KR100550494B1 - Composition comprising the amentoflavone derivatives isolated from Selaginella tamariscina - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention and treatment of cerebrovascular diseases, including amentoflavone derivative compounds isolated from baekbaek, in detail, the amentoflavone derivatives of the present invention are vascular smooth muscle Increase the synthesis of nitric oxide (NO), an endothelium-derived relaxing factor that is one of the most important mechanisms that regulate contraction and relaxation of c) and by activating guanylate cyclase. Increasing the production of cGMP to relax blood vessels, and also exhibit an inhibitory effect on vasoconstriction through angiotensin converting enzyme inhibitory action, the composition comprising amentoflavone derivatives isolated from the baekbaek of the present invention prevent and treat cerebrovascular disease It can be usefully used as a medicine or health food for.

Kwonbaek, amentoflavones, nitric oxide, vascular relaxation, cerebrovascular disease

Description

Composition comprising the amentoflavone derivatives isolated from Selaginella tamariscina}             

Figure 1a is a diagram showing the concentration-dependent curve for the relaxation effect of amentoflavones in the aorta without vascular endothelial cells or the aorta with vascular endothelial cells removed,

Figure 1b is a diagram showing a concentration-dependent curve for the relaxation effect of amentoflavones in the aorta without vascular endothelial cells or aorta without vascular endothelial cells in the presence of L-NAME,

FIG. 2A shows a concentration-dependent curve for the relaxation effect of amentoflavones in the aorta where vascular endothelial cells contracted with phenylephrine in the presence of methylene blue were not removed.

FIG. 2B shows a concentration-dependent curve for the relaxation effect of amentoflavones in the aorta where vascular endothelial cells contracted with phenylephrine in the presence of ODQ were not removed.

3 is a diagram showing a concentration-dependent curve on the relaxation effect of amentoflavones in the aorta where vascular endothelial cells contracted with phenylephrine in the presence of indomethacin are not removed.

Figure 4a is a diagram showing the concentration-dependent curve for the relaxation effect of amentoflavones in the aorta without vascular endothelial cells contracted with phenylephrine in the presence of glybenclamide,

Figure 4b is a diagram showing a concentration-dependent curve for the relaxation effect of amentoflavones in the aorta without vascular endothelial cells contracted with phenylephrine in the presence of TBA,

5 is a diagram showing a concentration-dependent curve for the relaxation effect of amentoflavones in the aorta where vascular endothelial cells contracted with phenylephrine in the presence of verapamil are not removed.

FIG. 6 shows a concentration-dependent curve for the vasorelaxant effect of amentoflavones in the aorta where the endothelial cells contracted with phenylephrine in the presence of atropine or propranolol are not removed.

FIG. 7 shows a concentration-dependent curve for the vasorelaxant effect of amentoflavones in the aorta without the removal of endothelial cells contracted with phenylephrine in the presence of IBMX or japrinat.

FIG. 8 shows a concentration-dependent curve for the vasorelaxant effect of amentoflavones in the aorta where the endothelial cells contracted with phenylephrine in the presence of Y-27632 (1 × 10 ⁻⁶ M) were not removed.

9 is a diagram showing the effect of amentoflavones on cGMP production in the thoracic aorta,

10 is a diagram showing the effect of amentoflavones in the thoracic aorta of rats dose-dependently contraction induced by phenylephrine,

Figure 11a is a diagram showing the inhibitory effect of amentoflavones on angiotensin conversion enzyme activity,

Figure 11b is a diagram showing the degree of inhibition of contraction induced by angiotensin I by amentoflavones.

The present invention relates to a pharmaceutical composition for the prevention and treatment of cerebrovascular diseases containing amentoflavone derivatives isolated from baekbaek as an active ingredient.

The mechanisms that control contraction and relaxation in vascular smooth muscles vary widely. One of the most important of these mechanisms is the endothelium-derived relaxing factor (vascular endothelial cell), the body is nitric oxide (NO). Nitric oxide in blood vessels is produced in vascular endothelial cells by nitric oxide synthase (NOS) using L-arginine as a substrate. There are at least three different isoforms of NOS, the first of which is brain-type nitric oxide synthase (bNOS, nNOS, NOS I), first discovered in brain tissue and primarily synthesizing nitric oxide as a neurotransmitter. Playing a role. The second isoenzyme is an inducible acid-nitrogen synthase (iNOS, NOS II), which synthesizes nitric oxide, which mainly acts on the immune system, and the third isozyme, which is mainly distributed in vascular endothelial cells, acts to relax blood vessels Endothelial cell type nitric oxide synthase (ecNOS, eNOS, NOS III) is activated by acetylcholine to relax blood vessels (Arnold WP., Mittal CK. Et al., Proc. Natl. Acad. Sci. USA) , 74 (8) , pp3203-3207, 1977). These nitric oxides activate cytoplasmic guanylate cyclase, increase the production of cGMP, and relax vascular smooth muscle through signaling pathways (Arnold WP, Mittal CK. Et al., Proc. Natl. Acad. Sci. USA, 74 (8) , pp3203-3207, 1977).

Vascular endothelial cell-derived relaxors are synthesized and secreted in endothelial cells even under normal conditions and synthesized and secreted by agonists such as acetylcholine, histamine, substance P, and isoproterenol. Is increased. Herbal medicines and herbal medicines that relax blood vessels by increasing the production of nitric oxide in vascular endothelial cells have also recently been reported, such as Kuramochi et al. (Kuramochi T. et al., Life Sci., 54 (26)). , pp2061-2069, 1994) reported that lyophilized solution increased nitric oxide-dependent vasorelaxant effect, and Goto et al. (Goto H. et al., Am. J. Chinese Med., 27) . (3-4) , pp339-345, 1999), in a series of studies reported that precoating fluids decreased the blood pressure of spontaneous hypertension and methanol extracts relaxed the blood vessels of the thoracic aorta in a nitric oxide-dependent manner. In addition, lithospermic acid B (lithospermic acid B) and protein components of Cordyceps sinensis are known to increase vascular relaxation depending on nitric oxide. In particular, Chen et al. (Chen ZY. Et al., Life Sci., 63 (22) , pp1983-1991, 1998) reported that ethanol extracts of the hawthorn relax the mesenteric artery nitric oxide-dependently. Et al. Reported that procyanidins isolated from hawthorn increased nitric oxide-dependent relaxation of blood vessels and increased the production of vascular cGMP.Kang et al. Also reported that berberine, an alkaloid compound isolated from rhubarb, is NO / It has been reported to relax blood vessels in a cGMP-dependent manner. Also, herbal extracts such as mistletoe, ginkgo biloba, windproof, Angelica, motherwort, etc. also exhibit vascular endothelial cell-dependent or independence, and glycoside ( glycoside, procyanidine, flavonoid and sesquiterpene lactone compounds have been identified (Nishida S. et al., Life Sci., 72 (23) , pp 2659-67, 2003; Lee TH. et al., Planta.Med ., 68 (6) , pp492-6, 2002; Yuzurihara M. et al., Eur.J. Pharmacol., 444 (3) , pp183- 9, 2002). The pharmacological significance of the vascular relaxation effect is not only to alleviate hypertension, arteriosclerosis, brain-vascular disease, heart disease, but also to inhibit kidney disease and erectile dysfunction.

Selaginella tamariscina BEAUV.SPRING refers to the outposts of the Buddha and rock hands belonging to the Buddha's Family, and are enshrined in the products of the Shinnongbonchobyeong. It is recorded to treat cold pain and the like. In addition, Myeong-Seok Myeongrok (醫 別 錄 名醫 別 錄) is said to stop severe coughing, to cure disinfection, to strengthen the sound, and to promote affection. Its medicinal effect is a bleeding effect when used in life, and it treats menstrual abolition, bruises, abdominal pain and asthma, and frys it when used to bleed and treat hemorrhage, bloody stools, hematuria, and prolapse. It is known (Information Sup, Shin Mingyo, Hyang-Yak Daejeon Dictionary, Younglim History, p88, 1990).

Studies on the components of Kwonbaek, Okigawa et al. (Okigawa M. et al., Phytoche., 10 , p286, 1971) have been conducted by amentoflavones, hinokiflavones and isoopoptomerins . (isocryoptomerin) was isolated, and Karyonone also separated amentoflavones and sotsutsuflavones. Among them, amentoflavones have antioxidant activity (Mora A. et al., Biochem.Pharmacol ., 40 (4) , pp793-7, 1990), and lymphocyte proliferation inhibitory activity (Lee SJ. Et al., Life) . Sci., 57 (6), pp551-558, 1995), inhibitory effect of phospholipase C-γ1 enzyme activity (Lee HS. Et al., Planta. Med., 62 (4) , pp293-296, 1996), anti-HIV action, anti-inflammatory action through inhibition of cyclooxygenase and lipoxygenase (Kim HP. Et al., Prostaglandins Leukot Essent Fatty Acids, 58 (1) , pp17- 24, 1998), cAMP-phosphodiesterase inhibitory activity, antibiotic activity against virus, antimicrobial activity, etc. have been studied (Kim HK. Et al., Arch. Pharm. Res., 21 (4)). , pp 406-410, 1998; Lin YM. et al., Planta.Med ., 65 (2) , pp120-125, 1999; Krauze-Baranowska M. et al., Planta.Med ., 65 (6) , pp572 -573, 1999).

However, no disclosure or teaching has been made about the vasorelaxant effect of amentoflavones isolated from baekbaek.

The inventors of the present invention, while searching for the vascular relaxation effect of about 20 kinds of herbal medicines having an effect of blood and squeezing, obtain amentoflavone derivatives, which are biflavonoid compounds, from the ethylacetate extract of Kwonbaek with excellent vascular relaxation effects. Vasorelaxant effect by various drugs such as phenylephrine, angiotensin I and indomethanecin, and the activity of cGMP and angiotensin I converting enzymes of the amentoflavone derivatives of the present invention were measured. As a result, the present invention was completed by confirming the excellent vasorelaxant effect of the amentoflavone of the present invention.

An object of the present invention is to increase the synthesis of vascular endothelial cell-derived nitric oxide (NO), which is one of the most important mechanisms regulating the contraction and relaxation of vascular smooth muscle, and activate guanylate cyclase Pharmacology for the prevention and treatment of cerebrovascular diseases containing amentoflavone derivatives isolated from baekbaek having an inhibitory effect on vasoconstriction through angiotensin converting enzyme inhibitory action by increasing cGMP production It is to provide a composition.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of cerebrovascular diseases containing the amentoflavone derivative of the general formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient.

(I)

(I)

Where

R ₁ to R ₆ are each independently hydrogen or a lower alkyl group of C ₁ to C ₄ .

Preferred among the compounds of the general formula (I) include amentoflavones of the following structural formula (Ia), which are separated from the roll and R ₁ to R ₆ are hydrogen.

(Ia)

(Ia)

The cerebrovascular disease includes transient cerebral ischemic attack, thrombotic cerebral infarction, embolic cerebral infarction, hemodynamic cerebral infarction, thermodynamic infarction or cerebral hemorrhage and the like.

Hereinafter, the present invention will be described in detail.

The amentoflavone derivatives of the general formula (I) isolated from the roll of the present invention can be obtained as follows.

The amentoflavone derivatives represented by the general formula (I) of the present invention may be prepared by various methods, and specifically may be obtained by chemical synthesis or extraction from plants.

Illustrating the method for producing the amentoflavone derivative compound of the present invention in detail, after gathering, drying and pulverizing the roll of the present invention, about 2 to 10 times the weight of the rolled sample, preferably about 3 to 5 A polar solvent of up to twice the volume of water and C ₁ to C ₄ lower alcohols such as methanol, ethanol, butanol or the like, or a mixed solvent having a mixing ratio of about 1: 0.1 to 1:10, preferably methanol, at room temperature The supernatant was recovered by vacuum filtration using an extraction method such as hot water extraction, cold needle extraction, reflux cooling extraction, or ultrasonic extraction for about 1 to 5 weeks, preferably 1 to 3 weeks. The process may be repeated 2 to 5 times, preferably 3 times to collect the supernatant and concentrated under reduced pressure to obtain a rolled crude crude extract.

In addition, the non-polar solvent soluble extract of the present invention is suspended in distilled water after the crude extract, the suspension is added to a nonpolar solvent, such as about 1 to 100 times, preferably about 1 to 5 times the volume of hexane, ethyl acetate, chloroform It can be obtained by extracting and separating the nonpolar solvent soluble layer 1 to 10 times, preferably 2 to 5 times. It is also possible to further carry out conventional fractionation processes (Harborne JB Phytochemical methods: A guide to modern techniques of plant analysis, 3rd Ed. , Pp 6-7, 1998).

More specifically, the crude white crude extract obtained by the above process, preferably organic solvents such as n-butanol, hexane, ethyl acetate, and the like, are prepared in the order of low polarity to high polarity, Solvent fractions were then sequentially concentrated under reduced pressure in the order of hexane, ethyl acetate, n-butanol, and water to obtain a rolled-up hexane, ethyl acetate, n-butanol fraction, and the ethyl acetate fraction having the highest activity among the obtained solvent fractions. Column chromatography was performed using a water: methanol mixed solvent as the elution solvent to separate the fractions into 5 to 11 fractions, and then, vascular relaxation experiments and TLC, ¹ H-NMR and ¹³ C-NMR were performed. After the activity of the fractions was searched, silica gel column chromatography was performed again on fraction 11, which had the highest activity, to 4 to 4 The amentoflavone derivative compound of the general formula (I) can be obtained by fractionation into 17 fractions and analysis by ¹ H-NMR and ¹³ C-NMR.

Amentoflavone derivatives having a prophylactic and therapeutic effect of cerebrovascular diseases of the present invention can also be synthesized through synthesis and fractionation of conventional substituents (Herbert O. House: Modern Synthetic Reactions , 2nd Ed ., The Benjamin / Cummings Publishing Co., 1972).

The compounds of the present invention represented by the general formula (I) may be prepared as pharmaceutically acceptable salts and solvates according to conventional methods in the art.

As the pharmaceutically acceptable salt, acid addition salts formed by free acid are useful. Acid addition salts are prepared by conventional methods, for example by dissolving a compound in an excess of aqueous acid solution and precipitating the salt using a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. Equal molar amounts of the compound and an alcohol such as acid or glycol monomethyl ether in water can be heated and then the mixture is evaporated to dryness or the precipitated salts can be suction filtered.

In this case, organic acids and inorganic acids may be used as the free acid, hydrochloric acid, phosphoric acid, sulfuric acid, nitric acid, tartaric acid, etc. may be used as the inorganic acid, and methanesulfonic acid, p -toluenesulfonic acid, acetic acid, trifluoroacetic acid, Citric acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, lactic acid, glycolic acid, gluconic acid ), Galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, and the like.

Bases can also be used to make pharmaceutically acceptable metal salts. An alkali metal or alkaline earth metal salt is obtained by, for example, dissolving a compound in an excess alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and then evaporating and drying the filtrate. At this time, as the metal salt, it is particularly suitable to prepare sodium, potassium or calcium salt, and the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt such as silver nitrate.

Pharmaceutically acceptable salts of the above general formula (I) include salts of acidic or basic groups which may be present in compounds of general formula (I) unless otherwise indicated. For example, pharmaceutically acceptable salts include sodium, calcium and potassium salts of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include hydrobromide, sulfate, hydrogen sulphate, phosphate, hydrogen phosphate, dihydrogen Phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p -toluenesulfonate (tosylate) salts, and methods or processes for preparing salts known in the art It can be prepared through.

As a result of experiments to determine the vasorelaxant effect and mechanism of the amentoflavone derivative compound of the general formula (I) of the present invention obtained by the above method, the amentoflavone derivative of the general formula (I) of the present invention Increases the synthesis of nitric oxide (NO), a vascular endothelial cell-releasing factor, activates guanylate cyclase, increases cGMP production, relaxes blood vessels, and also reduces vasoconstriction through angiotensin converting enzyme inhibition It was confirmed to exhibit an inhibitory effect.

The pharmaceutical composition for preventing and treating cerebrovascular diseases of the present invention comprises 0.1 to 50% by weight of the compound based on the total weight of the composition.

Pharmaceutical compositions comprising the compounds of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.

Pharmaceutical dosage forms of the compounds of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.

The pharmaceutical compositions comprising the compounds according to the present invention may be prepared in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Can be formulated and used. Carriers, excipients and diluents that may be included in the composition comprising the compound include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. When formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose, or the like. ) Or lactose, gelatin and the like are mixed. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. . Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

Preferred dosages of the compounds of the present invention depend on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, but may be appropriately selected by those skilled in the art. However, for the desired effect, the compound of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 10 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.

The compounds of the present invention can be administered to mammals such as mice, mice, livestock, humans, and the like by various routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The present invention provides a health functional food comprising the compound and a food acceptable food supplement additive exhibiting a prophylactic and improving effect of cerebrovascular disease. Examples of the food to which the compound of the present invention can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.

It may also be added to food or beverages for the purpose of preventing and improving cerebrovascular disease. At this time, the amount of the compound in the food or beverage may be added at 0.01 to 15% by weight of the total food weight, the health beverage composition may be added in a ratio of 0.02 to 5 g, preferably 0.3 to 1g based on 100 ml. have.

The health functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the compound as an essential ingredient in the indicated ratios, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those mentioned above, natural flavoring agents (tauumatin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. The proportion of natural carbohydrates is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.

In addition to the above, the compounds of the present invention include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese and chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like. In addition, the compounds of the present invention may contain flesh for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is usually selected in the range of 0 to about 20 parts by weight per 100 parts by weight of the compound of the present invention.

Hereinafter, the present invention will be described in detail by the following Examples and Experimental Examples.

However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.

Example 1 Isolation of Amentoflavone from Kwonbaek

1-1. Preparation of Kwonbaek crude extract

1.2 kg of Kwon-baek was purchased and ground into a grinder to be powdered. 5 L of methanol was added thereto, followed by extraction at room temperature for 3 weeks, followed by vacuum filtration to recover the supernatant. This process was repeated three times to collect the supernatant, and then concentrated under reduced pressure to give 63.2 g of a rolled methanol extract.

1-2. Preparation of Fractions by Solvent

After diluting 63.2 g of the rolled methanol extract obtained in Example 1-1 in 1 L of water, the solvents were sequentially fractionated from the solvent having the lowest polarity to the solvent having the highest polarity in the order of hexane, ethyl acetate, n-butanol, and water. . First, 1 liter of hexane was added to and dissolved in a white methanol extract suspended in 1 liter of water, and then vacuum-dried by separating only the components dissolved in the hexane layer in a fractionated filter. This process was repeated three times to obtain a hexane fraction (11.7 g). 1 liter of ethyl acetate was added to the remaining aqueous layer, and only the components dissolved in the ethyl acetate layer were separated from the fractional filter and dried in vacuo. This process was repeated three times to obtain an ethyl acetate fraction (15.3 g), and 1 liter of butanol was added to the remaining aqueous layer to obtain a butanol fraction (17.2 g). Finally, the remaining aqueous layer was concentrated under reduced pressure to give an aqueous layer fraction (12.4 g).

1-3. Preparation of ethyl acetate bottom fraction

The ethyl acetate fraction of the fractions obtained in step 1-2 was subjected to column chromatography to fractionate into 11 lower fractions as follows. That is, 400 g of C ₁₈ gel was placed in a column having a diameter of 7 cm and a length of 34 cm, distilled water was flowed to uniform the gel, and then 15 g of the sample was loaded. As the mobile phase, distilled water was first distilled off, and then eluted with 10% methanol to obtain 11 fractions. Vasorelaxation experiment and TLC on the obtained fraction. ^{As a} result of measuring activity by measuring ¹ H-NMR and ¹³ C-NMR spectra, it was confirmed that Fraction 11 was the best active fraction (hereinafter referred to as DH 110-2-11).

1-4. Isolation of Amentoflavones from Kwonbaek

AHentoflavone was isolated by performing silica gel chromatography on DH 110-2--11 obtained in Example 1-3. That is, the column was filled with silica gel and equilibrated with methylene chloride, followed by loading 4.9 g of DH 110-2-11. The mobile phase was initially effluent with methylene chloride and the proportion of methanol was increased by 1%. After the ratio of methanol became 10%, it was increased by 5% and flowed out to 45%, and separated into 17 fractions. Vasorelaxation was performed on the obtained fractions to confirm that fraction 10 was the highest active fraction, and then analyzed using ¹ H-NMR and ¹³ C-NMR spectra. (Amentoflavone) was isolated.

Melting Point (m.p.): 230-232 ℃

Molecular Weight: 538 g / mole

¹ H NMR: (500 MHz, DMSO- d 6): 13.11 (s, 1H), 12.98 (s, 1H), 10.81 (s, 1H), 10.27 (s, 1H), 8.00 (s, 2H), 7.58 (d, 2H, J = 8.3 Hz), 7.14 (d, 1H, J = 8.8 Hz), 6.85 (s, 1H), 6.80 (s, 1H), 6.72 (d, 2H, J = 8.3 Hz), 6.46 (s, 1H), 6.40 (s, 1H), 6.19 (s, 1H)

¹³ C NMR: (125 MHz, DMSO- d 6) 182.7, 182.3, 164.7, 164.3, 164.2, 162.3, 162.0, 161.8, 161.1, 160.0, 157.9, 155.0, 132.0, 128.7, 128.4, 121.9, 121.5, 120.5, 116.7 , 116.3, 104.5, 104.2, 104.1, 103.5, 103.2, 99.4, 99.1, 94.5.

Experimental Example 1. Reagent and Preparation

1-1. reagent

Phenylephrine HCl (phenylephrine HCl), NG-nitroarginine methyl ester (L-NAME), methylene blue (methylene blue), ODQ, indomethacin (glibenclamide), TEA, Verapamil, atropine, propranolol and yohimbin were purchased from Sigma Chemical CO., St. Louis, MO, USA and used Y-27632 dehydro. Chloride (trans-4-[(1R) -1-aminoethyl] -N-4-pyrididnylcyclo hexanecarbox amide dihydrochloride) and Zaprinast (2- (2-propyloxyphenyl) -8-azapurin-6-one) It was purchased from Tocris Cookson Ltd., Bristol, UK.

1-2. Isolation of the thoracic aorta

A healthy Sprague-Dawley white paper weighing about 250-300 g was consciously decapitated and the thoracic aorta isolated. The isolated thoracic aorta was placed in cold Krebs solution (pH 7.4) containing 118 mM NaCl, 4.7 mM KCl, 1.1 mM MgSO ₄ , 1.2 mM KH ₂ PO ₄ , 1.5 mM CaCl ₂ , 25 mM NaHCO ₃ , and 10 mM glucose. After removal of the connective tissue and fat, sections of about 3 mm in length were made.

Experimental Example 2 Measurement of Vascular Relaxation Effect of Amentoflavones by Various Drugs

 2-1. Measurement of blood vessel tension

The thoracic aortic section prepared in Experimental Example 1-2 was fixed in a Krebs solution at 37 ° C. saturated with 95% O ₂ and 5% CO ₂ gas, and then the isometric tension was force-displacement. Measurement was performed using a physiological recorder (Grass physiograph, Model 7E, USA) equipped with a transducer, Grass FT 03, USA. First, after shrinking with 1 x 10 ^-6 M of phenylephrine, 10 minutes later, by relaxing reaction with 1 x 10 ^-6 M of acetylcheoline (ACh) to measure the safety of vascular endothelial cells, then Krebs The solution was washed three times and the experiment was performed. Determination of changes in the vasorelaxant effect of amentoflavones by various drugs was first pretreated for 20 minutes and contracted with phenylephrine, followed by a concentration-dependent relaxation of amentoflavones. When vascular endothelial cell-independent experiments were performed, vascular endothelial cells were removed with a small cotton swab, and then phenylephrine contraction and ACh relaxation reactions were performed to confirm the removal of vascular endothelial cells.

When measuring the contraction inhibition reaction of amentoflavones by angiotensin I, contraction with phenylephrine (1 × 10 ^-6 M) and relaxation reaction with ACh, followed by washing three times with Krebs solution, followed by angiotensin I Shrinkage experiments were performed. The effects of amentoflavones and control solvents were measured in a concentration-dependent manner after the reaction to the thoracic aorta for 20 minutes, followed by angiotensin I contraction.

 2-2. Experiment result

2-2-1. Vascular Endothelial Dependent Relaxation of Amentoflavones

Concentration-dependent vasorelaxation effect of amentoflavones after contraction with phenylephrine (1 x 10 ^-6 M) was observed, starting with significant relaxation from the concentration of 1 x 10 ^-7 M and then 1 x 10 ^-5 M At the concentration of 70.1 ± 2.3% was relaxed. In order to determine whether the vascular relaxation effect of amentoflavone is vascular endothelial cell dependent, the vascular relaxation effect was measured after removing vascular endothelial cells, and it was confirmed that the vascular relaxation effect was almost completely suppressed (see FIG. 1A).

2-2-2. Relaxation Effect of Amentoflavones after Inhibition of Nitric Oxide Synthase

To determine whether the vasorelaxant effect of amentoflavones is related to the nitric oxide system, pretreatment with 1 x 10 ^-6 M L-NAME, a non-specific nitric oxide inhibitor, and the vasorelaxant effect of amentoflavones were measured. , It was confirmed that the vascular relaxation effect is completely suppressed (-4.3 ± 1.0% relaxation at amentoflavone concentration of 1 x 10 ^-5 M, see Figure 1b).

2-2-3. Vasorelaxant Effect by Amentoflavones after Inhibition of Guanylate Cycase

To determine whether the vasorelaxant effect of amentoflavones is related to cGMP, the vasorelaxant effect was measured after pretreatment with ODQ, a soluble guanylate cyclase inhibitor, and measured 1 x 10 ^-6 M and 1 x 10 ^-7 M. Vasorelaxant effect by amentoflavones disappeared completely at the ODQ concentration of, and similar results were obtained after pretreatment with 1 x 10 ^-5 M and 1 x 10 ^-6 M, another inhibitor of soluble guanylate cyclase. It can be seen that (see Fig. 2a and 2b).

2-2-4. Vascular Relaxation Effect by Amentoflavones after Cyclooxygenase Blocking

To determine whether prostacyclin with vasorelaxant effects on the vasorelaxant effect of amentoflavones, amentoflavones were treated with a nonspecific cyclooxygenase inhibitor indomethacin under 1 x 10 ^-5 M pretreatment. As a result of measuring the vasorelaxant effect, it was confirmed that there was no significant difference with the group without the indomethane pretreatment (see FIG. 3).

2-2-5. Vasorelaxant Effect by Amentoflavones after K ⁺ Channel Blocking

To determine whether blocking of K ⁺ channels affects the vasorelaxant effect of amentoflavones, Glybenclamide, an ATP-sensitive K ⁺ channel inhibitor, was measured using 1 x 10 ^-7 M and 1 x 10 ^-6 M, respectively. Pretreatment did not affect the vasorelaxant effect of amentoflavones, but partially inhibited the vasorelaxant effect by amentoflavones in the presence of the nonspecific K ⁺ channel inhibitor TEA and 1 x than at 1 x 10 ^-7 M In the presence of 10 ^-6 M TEA, the vasorelaxant effect by amentoflavones tends to be markedly reduced (see FIGS. 4A and 4B).

2-2-6. Vasorelaxant Effect by Amentoflavones after Ca ²⁺ Channel Blocking

In order to measure the Ca ²⁺ channel blocking effect on the vasodilator effect of amentoflavone, Ca ²⁺ channel blocker verapamil to 1 x 10 ^-7 M and 1 x 10 ^-6 Mentor O were pretreated with M flavone As a result of measuring the degree of relaxation by, it was confirmed that at low concentrations, there was no difference from the group that was not pretreated, but the relaxation effect was partially suppressed at an amentoflavone concentration of 3 × 10 ⁻⁶ M or more (see FIG. 5).

2-2-7. Vasorelaxant Effects by Amentoflavones After Blocking Sympathetic and Parasympathetic Nerve Receptors

In order to examine the association between the vasorelaxant effect of amentoflavones and the nervous system, pretreolol, a nonselective β-adrenergic antagonist, and atropine, an antimuscalinic drug, were pretreated with 1 x 10 ^-6 M, respectively, followed by amentoflavones. As a result of measuring the vasorelaxant effect, it was confirmed that there is no difference in the vasorelaxant effect compared to the control group without the drug (see FIG. 6).

2-2-8. Relaxation Effect of Amentoflavones after Inhibition of Phosphodiesterase

The host of Jaffe Lina (zaprinast) non-specific inhibitor, IBMX and cGMP-dependent PDE-5 inhibitor of PDE each of 1 x 10 ^-6 M and then a pre-processing results of the measurement of the vasodilator effect of amentoflavone, 3 x 10 ^-6 M In the above concentration of amentoflavones, it was confirmed that the vascular relaxation effect was partially suppressed compared to the control group (see FIG. 7).

2-2-9. Relaxation Effect of Amentoflavones after Rho kinase Inhibition

To observe whether the Rho kinase pathway affects the vasorelaxant effect of amentoflavones, pretreatment with 1 x 10 ^-6 M of Y-27632, a vasoconstrictor by amentoflavones, was performed. As a result of measuring the relaxation effect, it was confirmed that the vasorelaxation effect by amentoflavones was increased upon inhibiting kinases (see FIG. 8).

Experimental Example 3. Measurement of cGMP

After thoracic aortic section of Experimental Example 1-2 was equilibrated in Krebs solution for 30 minutes while supplying 95% O ₂ and 5% CO ₂ gas, 10 ^-5 M indomethacin and 10 ^-4 M IBMX (3-isobutyl -1-methylxanthin) was added and L-NAME and ODQ were added together according to the experiment. After equilibrating for 10 minutes by adding 10 ^-6 M of phenylephrine, reacting with 10 ^-4 M of amentoflavone for 2 minutes and stopping the reaction by immediately placing the vascular tissue in liquid nitrogen. The cGMP concentration was measured when the samples were collected while being stored in. The supernatant obtained by measuring the wet weight was homogenized in the presence of 6% trichloroacetic acid, followed by centrifugation at 13,000 g for 15 minutes, and extracted four times using ether saturated in water. Extracts were concentrated using a speed-vac concentrator and cGMP measurements were analyzed using radioimmunoassay (Kim SZ, Kim SH. Et al., J. Urol., 159 (5) , pp1741 -1746, 1998). 100 μl of diluted cGMP antibody (Calbiochem-Novabiochem Co., San Diego, Calif., USA) and sample and standard were added to 50 mM sodium acetate buffer (pH 4.8) to a final volume of 100 μl. CGMP (10000 cpm per 100 μl, specific activity 2200 Ci / mM, Dupont-New England Nuclear, Wilmington, DE, USA) combined with ¹²⁵ I was added together and reacted at 4 ° C. for 24 hours. Bound type and non-bound type were separated using charcoal, and radioactivity was measured using γ-counter.

As a result, cGMP of 211.1 ± 21.31 pM / min / g was produced in the group without amentoflavones, while 387.1 ± 28.4 pM / min / g in the group receiving 1 x 10 ^-5 M of amentoflavones. cGMP production was significantly increased in the amentoflavone-treated group (p <0.01), vascular endothelial cells removed, 1 x 10 ^-6 M L-NAME group And after the addition of 1 x 10 ^-6 M ODQ group was administered amentoflavones was confirmed that the production of cGMP is suppressed compared to the group administered only amentoflavones (p <0.01, see Figure 9). In addition, in order to observe whether amentoflavones affect the shrinkage effect of phenylephrine, the shrinkage effect of phenylephrine after pretreatment with 1 x 10 ^-6 M and 1 x 10 ^-5 M amentoflavones, respectively The results showed that 1 x 10 ^-6 M of amentoflavones did not affect the contraction, but 1 x 10 ^-5 M of amentoflavones showed a significant inhibition of phenylephrine vasoconstriction. Could be (see FIG. 10).

Experimental Example 4 Measurement of Activity of Angiotensin I Converting Enzyme

Angiotensin converting enzyme activity was used in combination with the method of Santos et al. (Santos RA. Et al., Hypertension, 7 (2) , pp244-252, 1985). 10 μl of plasma was added to 490 μl of assay buffer containing 5 mM Hip-His-Leu in 0.4 M sodium borate buffer and incubated at 37 ° C. for 15 minutes. To stop the reaction, 1.2 ml of 0.34N NaOH was added thereto, and 100 µl of O-phthaldialdehyde solution (dissolved in methanol at 20% concentration) was added to form a fluorescent substance, followed by stirring. After 10 minutes, 200 µl of 3N hydrochloric acid was added to complete the reaction. Centrifugation at 3,000 rpm for 10 minutes at room temperature to remove the final product, His-Leu, with a fluorescence spectrophotometer ((Hitachi, model F-2000, Tokyo, Japan) The standard curve was prepared using his-leu and the negative control was performed by first adding NaOH before reacting the plasma.

As a result of measuring the inhibition of angiotensin converting enzyme of amentoflavones, it was confirmed that amentoflavones inhibited angiotensin converting enzyme in a concentration-dependent manner, and that amentoflavones inhibit the angiotensin I-induced vasoconstriction effect in blood vessels. In order to measure the vasoconstriction effect of 1 x 10 ^-5 M amentoflavone and angiotensin I of 1 x 10 ^-7 M, the vasoconstriction effect was significantly higher than that of the control group without amentoflavone. Was inhibited (p <0.01, see FIGS. 11A and 11B).

Experimental Example 5. Statistical Processing

The significance of the experimental results was determined by the Student's t-test and one-way ANOVA test as a significant difference when p was less than 0.05.

Examples of the pharmaceutical compositions containing the compounds of the present invention will be described, but the present invention is not intended to be limited thereto, but is intended to be described in detail.

Formulation Example 1 Preparation of Powder

Amentoflavones 20 mg

Lactose 100 mg

Talc 10 mg

The above ingredients are mixed and filled in an airtight cloth to prepare a powder.

Formulation Example 2 Preparation of Tablet

Amentoflavone 10 mg

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

After mixing the above components, tablets are prepared by tableting according to a conventional method for preparing tablets.

Formulation Example 3 Preparation of Capsule

Amentoflavone 10 mg

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

According to a conventional capsule preparation method, the above ingredients are mixed and filled into gelatin capsules to prepare capsules.

Formulation Example 4 Preparation of Injection

Amentoflavone 10 mg

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO _4, 12H ₂ O 26 mg

According to the conventional method for preparing an injection, the amount of the above ingredient is prepared per ampoule (2 ml).

Formulation Example 5 Preparation of Liquid

Amentoflavones 20 mg

10 g of isomerized sugar

5 g of mannitol

Purified water

After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding lemon flavor appropriately, mixing the above components, adding purified water, adjusting the whole to 100 ml by adding purified water, and then filling into a brown bottle. The solution is prepared by sterilization.

Formulation Example 6 Preparation of Healthy Drink

Amentoflavones 100 mg

15 g of vitamin C

100 g of vitamin E (powder)

Iron lactate 19.75 g

3.5 g of zinc oxide

Nicotinamide 3.5 g

0.2 g of vitamin A

0.25 g of vitamin B ₁

0.3 g of vitamin B ₂

Water quantification

After mixing the above components according to a conventional healthy beverage manufacturing method, and then stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed and sterilized and then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.

Although the composition ratio is mixed with a component suitable for a favorite beverage in a preferred embodiment, the composition ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.

As described above, the amentoflavone derivative isolated from the baekbaek of the present invention is one of the most important mechanisms that regulate the contraction and relaxation of the vascular smooth muscle (vascular smooth endothelial cell derived from nitric oxide (NO) of the endothelial cell-releasing factor) Amentoflavone derivatives are increased by increasing synthesis, activating guanylate cyclase, increasing cGMP production, and relaxing blood vessels, and also inhibiting vasoconstriction through angiotensin converting enzyme inhibition. The composition containing as an active ingredient may be usefully used as a medicine and health functional food for the prevention and treatment of cerebrovascular diseases.

Claims (6)

A pharmaceutical composition for the prevention and treatment of cerebrovascular diseases containing the amentoflavone derivative of formula (I) or a pharmaceutically acceptable salt thereof as an active ingredient:

(I) Where R ₁ to R ₆ are each independently hydrogen or a lower alkyl group of C ₁ to C ₄ . The pharmaceutical composition according to claim 1, wherein the compound of formula (I) is amentoflavone, wherein R ₁ to R ₆ are hydrogen. The pharmaceutical composition according to claim 1 or 2, wherein the amentoflavone derivative is separated from the roll. The pharmaceutical composition of claim 1, wherein the cerebrovascular disease is a transient cerebral ischemic attack, thrombotic cerebral infarction, embolic cerebral infarction, hemodynamic cerebral infarction, thermodynamic infarction or cerebral hemorrhage. delete delete

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