KR102035281B1 - Composition with the ethyl acetate extract of Fomes fomentarius for the relaxation of blood vessel - Google Patents

Abstract

The present invention relates to a vascular relaxation composition comprising a horseshoe mushroom extract, horseshoe mushroom extract was confirmed to have the effect of treating the blood pressure by relaxing the blood vessels, the effect of the ethyl acetate fraction of the various fractions showed the greatest. In addition, the vasorelaxation effect of horseshoe mushroom extract appeared to be endothelial-dependent.

Description

Vascular relaxation composition comprising ethyl acetate extract of horseshoe mushroom {Composition with the ethyl acetate extract of Fomes fomentarius for the relaxation of blood vessel}

The present invention relates to a composition comprising a horseshoe mushroom extract, and more particularly to a vascular relaxation composition comprising an ethyl acetate extract of horseshoe mushroom.

Vasoconstriction (vasoconstriction) refers to a phenomenon in which blood vessels such as arteries or veins, including the aorta, narrow as the vessel wall contracts. Diseases caused by vasoconstriction include blood circulation disorders, arteriosclerosis, high blood pressure, stroke, cerebral infarction, cerebral hemorrhage, myocardial infarction and heart failure.

The most representative treatment for vascular disorders is nitric oxide (NO), a field of research that has been rapidly developing since its inception in 1980 by Furchgott and Zawadszki (Nature. 1980 Nov27; 288 (5789): 373). -6).

In general, the tension of vascular smooth muscle is influenced by several important factors, and the nitric oxide-cyclic guanosine monophosphate (NO-cGMP) pathway is a very important pathway in vascular relaxation.

The neurotransmitter nitric oxide (NO) is produced from L-arginine by nitric oxide synthase (NOS) in endothelial cells, which activates soluble guanulate cyclase (sGC), which in turn increases the production of cGMP. It relaxes vascular smooth muscle through signaling system. NO is currently being applied in all fields of medicine including autonomic nervous system, endocrine system, central nervous system and immune system. Therefore, it is necessary to develop various drugs capable of producing NO in order to relax blood vessels by controlling the NO-cGMP pathway.

However, conventional chemical vasodilators have a problem of causing side effects such as endothelial damage, hemolysis, acute toxicity or heart attack in vivo. Therefore, the development of a new technology that can control the NO-cGMP pathway by controlling the production of NO, while using natural materials as a material.

Meanwhile, Fomes fomentarius is a fungus belonging to Polyporaceae and Horseshoe, which is widely distributed in Korea, Japan, China, North America, the Philippines and Indonesia, and occurs in hardwood trees and trees. Park YH and Lee HD (1999) In Colored Korea Medical mushrooms, Kyohaksa, Korea.

The main ingredients are alkaloid, ergosta-7, 22-dien-3-one, 22-dien-3β-stearate, 22-dien-3β-ol, betulin, polysaccaride, lectin, fomentariol, fomentaric acid, agaritinic acid, agariolesin, carboxymethylcellulase, protease, β- (1-3) -D-giucan, D-parnkshion, Ge-132, which are known to be anti-cancer, inhibit gram positive bacterial growth, antioxidant, antipyretic, diuretic, and anti-diabetic ( Park YH and Lee HD (1999), In Colored Korea Medical mushrooms, Kyohaksa, Korea).

However, there are no studies related to the vasorelaxant effect of horseshoe mushrooms.

Korean Patent Publication No. 10-2005-0033022 (published date 2005.04.08) describes a functional beverage, a functional food, a functional confectionery, a functional bread and a functional liquor containing a horseshoe mushroom extract, and a method of preparing the same. . Korean Patent Publication No. 10-2008-0111955 (published date 2008.12.24) describes a vascular relaxation pharmaceutical composition containing Gomisin A as an active ingredient.

Young Hoon Kim et al., Immune-stimulatory Effects of Fomes fomentarius Extract in Murine Macrophages, J Appl Biol Chem (2014) 57 (4), 373-377. Park YH and Lee HD (1999), In Colored Korea Medical mushrooms, Kyohaksa, Korea.

The present invention is to prove the vascular relaxation effect of horseshoe mushroom, and based on this, to develop and provide a vascular relaxant, hypertension improvement, prevention, treatment.

The present invention provides a food composition for improving hypertension, comprising ethyl acetate extract of horseshoe mushroom.

The present invention provides a pharmaceutical composition for preventing and treating hypertension, comprising ethyl acetate extract of horseshoe mushroom.

In the composition of the present invention, the high blood pressure, preferably may be high blood pressure caused by vascular contraction.

The present invention provides a food composition for improving blood vessel contraction, comprising ethyl acetate extract of horseshoe mushroom.

In the composition of the present invention, the ethyl acetate extract of the horseshoe mushroom, preferably, after adding hexane and water to the ethanol extract of the horseshoe mushroom, fractionation (a); Adding ethyl acetate to the water layer obtained from step (a) and then fractionating (b); It is preferred that the ethyl acetate extract obtained from the process comprising.

The present invention provides a vasodilator comprising the ethyl acetate extract of horseshoe mushroom.

In the vasodilator of the present invention, the ethyl acetate extract of horseshoe mushroom, preferably, after adding hexane and water to the ethanol extract of horseshoe mushroom, fractionation (a); Adding ethyl acetate to the water layer obtained from step (a) and then fractionating (b); It is preferred that the ethyl acetate extract obtained from the process comprising.

Experimental results of the present invention, the vascular relaxation effect by the horseshoe mushroom was found to be higher in the order of ethyl acetate fraction, ethanol extract, butanol fraction and water fraction of horseshoe ethanol extract. Horseshoe mushrooms have the effect of relaxing blood vessels to treat hypertension, and the effect of ethyl acetate fraction was considered to be more effective.

In addition, horseshoe mushroom extract showed a vascular relaxation effect in the endothelial ratio.

1 is a view showing a process for obtaining extracts (fractions) for each solvent of horseshoe ethanol.
Figure 2 shows the vascular relaxation effect of the horseshoe mushroom extract in the blood vessels {ET (-)} endothelium removed.
Figure 3 shows the vascular relaxation effect of the horseshoe mushroom extract in the blood vessels {ET (+)} without removing the endothelium.
Figure 4 is the EC ₅₀ and E _max value of the horseshoe mushroom extract.

The present invention provides a food composition for improving hypertension, comprising ethyl acetate extract of horseshoe mushroom. In addition, the present invention provides a food composition for improving blood vessel contraction, comprising the ethyl acetate extract of horseshoe mushroom.

The present invention provides a food composition, which is not necessarily limited to a specific formulation, for example, meat, cereals, caffeine drinks, general beverages, chocolate, breads, snacks, confectionery, pizza, jelly, noodles, gums, ice creams, Alcoholic beverages, alcohol, vitamin complexes and other health supplements may be any one or more selected from.

On the other hand, the present invention provides a pharmaceutical composition for preventing and treating hypertension, comprising the ethyl acetate extract of horseshoe mushroom. The present invention also provides a vasodilator comprising the ethyl acetate extract of horseshoe mushroom.

Composition containing the horseshoe mushroom ethyl acetate extract of the present invention has a strong vasorelaxant effect as a preventive and therapeutic agent for diseases such as hypertension, arteriosclerosis, blood circulation disorders, diabetes, sexual dysfunction, memory disorders due to circulatory disorders It can be usefully used. When used clinically for this purpose, the composition of the present invention is combined with a conventional carrier in the pharmaceutical field and orally administered as a conventional agent in the pharmaceutical field, for example, tablets, capsules, troches, solutions, suspensions, and the like. Topical formulations, such as injectable preparations, ointments, creams, liquids, etc., in the form of preparations for injection, injectable solutions or suspensions, or ready-to-use injectable dry powders which can be prepared by injection with distilled water for injection. It can be formulated into a variety of formulations, such as.

Carriers that can be used in the compositions of the invention are conventional in the pharmaceutical art, for example in the case of oral preparations, binders, suspending agents, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, Pigments, perfumes, etc., and in the case of injectables, there are preservatives, analgesics, solubilizers, stabilizers, and the like. In the case of topical administration, there are bases, excipients, lubricants, and preservatives. The pharmaceutical preparations thus prepared may be administered orally or parenterally, for example, intravenously, subcutaneously, intraperitoneally, or topically. In addition, in order to prevent the decomposition of the drug by gastric acid during oral administration, an antacid may be used in combination, or a solid dosage form for oral administration such as tablets may be formulated into a coated formulation.

The dosage of horseshoe ethyl acetate extract according to the present invention to the human body is appropriately selected depending on the absorption rate, inactivation rate and excretion rate of the active ingredient in the body, the age, sex and condition of the patient, the severity of the disease to be treated, etc. As such, the adult is administered with an amount of 5 to 500 mg, preferably 10 to 200 mg per day. Therefore, when preparing the composition of the present invention in a unit dosage form, it may be formulated to contain 5 to 500 mg, preferably 10 to 200 mg of horseshoe mushroom ethyl acetate extract in consideration of the above-mentioned effective dose range. The unit dosage form thus formulated may be divided into several times, preferably 1 to 6 times, using specialized dosing methods or according to the needs of the individual who monitors or observes the administration of the drug as needed and according to the needs of the individual. May be administered.

Horseshoe mushroom ethyl acetate extract of the active ingredient of the composition according to the present invention exhibits the effect of relaxing the blood vessels without damaging the vascular endothelium, as evidenced from the experimental results described below, there is no side effects such as hemolysis or fish toxicity It does not show acute toxicity to experimental animals and can be used safely.

Meanwhile, in the composition of the present invention, the ethyl acetate extract of the horseshoe mushroom may be an extract extracted by adding ethyl acetate to a horseshoe mushroom as a solvent, or may be an ethyl acetate fraction prepared through a fractionation process.

Ethyl acetate fraction is preferably added to the ethanol extract of horseshoe mushroom hexane and water, followed by fractionation (a); Adding ethyl acetate to the water layer obtained from step (a) and then fractionating (b); It may be an ethyl acetate extract (fraction) obtained from the process comprising (see Figure 1). 1 is a view showing a process for obtaining extracts (fractions) for each solvent of horseshoe ethanol.

Hereinafter, the content of the present invention will be described in more detail through the following examples and experimental examples. However, the scope of the present invention is not limited to the following Examples and Experimental Examples, but includes modifications of equivalent technical ideas.

[ Example  One: Horseshoe Mushroom  Water Soluble of Ethanol Extract Fraction  Produce]

Horseshoe mushrooms were dried in the shade and then crushed, and extracted with a reflux extractor at 100 ° C. for 3 hours by adding 70% ethanol at a weight of 10 times. After cooling to room temperature, the extracted material was centrifuged at 3,000 × for 30 minutes, the supernatant was filtered through Whatman filter paper (Whatman International, Maidstone, UK), and then concentrated under reduced pressure (Eyela SB-1000). , Tokyo, Japan) and dried with a freeze dryer (FD 8508, Ilshin, Korea) to prepare a horseshoe ethanol extract powder. The extraction rate was 7.2% of the weight of raw materials.

The horseshoe mushroom ethanol extract powder was dissolved in water, and mixed with the same amount of hexane (hexane) and fractionated. The water layer was partitioned with ethyl acetate (EtOAC), and the remaining water layer was finally n-butanol (n -butanol) to obtain four fractions ( n- hexane, EtOAC, n -BuOH, water layer) with the final water layer (see Figure 1). 1 is a view showing a process for obtaining a fraction by solvent of horseshoe mushroom ethanol.

Hexane (hexane) fraction of the fraction is very small, except in the experiment ethanol extract, ethyl acetate (ethyl acetate), n-butanol (n-butanol), the final water layer was used in the following experimental example.

[ Experimental Example  One: Horseshoe Mushroom  Of ethanol extract Vascular tension  Measure]

In this experiment, male Sprague-Dawley Rat, 10 weeks old, weighing 320-350 g was used. Each rat was fed with sufficient water while freely feeding the solid feed until the day of each experiment. After adjusting to room temperature 22 ± 2 ℃, relative humidity 50 ± 10%, lighting time 12 hours (07: 00 ~ 19: 00), illuminance 150 ~ 300 Lux and adapting to laboratory environment for 1 week, weight change is constant, Only healthy rats were selected and used in the experiments. The experiments were conducted in compliance with the Animal Experiment Ethics Regulations of the Ministry of Food and Drug Safety.

Solid feed composition components and amounts used in this experiment are shown in Table 1 below.

ingredient Content (% by weight) Crude protein 22.1% or more Crude fat 3.5% or more Crude fiber 5.0% or less View minutes 8.0% or less calcium 0.6% or more sign 0.4% or more

Vascular tension measurement of horseshoe ethanol extract was performed by the following method.

Male Sprague-Dawley rats weighing between 320 and 350 g were anesthetized with sodium pentobarbital (50 mg kg ^-1 ip) and immediately extracted from the thoracic aorta and submerged in cold nutrient solution. The composition (mM) of the nutrient solution used in this experiment was NaCl 115.0, KCl 4.7, CaCl ₂ 2.5, MgCl ₂ 1.2, NaHCO ₃ 25.0, KH ₂ PO ₄ 1.2, dextrose 10.0.

The vascular aorta was immersed in Krebs-bicarbonate solution according to Seok et al., Removed the connective tissue attached to the blood vessels on the wet filter paper, and a 3.5 mm loop was made (Seok YM et al., Enhanced Ca ²⁺ -dependent activation of phosphoinositide 3-kinase class IIα isoform-Rho axis in blood vessels of spontaneously hypertensive rats.Hypertension. 2010 Nov; 56 (5): 934-41).

Endothelial cells were removed by gently rubbing the inner surface of the vascular ring with the end of the forcep for vascular endothelial removal. The vascular ring was suspended in a tissue bath (6 ml), the temperature of the tissue bath was maintained at 37 ° C., and a 95% O ₂ and 5% CO ₂ mixed gas was supplied.

Each vascular ring was connected to the lower fixed axis and the other to an isotonic transducer (Danish Myo Technology, Skejbyparken, Aarhus N, Denmark). At this time, care was taken to prevent vascular damage. The vessel rings were stretched manually by applying 2.0 g of tension and maintained in tension throughout the experiment.

Each vascular ring was stabilized in the tissue bath for 60 minutes before inducing a contractile response to 50 mM KCl. Tension for each aortic ring was recorded using a computerized data acquisition system (PowerLab / 8SP, AD Instruments, Castle Hill, NSW, Australia).

On the other hand, in order to confirm the vasorelaxation effect by horseshoe mushrooms, 70% ethanol extract and fractions of ethyl acetate, ethyl acetate, n-butanol, and the final water layer were used.

The drug was dissolved in 100 mg / ml stock at a ratio of 1: 3 in DMSO and distilled water. First, endothelial vessels (FIG. 2, ET (-)) and non-removed vessels (FIG. 3, ET (+)) were treated with U46619 (30nM, thromboxane A2 receptor agonist), which is a vasoconstrictor, and contracted. When the contraction is stabilized, ethanol extract, ethyl acetate, n-butanol, and final water layer are accumulated cumulatively (0.1, 0.2, 0.3, 0.4, 0.5 mg / mL) in the vascular ring. After work up.

The vascular tension was expressed as relaxation by measuring the percentage of relaxation at the maximum contraction value by treatment of U46619.

As a result, it was found that the vasorelaxation effect by the horseshoe mushroom was the highest in the ethyl acetate layer of the horseshoe ethanol extract. Specifically, horseshoe mushrooms relaxedly accumulated at a concentration of blood vessels induced by thromboxane A2 receptor agonist U46619.

Vascular relaxation was increased in the order of ethyl acetate layer, 70% ethanol extract, butanol layer and water layer of horseshoe mushroom. Here, the EC ₅₀ values of the drug for the relaxation of blood vessels treated with ethyl acetate layer were 0.23 mg / ml and 0.25 ± 0.05 mg / ml, respectively, when the endothelium was present (ET +) and when removed (ET−). . The EC ₅₀ values of the 70% ethanol extract were 0.40 mg / ml and 34 ± 0.07 mg / ml, but the EC ₅₀ values of the butanol layer were 0.40 mg / ml and 0.43 ± 0.03 mg / ml, and the water layer was 0.40 mg / ml. 0.44 ± 0.04 mg / ml (FIG. 4).

Therefore, comparing the EC ₅₀ value, the effect of the drug on the relaxation of the blood vessels in the ethyl acetate layer, it can be seen that higher than the 70% ethanol extract, butanol layer and water layer. Vascular relaxation for each drug did not differ with or without endothelial. That is, endothelial non-endovascular vascular relaxation was shown.

The E _max value, which indicates the maximum relaxation of the drug, was found in the presence of endothelium (ET +), removal (ET-), 70% ethanol extract (112.8%, 104.2 ± 3.0%), ethyl acetate layer (100.1%, 100.1 ± 0.1 %), Butanol layer (96.1%, 117.0 ± 26.2%) and water layer (23.7%, 41.4 ± 17.0%) showed the lowest E _max in the water layer, from which the relaxation effect of the drug in the water layer was low. It could be seen (Fig. 4).

In summary, the vasorelaxation effect of horseshoe mushroom was higher in order of ethyl acetate fraction, ethanol extract, butanol fraction and water fraction of horseshoe ethanol extract.

Horseshoe mushrooms have the effect of relaxing blood vessels to treat hypertension. Ethyl acetate fraction was considered to have a greater effect. In addition, the vasorelaxation effect of horseshoe mushroom extract was shown to be endothelial dependent.

Claims (7)

Adding hexane and water to the 70% ethanol extract of the horseshoe mushroom, and then fractionating (a); Adding ethyl acetate to the water layer obtained from step (a) and then fractionating (b); Horseshoe mushroom ethyl acetate obtained from the process comprising a fraction,
A food composition for improving hypertension, which has an effect of improving hypertension caused by vasoconstriction.
Adding hexane and water to the 70% ethanol extract of the horseshoe mushroom, and then fractionating (a); Adding ethyl acetate to the water layer obtained from step (a) and then fractionating (b); Horseshoe mushroom ethyl acetate obtained from the process comprising a fraction,
A pharmaceutical composition for preventing and treating hypertension, characterized in that it has an effect of preventing and treating hypertension caused by vasoconstriction.
delete Adding hexane and water to the 70% ethanol extract of the horseshoe mushroom, and then fractionating (a); Adding ethyl acetate to the water layer obtained from step (a) and then fractionating (b); Food composition for improving blood vessel contraction, comprising a horseshoe mushroom ethyl acetate fraction obtained from the process comprising a.
delete Adding hexane and water to the 70% ethanol extract of the horseshoe mushroom, and then fractionating (a); Adding ethyl acetate to the water layer obtained from step (a) and then fractionating (b); Vascular relaxation agent comprising a horseshoe mushroom ethyl acetate fraction obtained from the process comprising a. delete

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