamoto K., Ohmichi M., Kanda Y., Adachi K., Nishio Y., Hayakawa J., Mabuchi S., Takahashi K., Tasaka K., Miyamoto Y., Taniguchi N., Murata Y., Induction of endothelial nitric-oxide synthase phosphorylation by the raloxifene analog LY117018 is differentially mediated by Akt and extracellular signal-regulated protein kinase in vascular endothelial cells., J. Biol. Chem., 2001, 276 (50), 47642-47649 Morrow V.A., Foufelle F., Connell J.M., Petrie J.R., Gould G.W., Salt I.P., Direct activation of AMP-activated protein kinase stimulates nitric-oxide synthesis in human aortic endothelial cells., J. Biol. Chem., 2003, 278 (34), 31629-31639 Maeda A., Tanimoto S., Abe T., Kazama S., Tanizawa H., Nomura M., Chemical constituents of Myristica fragrans Houttuyn seed and their physiological activities., Yagaku Zasshi, 128 (1), 2008, 129--133 Hallstrom H., Thuvander A., Toxicological evaluation of myristicin., Natural Toxins, 5 (5), 1997, 186--192

An object of the present invention is nutmeg ( Myristica fragrans ) extract or a composition for the prevention or treatment of vascular diseases containing a lignan compound isolated therefrom. Still another object of the present invention is a 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan-based compound (2,5-bis-aryl-3) extracted from 10-30% ethanol aqueous solution of nutmeg or separated therefrom It is to provide a composition for the prevention or treatment of vascular diseases containing, 4-dimethyltetrahydrofuran lignan compounds) as an active ingredient.

The present invention relates to a nutritional supplement ( Myristica fragrans ) extract or a composition for the prevention or treatment of vascular diseases containing a lignan compound isolated therefrom. The vascular disease may be a disease selected from the group consisting of atherosclerosis, hypertension, angina pectoris, myocardial infarction, ischemic heart disease, heart failure, complications occurring after carotid angioplasty, cerebral infarction, cerebral hemorrhage, and stroke.

The nutmeg extract is prepared in a state in which toxic substances such as myristicin are removed by extracting nutmeg with 10-30% ethanol aqueous solution extract and having high stability.

Therefore, preferably, the present invention has a myristicin content of 0.5% by weight or less, and the 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan compound (2,5-bis-) of Formula 1 Nectandrin B, Nectandrin A, Fragransin C1, Verrucosin, Saucernetinol, aryl-3,4-dimethyltetrahydrofuran lignan compounds And tetrahydrofuroguaiacin (Tetrahydrofuroguaiacin) relates to a pharmaceutical composition for the prevention or treatment of vascular disease, characterized in that it contains an extract of 10-30% ethanol aqueous solution of nutmeg containing at least one compound selected from the group consisting of. .

[Formula 1]

In addition, the present invention is a 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan compound (2,5-bis-aryl-3) of Formula 1 isolated from the extract of 10-30% ethanol aqueous nutmeg Nectandrin B, Nectandrin A, Fragransin C1, Verrucosin, Saucernetinol, and tetrahydro, which are, 4-dimethyltetrahydrofuran lignan compounds It relates to a pharmaceutical composition for the prevention or treatment of vascular diseases, characterized in that it contains at least one compound selected from the group consisting of puroguaiacin (Tetrahydrofuroguaiacin).

The 2,5-bisaryltetrahydrofuran lignan compound is obtained by grinding nutmeg and extracting it with 10 to 30% aqueous solution of ethanol; And purely separating and purifying the 2,5-bisaryl tetrahydrofuran lignan compound using chromatography.

The 10 to 30% ethanol aqueous solution extract of the nutmeg may further be used to remove the remaining trace amount of myristidine and concentrate the 2,5-bisaryltetrahydrofuran lignan compound using an ion exchange resin. Thus, the ion exchange resin eluate is a 2,5-bisaryl tetrahydrofuran lignan compound of Formula 1, Nectandrin B, Nectandrin A (Nectandrin A) ), Fragransin C1 (Fragransin C1), berucosin (Verrucosin), Sausnetinediol (Saucernetindiol) and tetrahydrofuroguaiacin (Tetrahydrofuroguaiacin) at least one compound selected from the group comprising as an active ingredient It is characterized by. In addition, the myristidine content of the ion exchange resin eluate of the nutmeg can be 0.1% by weight or less. More specifically, the ion exchange resin may be one of aromatic non-substituted resins (Diaion HP-20, SP825, AXT204, XAD1600T, and MN200) as a synthetic adsorbent.

Nectandrin B, Nectandrin A, Fragransin C1, Verrucosin, and Suchenetin, which are 2,5-bisaryltetrahydrofuran lignan compounds of the present invention, Saucernetindiol and Tetrahydrofuroguaiacin can be separated and extracted from nutmeg by extraction with an organic solvent (alcohol, ether, acetone, etc.), hexane and water, and column chromatography. Known methods used can be easily obtained by using alone or in suitable combination. The crude extract can be further purified according to the conventional method, if necessary.

The chromatography used in the present invention includes silica gel column chromatography, LH-20 column chromatography, ion exchange resin chromatography, thin layer chromatography Thin layer chromatography (TLC) and high performance liquid chromatography may be used.

In addition, the 10 to 30% ethanol aqueous solution extract of the nutmeg of the present invention or the 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan compound isolated therefrom not only can be easily separated from nutmeg, Therefore, it can be used as an additive for foods and medicines.

The pharmaceutical composition comprising the aqueous solution of 10 to 30% ethanol in nutmeg or the 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan compound isolated therefrom may be formulated into powders, granules, And may be formulated in the form of tablets, capsules, suspensions, emulsions, oral preparations such as syrups and aerosols, external preparations, suppositories, and sterilized injection solutions. Examples of carriers, excipients and diluents that can be contained in the pharmaceutical composition 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. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may be prepared by mixing the aqueous solution of 10 to 30% ethanol in the nutmeg of the present invention or the 2,5-bis- Dimethyltetrahydrofuran lignan compound is prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Examples of the liquid preparation for oral use include suspensions, solutions, emulsions, and syrups. In addition to water and liquid paraffin, simple diluents commonly used, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like may be included . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The dosage of the pharmaceutical composition containing the 10 to 30% ethanol aqueous solution extract of the nutmeg or the 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan compound isolated therefrom of the present invention may vary depending on the age, Sex, weight and the particular disease or condition being treated, the severity of the disease or condition, the route of administration, and the judgment of the prescriber. Dosage determinations based on these factors are within the level of ordinary skill in the art and generally the dosage ranges from 0.01 mg / kg / day to approximately 2000 mg / kg / day. A more preferable dosage is 1 mg / kg / day to 500 mg / kg / day. 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.

The pharmaceutical composition containing the extract of aqueous solution of 10 to 30% ethanol in nutmeg or the 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan compound isolated therefrom of the present invention can be administered to mammals such as rats, ≪ / RTI > by various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection. Since the extract of the present invention has little toxicity and side effects, it can be safely used even for long-term administration for the purpose of prevention.

The present invention also provides a health functional food comprising 10 to 30% ethanol aqueous solution extract of nutmeg and a food acceptable food supplementary additive. The health functional food of the present invention includes forms such as tablets, capsules, pills, and liquids. Examples of the foods to which the extract of the present invention can be added include various foods, beverages, gums, tea, vitamins , And health functional foods. Specifically, the present invention provides a health functional food for the prevention or improvement of vascular diseases, including nutmeg 10-30% ethanol aqueous solution extract and food acceptable food supplement additives.

Nutmeg 10 ~ 30% ethanol aqueous solution extract according to the present invention is high in the concentration of 2,5-bisaryl tetrahydrofuran lignan-based compound with a low content of myristicin, a toxic substance, more toxic and safer than conventional nutmeg extracts. High activating effect In addition, the nutmeg 10-30% aqueous ethanol extract or 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan-based compounds isolated from the NO production activation effect through the eNOS phosphorylation of vascular endothelial cells Inhibition of vascular wall thickening by inhibiting vascular smooth muscle cell proliferation. It can be usefully used as a composition that can effectively prevent or inhibit vascular diseases, including.

FIG. 1 is a graph showing the spectral results of HPLC analysis of 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan compounds separated from an extract obtained by extracting nutmeg with 30% ethanol.
FIG. 2a shows the result of confirming the increase of eNOS phosphorylation using Western blot after treating Nectandrin B to ECV 304 cells (presumptive endothelial cell line). FIG. 2b shows Nectandrin B for 24 hours. The result which confirmed the increase of NO production | generation in the cultured medium using the Gries reaction liquid is shown.
Figure 3 is a 30% ethanol aqueous solution extract of nutmeg, tetrahydrofuroguaiacin B (Verucosin), Fragransin (Fragransin C1), Sauceretine diol (Saucernetindiol) to increase eNOS phosphorylation Western blot analysis to confirm that.
Figure 4a is a graph confirming the vascular relaxation effect compared to acetylcholine by treating Nectandrin B in the aortic vascular ring, Figure 4b is due to the treatment of Nectandrin B (Nectandrin B) under the same conditions Western blot analysis confirmed that the eNOS phosphorylation in the vascular ring increased.
FIG. 5A is a result of confirming through MTT assay that Nectandrin B is treated to vascular smooth muscle cells to inhibit proliferation of increased vascular smooth muscle cells by induction of platelet-derived growth factor (PDGF), FIG. 5B In the same conditions, it was confirmed by BrdU insertion method that DNA synthesis is inhibited by the treatment of Nectandrin B (Nectandrin B).
Figure 6 shows the results confirming that the vascular inner wall thickening is inhibited when Nectandrin B (Nectandrin B) administration to the wounded blood vessel wall.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein but may be embodied in other forms. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

< Example  One : Myristicine  Low in content nutmeg  Preparation of ethanol aqueous solution extract &gt;

100 g of crushed nutmeg was placed in 500 ml of each solvent (see Table 1), and an extract was prepared for each solvent condition using an ultrasonic wave extender three times for 2 hours. The extracted extracts were used at the same concentration and the Myristicine standard was purchased from Sigma (product number: M9237). Each nutmeg solvent extract and myristicin standard were HPLC (Optima Pak C ₁₈ column 4.6 × 250mm, 5μm) in MeOH-H ₂ O (0-32min: 63% MeOH, 32-37min: 63 → 100% MeOH) Particle size, flow rate 1ml / min, UV detection: 260nm], and the myristicin content of each nutmeg extract is shown in Table 1.

Alcohol extraction solvent Among the extracts
Myristicin content (%) Among the extracts
Nectarine B content (%) Water extract 0.19% 0.62 10% aqueous solution of ethanol 0.31% 0.94 20% ethanol aqueous solution extract 0.33% 2.98 30% ethanol aqueous solution extract 0.45% 7.48 40% ethanol aqueous solution extract 1.34% 1.95 50% ethanol aqueous solution extract 1.48% 2.77 75% Ethanol Aqueous Extract 1.27% 2.95 75% aqueous methanol solution extract 1.85% 7.47

As shown in Table 1, it was confirmed that the myristicin content of the 10-30% ethanol aqueous solution extract was extracted on average three times less than that of the extract under other conditions. In addition, the nutmeg solvent extract under each condition was purified by HPLC [OptimaPak_C ₁₈ ] under MeOH-H ₂ O (0-35 min: 60% MeOH, 35-60 min: 60 → 100% MeOH). column 4.6 × 250mm, 5μm particle size, flow rate 1ml / min, UV detection: 205, 280nm] and analyzed the content of nectanrin B (see Table 1). It was found that the most extracted. Therefore, it was confirmed that the nutmeg extract was extracted with an ethanol aqueous solution of 30% or less while maximizing the amount of the active substance and minimizing the toxic substance content.

< Example  2 : nutmeg  Toxic from Ethanol Aqueous Extract Myristicin  Add remove>

100 g of nutmeg was extracted with 500 ml of a 30% aqueous ethanol solution and then adsorbed through 500 g of Diaion HP-20, one of ion exchange resins. Thereafter, the adsorbates were eluted using 500 mL of 30-90% ethanol aqueous solution, 100% ethanol, and 100% acetone, respectively, and the contents of myristicine and nectandrin B contained in the eluate are shown in Table 2. Referring to Table 2, it was confirmed that myristicine was hardly detected when the adsorbate was eluted with an ethanol aqueous solution of 80% or less. In addition, it was confirmed that the use of 80% ethanol aqueous solution as the solvent elicited a large amount of nethandrine B.

Eluting solvent condition 30% aqueous ethanol solution 50% aqueous ethanol solution 70% ethanol aqueous solution 80% aqueous ethanol solution 90% aqueous ethanol solution 100% ethanol Acetone The content of nectandrin B in the eluate 0.01%   0.1% 24.2%   61.0%   13.9% 1.0%    3.0% The content of myristidine in the eluate 0.04% 0.04% 0.04% 0.04% 0.66% 20.77% 13.71%

< Example  3: From nutmeg  Identification of extracted compounds &gt;

The 2,5-bis-aryl-3,4-dimethyltetrahydrofuran lignan compound was isolated from the nutmeg extract of 30% aqueous ethanol solution of Example 1 by HPLC, and the compound was analyzed by HPLC (see FIG. 1) ¹ H, and ¹³ C-NMR. The physicochemical properties of the compound are as follows.

3-1. Tetrahydrofuroguanidine  B ( Tetrahydrofuroguaiacin  B, compound 1)

Colorless powder, hydrogen nuclear magnetic resonance spectrum: ppm (500 MHz, CDCl ₃ ): δ 0.61 (6H, d, J = 6.0 Hz, 3- and 4-Me), 2.67 (2H, m, 3- and 4- H), 3.91 (6H, 3'- and 3 "-OMe), 5.12 (2H, d, J = 6.6 Hz, 2- and 5-H), 5.59 (2H, s, 4'- and 4" -OH ), 6.90-6.99 (6H, m, 2'-, 5'-, 6'-, 2 "-, 5"-and 6 "-H), Carbon nuclear magnetic resonance spectra: ppm (125 MHz, CDCl ₃ ) δ 11.7 (3- and 4-Me), 41.5 (C-3 and C-4), 55.8 (2 × OMe), 82.7 (C-2 and C-5), 109.0 (C-2 'and C- 2 "), 113.9 (C-5 'and C-5"), 119.3 (C-6' and C-6 "), 132.5 (C-1 'and C-1"), 144.3 (C-4' and C-4 ″), 146.2 (C-3 ′ and C-3 ″).

3-2. Sausage net dior  ( Saucernetindiol , Compound 2)

As a colorless oil, hydrogen nuclear magnetic resonance _{spectrum: ppm (500 MHz, CDCl 3} ): δ 0.63 (3H, d, J = 6.5 Hz, 4-Me), 1.01 (3H, d, J = 6.5 Hz, 3-Me ), 2.33 (2H, m, 3- and 4-H), 3.90-3.92 (6H, 3'- and 3'-OMe), 4.65 (1H, d, J = 9.5 Hz, 1H, d, J = 4.5 Hz, 5-H), 5.58 (2H, s, 4'- and 4'-OH), 6.77-6.99 (6H, m, 2 "-, 5" - and 6 "-H), carbon nuclear magnetic resonance _{spectrum: ppm (125 MHz, CDCl 3} ): δ 9.42 (3-Me), 11.83 (4-Me), 43.4 (C-3) (C-4), 55.8 (2xMe), 84.8 (C-2), 85.7 (C-5), 108.3 ), 114.0 (C-5), 118.8 (C-6), 119.3 (C-6) , 145.0 (C-4 "), 146.3 (C-3"), 146.6 (C-3 ').

3-3. Berukosin  ( Verrucosin , Compound 3)

As a colorless oil, hydrogen nuclear magnetic resonance _{spectrum: ppm (500 MHz, CDCl 3} ): δ 0.67 (3H, d, J = 6.5 Hz, 4-Me), 1.06 (3H, d, J = 6.5 Hz, 3-Me ), 1.79 (1H, m, 3-H), 2.25 (1H, m, 4-H), 3.87-3.89 (6H, 3'- and 3 "-OMe), 4.36 (1H, d, J = 9.5 Hz , 2'-, 5'-, 6'-, 2'-, 5''- and 2'H), 5.10 (1H, d, J = 9.0Hz, 5H), 6.80-6.96 6 "-H), Carbon Nuclear Magnetic Resonance Spectrum: ppm (125 MHz, CDCl ₃ ):? 14.8 (3-Me), 15.2 (4-Me), 45.9 C-2 '), 111.6 (C-2'), 115.7 (C-5 '), 116.1 (C- C-4 '', 146.8 (C-4 ''), 120.7 (C-6 ' , 148.6 (C-3 "), 149.0 (C-3 &apos;).

3-4. Nectandrine  B ( Nectandrin  B, compound 4)

(600 MHz, CDCl ₃ ):? 1.05 (6H, d, J = 6.0 Hz, 3- and 4-Me), 2.35 H), 3.85 (6H, 3'- and 3 "-OMe), 4.53 (2H, d, J = 5.4 Hz, 2- and 5-H), 5.74 (2H, brs, 4'- and 4" -OH ), 6.91 (2H, d, J = 7.8Hz, 5'- and 5''H), 6.93 (2H, dd, J = 1.8, 7.8Hz, d, J = 1.8 Hz, 2'- and 2 "-H), carbon nuclear magnetic resonance _{spectrum: ppm (200 MHz, CDCl 3} ): δ 133.9 (C-1 'and C-1"), 114,1 ( C-2 'and C-2 "), 146.4 (C-3' and C-3"), 144.9 (C-6 and C-6), 87.2 (C-2 and C-5), 44.1 (C-3 and C-4), 12.7 × 2).

3-5. Nectandrine  A ( Nectandrin  A, compound 5)

White crystals, hydrogen nuclear magnetic resonance _{spectrum: ppm (500 MHz, CDCL 3} ) δ: 1.00 (3H, d, J = 3.5 Hz, 4-Me), 1.02 (3H, d, J = 3.5 Hz, 3-Me) (1H, d, J = 7.5 Hz, 2-H), 2.27 (2H, m, 3- and 4-H), 3.80-3.86 J = 7.5 Hz, 5-H), 6.81-7.09 (6H, m, Ar-H); Carbon Nuclear Magnetic Resonance Spectrum: ppm (125 MHz, CDCl ₃ )?: 13.1 (3-Me), 13.2 (4-Me), 45.6 (C-4), 45.5 OMe), 88.0 (C-5), 88.2 (C-2), 110.9 (C-2 '), 111.3 (C-6), 120.0 (C-6), 135.1 (C-1 '), 136.4 -3 "), 150.4 (C-3 &apos;).

3-6. Praagan Shin C1  ( Fragrance C1 , Compound 6)

As a colorless oil, hydrogen nuclear magnetic resonance _{spectrum: ppm (500 MHz, CDCl 3} ): δ 1.04 (3H, d, J = 6.6 Hz, 3-Me), 1.06 (3H, d, J = 7.2 Hz, 4-H ), 2.32 (1H, m, 3-H), 2.34 (1H, m, 4-H), 3.88 (9H, 3'-, 5'- and 3 "-OMe), 4.50 (1H, d, J = 7.2 Hz, 2H), 4.52 ( 1H, d, J = 7.2 Hz, 5-H), 5.47-5.58 (2H, br, s, 4'- and 4 "-OH), 6.67 (2H, br, s, 2'- and 6'-H) , 6.91-6.97 (3H, m, 2 "-, 5" - and 6 "-H), carbon nuclear magnetic resonance _{spectrum: ppm (125 MHz, CDCl 3} ): δ (C-3), 44.5 (C-4), 56.3-55.8 (3xMe), 87.2 (C-2), 87.5 , 103.1 (C-2 'and C-6'), 102.9 (C-2 "), 114.1 1 "), 145.1 (C-4 '), 146.4 (C-4"), 146.9 (C-3', C-5 'and C-3 ").

< Example  4: in vascular endothelial cells eNOS  Phosphorylation and NO  Confirm creation

In order to confirm eNOS phosphorylation and NO production in vascular endothelial cells, ECV 304 cells (presumptive endothelial cell line), a human umbilical vein endothelial cell-derived cell line, were used. The cells were cultured by passage using trypsin-ethylenediaminetetraacetic acid (trypsin-ethylenediaminetetraacetic acid) in Dulbecco's Modified Eagles Medium (DMEM) containing 10% FBS (fetal bovine serum) and 1% antibiotic. To this end, nectandrin B was treated with FBS (fetal bovine serum) depleted conditions for 1 hour to obtain total cell lysates and phosphorylated eNOS in the cell lysates using Western blot analysis. The expression level of was confirmed. More specifically, each cell was collected by treating with 100 µl of lysis buffer [120 mM NaCl, 40 mM Tris (pH 8), 0.1% NP40 (Nonidet P-40)], followed by centrifugation at 10,000 × g for total cells. A solution was obtained. Thereafter, 10% SDS-PAGE (sodium dodecyl sulfate poly acrylamide gel electrophoresis) electrophoresis was performed using the cell lysate, and the protein was fixed to the nitrocellulose transfer membrane using a transfer kit. The membrane was blocked at room temperature for 1 hour in 5% skim milk, and after reacting the primary and secondary antibodies to each protein, the change in the amount of expression of each protein was observed. It confirmed, and the result is shown in FIG. Referring to the results of Figure 2a, it can be seen that due to the treatment of nectandrin B (1 ~ 10㎍ / ㎖), phosphorylation of eNOS (p-eNOS) was increased than the nectandrin untreated group. Insulin, used as a control, is well known for its hypoglycemic effect as well as vascular relaxation through phosphorylation of eNOS. It was confirmed that the compounds of the present invention have eNOS phosphorylation effect similar to insulin 100nM treatment group.

On the other hand, NO formation by eNOS phosphorylation and activation was measured by the reaction of Assay Design's Griess with the content of nitrite, which is a NO metabolite produced through nitrite reductase reaction after collection of experimental medium. The degree of color development coming through was quantified at 540 nm and is shown in FIG. 2B. The result was expressed as a fold value for the control group without sample treatment (nextandrin treated group) as a reference (1.00). Referring to the results of Figure 2b, it can be seen that the production of NO increased with the treatment concentration (1 ~ 10㎍ / ㎖) of nectanrin B.

In addition to nectandrin B, eNOS of 30% ethanol extract of nutmeg, tetrahydrofuroguaiacin B, verrucosin, fragransin C1, saucernetinol, and eNOS Also, the degree of phosphorylation was confirmed under the same conditions as that of nectandrin B. Similar to the result of nectandrin B, it was confirmed that the phosphorylation of eNOS increased by the treatment concentration of each compound (1 ~ 10㎍ / ㎖). , 30% ethanol aqueous solution extract of the nutmeg (30 ㎍ / ㎖) also showed that phosphorylation of eNOS also appeared (see Figure 3). In addition, although not included in the above results, it was confirmed that nectanrin A showed similar results with the compounds of FIG. 3.

< Example  5: vascular relaxation effect>

In order to confirm whether nectanrin B, which is contained as an active ingredient, in the nutmeg 10-30% ethanol aqueous solution of the present invention, causes actual vascular tissue relaxation, a tissue chamber using a rat aortic vascular ring in which the endothelial is not damaged. It was evaluated by the experimental method (Fig. 4). To this end, the aorta of a male rat (weight 300g) was extracted, and a sample of a 2 to 3 mm diameter vascular ring was produced, and then suspended on a ring connected to an isometric transducer to measure contraction and relaxation. The degree of relaxation was expressed as percent relaxation relative to acetylcholine (1 μM), which is known to induce NO production. In the case of acetylcholine, 5 μl (final concentration 1 μM) of 1 mM concentrated solution dissolved in distilled water was added to the tissue chamber, and nectandrin B was dissolved in DMSO (dimethylsulfoxide) to make 10 mg / ml, and the final concentration was 10 μg / in the tissue chamber. Treated to ml.

In addition, in order to evaluate whether nectanrin B causes phosphorylation of eNOS in the actual aorta, Western blotting was performed in the same manner as in Example 4 using the supernatant centrifuged at 10,000 × g after crushing the vascular ring specimen. Phosphorylation of eNOS over time was confirmed. The results are shown in FIG. 4. Referring to FIG. 4, 63.5% of the blood vessels treated with nectandrin B (10 μg / ml) converted to 100% of the relaxation reaction caused by acetylcholine [1 μM]. It was confirmed that there is a relaxation effect, it was confirmed that the phosphorylation of eNOS also increased.

< Example  6: vascular smooth muscle cell proliferation and DNA  Composite Effects>

To determine how nectanrin B compounds affect vascular smooth muscle cell proliferation, nectedrin B and PDGF (platelet-) were used in primary cultured vascular smooth muscle cells isolated from rat aorta (using cells within 4-10 passages). the degree of cell proliferation and the degree of DNA synthesis after treatment with the drived growth factor, platelet-derived growth factor, and blood vessel lining) were determined by MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl- tetrazolium bromide] and BrdU insertion (5-bromo-2'-deoxy-uridine uptake). To this end, the vascular smooth muscle cells were treated with nectanrin B for 1 hour and then incubated with PDGF (30 ng / ml) for 24 hours.

MTT assay was performed by incubating 200 μl of MTT reagent (2 mg / ml) on vascular smooth muscle cells cultured in 96 well plate for 4 hours, then removing the cell culture medium and using 200 μl of DMSO (dimethyl sulfoxide). Was dissolved and quantified by measuring absorbance at 540 nm. The results are shown in FIG. 5A.

Referring to FIG. 5A, it was confirmed that the proliferation of vascular smooth muscle cells was suppressed according to the treatment concentration of nectanrin B (1 to 10 µg / ml).

The degree of BrdU insertion was quantified using a Roche kit. Vascular smooth muscle cells were exposed to 10 μM BrdU staining solution for 2 hours, and then the cells were fixed with fixed solution for 30 minutes. 100 μl of BrdU antibody (anti-BrdU) peroxidase-labeled antibody) for 90 minutes and washed three times with PBS. Subsequently, 100 μl / ml of the substrate solution was added, and the color development was confirmed at 405 nm using a microtiter plate reader (Berthold Tech., Bad Wildbad, Germany). The results are shown in FIG. 5B.

Referring to FIG. 5B, it was confirmed that DNA synthesis in vascular smooth muscle cells was suppressed according to the treated concentration of nectanrin B (1 to 10 µg / ml).

< Example  7: Femoral Artery  Vascular lining after injury After rain  Affection Nectandrine  Effect of B>

In order to determine how the compound of the present invention affects the vascular wall thickening, a 7-week-old male ICR mouse (30g) was anesthetized with pentobarbital, and then exposed and damaged the femoral artery, followed by a guide wire. Was inserted into the vessel for 2 minutes. After removing the guide wire, the vessel was sewn. After vascular seaming, mice were treated with nectanrin B (20 mg / kg, 30% polyethylene glycol 400, dissolved in 1% ethanol solvent) twice a week (Mon, Thursday) for 3 weeks, and nectanrin B was used as a control. Only dissolved solvents (30% polyethylene glycol 400, 1% ethanol solvent) were administered. Three weeks later, the femoral blood vessels were extracted from ether anesthetized mice, and the blood vessel tissue samples were fixed by fixing the femoral blood vessels in neutral formalin solution. The thickness of the inner wall of the blood vessel was confirmed and measured by a skilled pathologist, and the measurement results thereof are shown in FIG. 6.

Referring to FIG. 6, the inner wall of the blood vessel of the wire-injured rat was significantly thickened compared to the blood vessel of the control rat (Sham) rat. However, after the guard wire treatment, nectanrin B was administered. The vascular lining of one mouse (+ Nectandrin B, 20 mg / kg) was found to have a thickness similar to that of the control group.

< Experimental Example  8: Toxicity test>

8-1. Acute toxicity

This experiment was conducted to investigate the toxicity of a 10 to 30% aqueous ethanol solution of nutmeg and nexandrine B of the present invention to an animal body in an acute (within 24 hours) when an excessive amount of the extract was administered in a short period of time and to determine the mortality rate . Twenty mice of the general mouse ICR mouse line were assigned to each of the control and experimental groups. In the control group, only PEG-400 / tween-80 / ethanol (8/1/1, v / v / v) was administered, and the experimental group added nutmeg 10-30% ethanol extract and nectandrin B to the PEG-400 / tween-80. It was dissolved in / ethanol (8/1/1, v / v / v) and orally administered at a concentration of 2g / kg / day, respectively. After 24 hours of administration, the mice were found to survive in the control group and in the test group administered with 2 g / kg / day of nutmeg extract and nectandrin B, respectively.

8-2. Experimental group  And control organ organs and tissue toxicity experiments

Long-term toxicity experiment was conducted by administering the nutmeg 10-30% ethanol aqueous solution extract of the present invention and nectandrin B to C57BL / 6J mice (10 per group) with a final concentration of 2g / kg / day. In order to investigate the effects on the organs (tissues) of the animals, the experimental group administered with nutmeg 10-30% ethanol aqueous solution extract and nectandrin B and PEG-400 / tween-80 / ethanol (8/1/1, Blood was collected after 8 weeks from animals in the control group that received only v / v / v), and blood concentrations of glutamate-pyruvate transferase (GPT) and blood urea nitrogen (BUN) were selected from the Select E (Vital Scientific NV, Netherland) instrument. Measured using. As a result, GPT, which is known to be related to hepatotoxicity, and BUN, which is known to be related to renal toxicity, showed no significant difference compared to the control group. In addition, liver and kidney were sampled from each animal, and histological observation was performed with an optical microscope through a conventional tissue section production process. No abnormalities were observed in all tissues.

< Formulation example  1: Pharmaceutical preparations>

1-1. Manufacture of tablets

200 g of nectandrin B was mixed with 175.9 g of lactose, 180 g of potato starch and 32 g of colloidal silicic acid. To this mixture was added a 10% gelatin solution, which was pulverized and passed through a 14-mesh sieve. This was dried, and a mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into tablets.

1-2. Injection preparation

1 g of nectandrin B, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to make 100 ml. This solution was placed in a bottle and sterilized by heating at 20 DEG C for 30 minutes.

< Formulation example  2: Food manufacturing>

2-1. Manufacture of cooking seasonings

The spice for health promotion was prepared by adding 1% by weight of the nutmeg 30% ethanol aqueous solution extract of the present invention to the cooking seasoning.

2-2. Manufacture of flour food products

The nutritional enhancer was prepared by adding 0.1% by weight of the nutmeg 30% ethanol aqueous solution extract of the present invention to wheat flour and preparing bread, cake, cookies, crackers and noodles using the mixture.

2-3. soup  And juicy ( gravies Manufacturing

Health enhancing soup and juice were prepared by adding a 30% ethanol aqueous solution extract of nutmeg according to the present invention to soup and juice at 0.1 wt%.

2-4. dairy product( dairy products Manufacturing

The nutmeg 30% ethanol aqueous solution extract of the present invention was added to milk in an amount of 0.1% by weight and various dairy products such as butter and ice cream were prepared using the milk.

2-5. Vegetable juice  Produce

Healthy vegetable juice was prepared by adding 0.5 g of the nutmeg 30% ethanol aqueous solution extract of the present invention to 1,000 ml of tomato juice or carrot juice.

2-6. Fruit juice  Produce

0.1 g of nutmeg 30% ethanol aqueous solution extract of the present invention was added to 1,000 ml of apple juice or wine to prepare fruit juice for health promotion.

Claims (8)

Myristicin content is 0.5% by weight or less, Nectandrin B (Nectandrin B), Nectandrin A (Fragransin C1), Verrucosin (Verrucosin), Saucerinetinol (Saucernetindiol) and tetrahydrofuroguaiacin (Tetrahydrofuroguaiacin) pharmaceutical composition for the prevention or treatment of vascular diseases, characterized in that it contains an extract of 10-30% ethanol aqueous solution containing nutmeg containing one or more compounds selected from the group consisting of .
[Formula 1]

The method of claim 1,
The nutmeg 10-30% aqueous solution of ethanol extract, the content of myristicin, which is a toxic substance using an ion exchange resin, 0.1 wt% or less, Nectandrin B of Formula 1, Nectandrin A (Nectandrin) A), fragransin C1 (Fragransin C1), berucosin (Verrucosin), Sausnetinediol (Saucernetindiol) and tetrahydrofuroguaiacin (Tetrahydrofuroguaiacin) is characterized in that it contains at least one compound selected from the group consisting of Pharmaceutical composition for the prevention or treatment of vascular diseases.
[Formula 1]

3. The method according to claim 1 or 2,
The pharmaceutical composition is a pharmaceutical composition for the prevention or treatment of vascular diseases, characterized in that it has an effect of increasing the formation of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) phosphorylation and inhibiting vascular wall thickening. 3. The method according to claim 1 or 2,
The vascular disease is a disease selected from atherosclerosis, hypertension, angina pectoris, myocardial infarction, ischemic heart disease, heart failure, complications after angioplasty, cerebral infarction, cerebral hemorrhage, and stroke. Therapeutic composition. Mysticin is a toxic substance is 0.5% by weight or less, Nectandrin B (Nectandrin B), Nectandrin A (Fragransin C1), Verrucosin (Verrucosin), Sau Prevention or improvement of vascular disease characterized by containing 10-30% ethanol aqueous solution of nutmeg containing one or more compounds selected from the group consisting of susnetinediol and tetrahydrofuroguaiacin Dietary supplements.
[Formula 1]

The method of claim 5, wherein
The vascular disease is a disease selected from atherosclerosis, hypertension, angina pectoris, myocardial infarction, ischemic heart disease, heart failure, complications after angioplasty, cerebral infarction, cerebral hemorrhage, and stroke. Health functional food for improvement. Nectandrin B, Nectandrin A, Fragransin C1, Verrucosin, Sauceretinediol and tetrahydrofuroguacin of Formula 1 Tetrahydrofuroguaiacin) pharmaceutical composition for the prevention or treatment of vascular diseases, characterized in that it contains at least one compound selected from the group consisting of as an active ingredient.
[Formula 1]

The method of claim 7, wherein
The pharmaceutical composition is a pharmaceutical composition for the prevention or treatment of vascular diseases, characterized in that it has an effect of increasing the formation of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS) phosphorylation and inhibiting vascular wall thickening.

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