KR102140965B1 - Composition comprising extract of dendropanax mobifera and rubus coreanus for preventing and treating vascular disorder - Google Patents

Abstract

The present invention relates to a composition for the prevention and treatment of vascular diseases containing a complex extract as an active ingredient, specifically, a hwangchil tree extract subjected to primary hydrolysis treatment with protease and secondary hydrolysis treatment with aminopeptide enzyme and The present invention relates to a composition for preventing and treating vascular diseases, which can exhibit excellent vasodilating effects by containing a complex extract of immature and biscuit extract as an active ingredient.

Description

Composition for prevention and treatment of vascular diseases containing hwangchil tree and bokbunja extract as an active ingredient {COMPOSITION COMPRISING EXTRACT OF DENDROPANAX MOBIFERA AND RUBUS COREANUS FOR PREVENTING AND TREATING VASCULAR DISORDER}

The present invention relates to a composition for the prevention and treatment of vascular diseases containing hwangchil tree and bokbunja extract as an active ingredient, and in detail, a complex extract of hwangchil tree and immature and bokbunja molecules with increased active ingredient content treated with hydrolase. It relates to a composition for preventing and treating vascular diseases containing as an active ingredient.

Hypertension is the cause of all circulatory system diseases, and is a chronic degenerative disease with a very high mortality rate in case of complications such as brain hemorrhage, heart disease, and kidney disease. The prevalence is estimated to be 15-20% in middle-aged and older people in their 40s. Many antihypertensive drugs are currently being developed and used for the treatment of hypertension, one of adult diseases, and depending on the mechanism of action and the point of action, diuretics, sympathetic nervous system drugs (α,2-adrenergic antagonists, β-adrenergic antagonists), vasodilators, calcium It is classified as a channel antagonist (calcium channel blockers), an angiotensin converting enzyme (ACE) inhibitor, and the like.

Circulatory diseases, including hypertension and ischemic heart disease, are mainly due to atherosclerosis and vascular relaxation. Among the factors involved in the physiological regulation of blood vessels, especially arteries, the balance between vasodilator and contractile substances is very important. The contraction of vascular smooth muscle is controlled by the concentration of free calcium ions in the cell, and vasoconstrictor substances (Ang II, endothelin-1, norepinephrine, etc.) increase the concentration of intracellular calcium ions. On the other hand, it is well known that the role of NO/cGMP is very important in the mechanism of regulating blood vessel relaxation. NO secreted by various in vitro and extracorporeal substances activates soluble guanylyl cyclase (sGC) to promote cGMP production, and the produced cGMP inhibits calcium ion free from sarcoplasmic reticulum and degrades calcium ion permeability outside the cell It expands blood vessels by reducing the concentration of calcium ions in the cells.

NO is known to reduce vascular smooth muscle and expand blood vessels to regulate blood pressure to prevent hypertension, prevent blood clots that cause strokes and heart attacks, and soften blood vessels to reduce arteriosclerosis. This NO is produced by the decomposition of L-arginine by NOS synthase (Nitric Oxide Synthase).

Gamma-Amino Butyric Acid (GABA) is a non-protein amino acid and is a neurotransmitter of mammals. It has been reported that it improves blood flow in the brain and suppresses the secretion of antidiuretic hormone by acting on the vascular center and prevents hypertension by lowering blood vessels by expanding blood vessels.

Vascular contraction is a phenomenon in which blood vessels, including the aorta, small arteries, and veins, become narrower as the walls of the blood vessels contract. Vascular disorder diseases caused by contraction of blood vessels include arteriosclerosis, blood circulation disorders, hypertension, headache, stroke, cerebral infarction, brain hemorrhage, myocardial infarction and heart failure. For the treatment of these diseases, vasodilators are administered. However, conventional chemical vasodilators sometimes cause side effects such as endothelial damage, hemolysis, acute toxicity or fish toxicity, acute nephritis or heart attack in the living body, and there is a problem in that mass production is not easy due to a difficult production process.

Crude drugs are a kind of medicines, and natural products produced in nature are used as raw materials or processed as medicines or used as raw materials. It is known that unlike pharmaceuticals made of single-component compounds, many types of ingredients are mixed, so the effect is complex and has relatively few side effects. Hwangchil tree ( Dendropanax morbifera Lev. ) is a dicotyledonous plant native to the southern coast of Korea and Jeju Island, and belongs to the evergreen tree. Hwangchil trees do not fall leaves even in winter, and when the bark is damaged, a yellow resin solution appears, which is called Hwangchil. Physiological activity has been reported, such as anticancer activity of hwangchil tree extract. Bokbunja ( Rubus coreanus M. ) has the efficacy of interpolation, nominal, and diuretics in oriental medicine, and is known to treat stamina, oil well, and urinary frequency, and includes ingredients such as carvone acid, vitamin C, and polysaccharides. This is known.

KR 100845338 B1 KR 101510092 B1

An object of the present invention is to provide a composition for preventing, improving or treating vascular disease, which contains a hwangchil tree extract and a bokbunja extract, which are treated with a hydrolytic enzyme and have an increased active ingredient content, as active ingredients.

According to one aspect of the invention, the first hydrolysis treatment with proteolytic enzymes and secondary hydrolysis treatment with a hydrolysis enzyme containing bromelain and papain, Dendropanax Morbifera extract and immature and peritoneum ( Rubus coreanus ) A composition for preventing, improving or treating vascular diseases, including a complex extract of an extract, may be provided.

In addition, the protease may include protease and trypsin.

delete

In addition, the composite extract may include the hwangchil tree extract and the immature fruit extract in a mixed weight ratio of 1:0.4 to 0.6.

In addition, the vascular disease may be at least one selected from the group consisting of hypertension, arteriosclerosis, peripheral blood circulation disorder, vascular stenosis, cerebral infarction, angina pectoris, myocardial infarction and ischemic brain disease.

In addition, the complex extract may be to act to relax the blood vessels.

According to another aspect of the present invention, a pharmaceutical agent for preventing or treating vascular disease comprising the composition may be provided.

According to another aspect of the present invention, a functional food for preventing or improving vascular disease comprising the composition may be provided.

The composition comprising the Hwangchil tree treated with the hydrolytic enzyme of the present invention and the complex molecule extract may exhibit excellent vasodilating effects. Through this, it can be used as a pharmaceutical composition or health supplement for the prevention, improvement or treatment of vascular disease.

1 is a flow chart of an embodiment showing a process for producing a hydrolytic enzyme-treated hwangchil tree extract contained in the complex extract of the present invention,
Figure 2 confirms that the complex extract of the present invention shows an excellent inhibitory effect on cGMP selective PDE,
Figure 3 confirms that the complex extract of the present invention shows an excellent inhibitory effect on cAMP selective PDE,
Figure 4 confirms that the composite extract of the present invention shows excellent angiotensin inhibitory activity,
Figure 5 is a hydrolytic enzyme contained in the complex extract of the present invention to confirm the vasodilating effect of Hwangchil tree extract and bokbunja extract, respectively,
Figure 6 confirms the vasodilator effect of the complex extract of the present invention.

The present invention relates to a composition for preventing, improving or treating vascular diseases, including a complex extract of a hydrolyzed hwangchil tree extract and an immature and biplane extract.

Hereinafter, the present invention will be described in more detail. In describing the present invention, when it is determined that a detailed description of known technology related to the present invention may unnecessarily obscure the subject matter of the present invention, the detailed description will be omitted.

The present invention contains a hydrolyzed hwangchil tree extract and a complex extract of immature and bokbunja extracts as active ingredients.

The hwangchil tree extract may be hydrolyzed with an enzyme to increase L-arginine and GABA. 1 is a flowchart of a method for manufacturing a hwangchil tree extract according to a preferred embodiment of the present invention.

Referring to FIG. 1, first, the raw material of hwangchil tree is immersed in water to extract, and at the same time, proteolytic enzyme treatment may be performed to perform primary hydrolysis.

The raw material of the hwangchil tree is for separating the hwangchil tree extract, and may be at least one selected from the group consisting of hwangchil tree leaves, branches, roots, and fruits, but is not limited thereto. Preferably, the leaves of hwangchil tree are preferably selected. This can be used by cutting, drying or pulverizing by a conventional method. Preferably, it is good to use it by cutting and drying in terms of improving the yield of the hwangchil tree extract produced. The drying method is not particularly limited, and a conventional method can be used. For example, natural drying, hot air drying or freeze drying can be used.

The extraction method for producing the hwangchil tree extract is not particularly limited, and a method commonly used in the art may be used to prepare the extract. For example, a method using an extraction device such as hot water extraction, immersion extraction, reflux cooling extraction, supercritical extraction, subcritical extraction, high temperature extraction, high pressure extraction, ultrasonic extraction or using an adsorption resin containing XAD and HP-20 Method may be used, but is not limited thereto. Preferably, it can be obtained by treating and extracting an extraction solvent. The extraction solvent is not particularly limited, and may be selected in consideration of enzyme activity. In one preferred embodiment of the present invention, it will be described as using water. When treating the extraction solvent to separate the extract, the extraction solvent may be added at 1 to 100 times the total weight of the hwangchil tree. Preferably, 5 to 20 times the total dry weight of hwangchil tree is added.

In this step, by adding the enzyme to the solvent, extraction and enzyme treatment of hwangchil tree can be simultaneously performed. In this step, the enzyme to be treated may be a proteolytic enzyme. Specifically, it may be a protease (protease) or trypsin (trypsin). Preferably, the protease and trypsin are treated together. The proteolytic enzyme may be processed to increase the amount of protein eluted from hwangchil tree. The protease is preferably an enzyme derived from Bacillus subtilis . The enzyme to be added at this stage may be treated in the form of an enzyme solution dissolved in a solvent.

This step of separating the extract by treating the extraction solvent may be performed at a temperature of 30 to 60° C. in consideration of the extraction yield and the activity temperature of the enzyme to be hydrolyzed. Preferably, it can be carried out at 40 to 50 ℃. If the temperature at which this step is performed exceeds 60°C, the activity of the enzyme to be treated decreases, and if it is less than 30°C, the yield of hwangchil tree extract may decrease and enzyme activity may decrease. Separation and primary hydrolysis treatment of the hwangchil tree extract may be performed by treating for 1 to 12 hours within the temperature range. Preferably, the treatment is performed for 6 to 10 hours. If necessary, the pH can be adjusted. In this step, the pH is preferably neutral to weakly basic. Specifically, it is preferable that it is pH 7-8. Through this step, it is possible to obtain a primary hydrolyzed hwangchil tree extract.

Next, secondary hydrolysis may be performed by treating the degrading enzyme. This step can be performed by treating the enzyme in the extract solution immersed in the hwangchil wood of the previous step.

In this step, the degrading enzyme can be treated to degrade the peptide using the primary hydrolyzed extract and convert it into amino acids. The degrading enzyme may include bromelain or papain. Preferably, the primary hydrolyzed hwangchil tree extract is preferably treated with bromelain and papain. The enzyme processed in this step can be processed in the form of an enzyme solution dissolved in a solvent.

In the second hydrolysis performed in this step, an enzyme is added to the hwangchil tree extract that is first hydrolyzed in the previous step and 10 to 20 hours at a temperature of 40 to 60°C, preferably 50 to 60°C, preferably 14 It can be carried out by treatment for 18 hours. If necessary, the pH can be adjusted. In this step, the pH is preferably weakly acidic. Specifically, it is preferable that it is pH 5-6.

Next, the enzyme treated with the hwangchil tree extract can be inactivated.

This step can be carried out by heating the hwangchil wood immersed extract solution, which has been subjected to primary and secondary hydrolysis, with the enzyme added in the state where the hwangchil wood raw material is immersed for 30 minutes to 6 hours at a temperature of 100 to 120°C. . Through this, it is possible to inactivate the enzyme treated in the previous step while simultaneously extracting the active ingredient of hwangchil tree.

If necessary, the method for preparing a hwangchil tree extract according to an embodiment of the present invention may further include removing a solvent through a filtration, concentration, or drying process. Through this, the hwangchil tree extract can be provided in the form of a liquid or solid powder with a controlled concentration. Preferably, in order to increase the concentration of useful components and to improve storage, it is better to be freeze-dried and provided in the form of a powder. The filtration, concentration or drying method is not particularly limited, and may be performed by a conventional method.

The hydrolyzed hwangchil tree extract of the present invention prepared by the above manufacturing method has a significant increase in the L-arginine and gaba (GABA) contents as useful components. In general, the production of gaba and the manufacturing technique for amplifying the content of gaba require a vacuum treatment process, but the present invention can amplify the gaba content except for such a vacuum treatment process.

The immature fruit bokbunja extract can be prepared by extracting the immature fruit of the bokbunja as a raw material. The bokbunja immature fruit refers to the fruit of the green rind formed by bokbunja flowers, which may be obtained from April to September. Preferably, it may be obtained from May to August, more preferably from May to July. It can be used by cutting, drying, and grinding by a conventional method. The drying method is not particularly limited, and for example, natural drying or freeze drying may be used.

The method for separating the immature and bokbunja extract is not particularly limited, and may be extracted in the same manner as the method for separating the aforementioned hwangchil tree extract. Preferably, it can be obtained by treating and extracting an extraction solvent. The extraction solvent is not particularly limited, and for example, water or alcohol may be used. Preferably, water is used.

When extracting is separated by treating the extraction solvent, the extraction solvent may be added at 1 to 100 times the total dry weight of the raw materials. Preferably, it is preferably added at 5 to 20 times the total dry weight of the raw material. The temperature at the time of separating the extract by treating the extraction solvent may be 10 to 120°C. Preferably, it may be 30 to 100 ℃. It can be extracted for 1 to 72 hours within the above temperature range to separate the immature and bokbunja extract. Preferably, it is extracted for 2 to 12 hours, and more preferably for 3 to 7 hours to separate the immature and bokbunja extracts.

The composite extract of the present invention may be prepared by mixing the hydrolyzed hwangchil tree extract and the immature and bokbunja extract. The composite extract may include the hwangchil tree extract and the immature fruit extract in a mixed weight ratio of 1:0.4 to 3, preferably 1:0.4 to 0.6.

The composition comprising the complex extract of the present invention can relax blood vessels. Hwangchil tree extract contained in the complex extract of the present invention is hydrolyzed using an enzymatic reaction to increase the content of L-Arginine of Hwangchil tree to increase the useful components of the Hwangchil tree extract and thereby reduce blood vessel relaxation effects. Can be further improved. In addition, the complex extract of the present invention can exhibit a markedly enhanced vasodilator effect by including both hwangchil tree extract and immature and bi-molecule extracts, thereby preventing and treating vascular disorder diseases caused by constriction of blood vessels. Or it can be used as a high-efficiency pharmaceutical or functional composition for improvement. The vascular disorder diseases include, for example, hypertension, arteriosclerosis, (peripheral) blood circulation disorder, vascular stenosis, stroke, cerebral infarction, brain hemorrhage, angina pectoris, myocardial infarction or ischemic brain disease.

The composition containing the complex extract of the present invention as an active ingredient may be prepared as a pharmaceutical preparation for application to prevent or treat vascular diseases. The pharmaceutical preparation of the present invention uses at least one selected from the group consisting of pharmaceutically acceptable carriers, diluents and excipients, according to a method that can be easily carried out by those skilled in the art to which the present invention pertains. It can be prepared in unit dosage form by formulating it, or by incorporating it into a multi-dose container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of ex, powder, granule, tablet or capsule. The formulation may further include a dispersing agent or a stabilizing agent.

If necessary, the pharmaceutical preparation may further include a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is commonly used in preparation, for example, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, calcium silicate, fine Crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, but is not limited thereto.

If necessary, the pharmaceutical formulation of the present invention may further include additives known in the art in addition to the above components. For example, it may further include at least one selected from the group consisting of lubricants, wetting agents, sweeteners, flavoring agents, emulsifying agents, suspending agents and preservatives, but is not limited thereto.

The pharmaceutical preparation of the present invention may be administered orally or parenterally, and for parenteral administration, it may be administered by skin application, rectal injection, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, and the like. Suitable dosages of the pharmaceutical compositions of the present invention can be controlled by factors such as formulation method, mode of administration, patient's age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion, and response sensitivity. In general, a skilled doctor can easily determine and prescribe a dose effective for the desired treatment or prevention. The daily dosage of the pharmaceutical composition of the present invention may be 0.001 mg/kg to 10 g/kg, preferably 1 mg/kg to 1 g/kg. If necessary, the dosage may be divided into several times and administered at regular time intervals. The daily dosage of the pharmaceutical composition of the present invention can be adjusted by a skilled physician.

The composition containing the complex extract of the present invention as an active ingredient may be prepared as a functional food for preventing or improving vascular disease. The functional food is a food prepared by adding a complex extract to a food material, or a food prepared by capsules, powders, suspensions, etc., and includes healthy foods having a general meaning in health when ingested. Examples of the types of foods include drinks, meat, sausage, bread, biscuits, rice cakes, chocolate, candy, snacks, snacks, pizza, noodles, chewing gum, ice cream, soups, drinks, tea, vitamin complexes, etc. It is not limited to this.

If necessary, the functional food of the present invention may further include a component that is conventionally added during food production. For example, nutrients, vitamins, minerals (electrolytes), flavors, colorants, neutralizers, pectic acid, pectates, alginic acid, alginates, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol , Carbonic acid, pulp, and the like, but is not limited thereto.

In one embodiment of the present invention, the functional food may be a drink agent. The drink agent may further include a flavoring agent or natural carbohydrates. The natural carbohydrates include monosaccharides (eg, glucose, fructose, etc.); Disaccharides (eg, maltose, sucrose, etc.); oligosaccharide; Polysaccharides (eg, dextrin, cyclodextrin, etc.); And sugar alcohols (eg, xylitol, sorbitol, erythritol, etc.). As the flavoring agent, a natural flavoring agent (for example, taumatin, stevia extract, etc.) and a synthetic flavoring agent (for example, saccharin, aspartame, etc.) may be used alone or in combination.

Hereinafter, the present invention will be described in more detail based on the following examples in order to help the understanding of the present invention. However, these examples are only illustrative of the present invention and are not intended to limit the scope of the appended claims, and it is apparent to those skilled in the art that various changes and modifications to the embodiments are possible within the scope of the present invention and the scope of technical thought. It is also natural that such modifications and modifications fall within the scope of the appended claims.

Example

<Manufacture of general hwangchil tree extract>

Hot air drying was performed by cutting the leaves up to the green area within 50 cm based on the end of hwangchil tree leaves into 2 cm or so as raw materials. The extract of hwangchil tree was added with 20 times distilled water based on the weight of the raw material, extracted at 100°C for 4 hours, concentrated over 30 brix, and then freeze-dried to obtain an extract of hwangchil tree leaves in the form of lyophilized powder. The extraction amount and yield are shown in Table 1 below.

division Raw material Raw water volume(Kg) Extraction amount (g) yield(%) Preparation Example 1 Finely dried leaves 2.5 521.55 20.9

<Preparation of unripe and bokbunja extract>

The immature fruit of the green leafy bokbunja was selected and used as a raw material. The raw material was prepared by performing hot air drying immediately after harvesting the fruit. Distilled water containing 5% (v/v) alcohol was added 20 times based on the weight of the raw material, extracted at 100°C for 4 hours, concentrated over 30 brix, and then freeze-dried to obtain immature and bokbunja extracts in the form of lyophilized powder. .

<Preparation of hydrolyzed hwangchil tree extract>

The dried fine hwangchil tree raw material was prepared and used in the same manner as the general hwangchil tree extract. The final water amount is set to 20 multiples with respect to 100 parts by weight of hwangchil tree raw materials, and 9 times the amount of the water is first added to the finish, and then 1 g of protease and 1 g of trypsin are dissolved and enzyme solution prepared is added and treated at 45° C. for 8 hours. So, immersion and primary hydrolysis were performed simultaneously. After the completion of the first hydrolysis, 1 g of bromelain and 1 g of papain were dissolved in separate water, and an enzyme solution was prepared, added to the immersion solution, and treated at 55° C. for 16 hours to perform secondary hydrolysis. After the completion of the second hydrolysis, the remaining 10 times of the unadded water is additionally added from the amount of 20 times water initially set, extracted for 4 hours at 100°C, and then subjected to an enzyme deactivation process, then concentrated to 30 brix. The final sample was lyophilized to obtain an extract of hydrolyzed hwangchil tree leaves. Hereinafter, this is referred to as Preparation Example 2.

<Check the content of L-arginine and gaba>

The content of L-arginine and gaba in the prepared hwangchil tree extract was checked, and an amplification factor of an active ingredient due to hydrolysis treatment was analyzed. As an example, a sample of Preparation Example 2 was prepared, and as a comparative example, a sample obtained by performing only hot water extraction using the dried fine hwangchil tree of Preparation Example 1 as a raw material was prepared as a control.

For L-arginine and gaba analysis, a 6410A triple quadrupole mass spectroscopy (Agilent, USA) combined with 1200 series LC (Agilent, USA) was used. After ionizing the sample with a positive electro-spray ionization (+ESI) device, analysis is performed in multiple reaction monitoring (MRM) mode, and the nebulizer pressure, N ₂ gas flow rate and temperature are 40 psi, 10 L/min, 310℃, respectively. And capillary voltage was maintained at 4 kV. Gemini-NX C18 (4.6 × 150 mm, 3 μm, Phenomenex) was used as the column, and the sample injection amount was 5 μl and the column oven was maintained at 35°C. 5% ammonium acetate and 0.1% (v/v) formic acid mixed with mobile phase using water (A) and methanol (B) starting at the initial 0% (v/v) B and 95% (v/v) After sequentially changing to B, it was again lowered to 0% (v/v) B for 30 minutes to analyze, and the flow rate was maintained at 0.5 ml/min. Analysis for each Example and Comparative Example was measured twice, and the results are shown in Table 2 below.

Condition Measure
repeat L-arginine
(Mg/g) Average amplification factor
(%) Gaba
(Mg/g) Average amplification factor
(%) Comparative example 1 time 3.45 100% 19.975 100% Episode 2 2.903 18.017 Average 3.177 18.996 Example 1 time 16.840 559% 25.176 146% Episode 2 18.730 30.450 Average 17.770 27.813

Looking at Table 2, the hydrolyzed hwangchil tree extract according to the present invention is shown to contain in an amount of L-arginine 15 to 20 mg/g, Gaba 25 to 35 mg/g, and significantly improved L- compared to the comparative example It can be seen that it has arginine and gaba content.

<CGMP selective phosphodiesterase (PDE) inhibitory activity confirmation>

The cGMP-specific PDE inhibiting activity assay (cGMP-specific PDE inhibiting activity assay) was performed using the PDE Activity Assay Kit (Colorimetric) (ab139460, Cambridge, UK). For the analysis of cGMP selective phosphodiesterase (PDE) inhibitory activity, a method of measuring the degradation of cGMP by cyclic nucleotide phosphodiesterase of cGMP was used. The 5'-nucleotide released by the phosphodiesterase is further cleaved into nucleosides and phosphates by the 5'-nucleotide enzyme, and the phosphate released by the enzyme cleavage is converted to green color as Green Resay Reagent It was quantified by measuring the absorbance at 620nm.

As a control, 3-isobutyl-1-methylxanthine (IBMX), a non-specific cyclic nucleotidephosphodiester inhibitor inhibitor, was used as a control for comparison of enzyme activity inhibitory effects. IC ₅₀ , an inhibitory effect of IBMX as a control, was about 25 μM.

CGMP selective phosphodiesterase activity inhibitory effect of the complex extract prepared by mixing the hwangchil tree extract and the immature and bokbunja extracts of Preparation Example 2 in various mixing weight ratios were measured at concentrations of 0.3 and 1 μg/ml, respectively, and quantitative analysis was performed to suppress the inhibitory effect. It was converted to% and shown in FIG. 2. In FIG. 2, Hy-Dp means hydrolyzed hwangchil tree extract of Preparation Example 2, uRO means immature and bokbunja extract, and each sample number is hydrolyzed hwangchil tree extract and immature and bokbunja extract It means the mixed weight ratio of. For example, in the sample represented by 1:2 in FIG. 2(A), the complex extract prepared by mixing in a weight ratio of hydrolyzed hwangchil tree extract: immature and bokbunja = 1:2 was measured at a concentration of 0.3 ml. Means

Referring to Figure 2, it can be seen that the hydrolyzed hwangchil tree extract and the complex extract prepared by mixing immature and bokbunja in a weight ratio of 1:0.5 show the best effect.

<CAMP selective phosphodiesterase (PDE) inhibitory activity confirmation>

The cAMP selective phosphodiesterase (PDE) inhibitory activity experiment was performed using the PDE Activity Assay Kit (Colorimetric) (ab139460, Cambridge, UK). For the analysis of cAMP selective phosphodiesterase (PDE) inhibitory activity, a method of measuring the degradation of cAMP by cyclic nucleotide phosphodiesterase of cAMP was used. The 5'-nucleotide released by the phosphodiesterase is further cleaved into nucleosides and phosphates by the 5'-nucleotide enzyme, and the phosphate released by the enzyme cleavage is converted to green color as Green Resay Reagent It was quantified by measuring the absorbance at 620nm.

For comparison of the inhibitory effect of enzyme activity, 3-isobutyl-1-methylxanthine (IBMX), a non-specific cyclic nucleotide phosphodiester inhibitor inhibitor, was used as a control, and IC ₅₀ , an inhibitory effect of IBMX, as a control It was about 25 μM.

Quantitative analysis was performed by measuring the inhibitory effect of cAMP-selective phosphodiesterase activity on the hydrolyzed hwangchil tree extract of Preparation Example 2 and the complex extract of the immature and biplane extract at 0.3, 1 μg/ml, and the inhibitory effect was converted to %. It is shown in FIG. 3.

Referring to FIG. 3, in the composite extract, the mixed weight ratio of the hydrolyzed hwangchil tree extract and the immature and bokbunja extract shows excellent inhibitory effect even when 1:1 and 1:2, and when 1:0.5, more It can be seen that it exhibits an enhanced inhibitory effect.

<Confirmation of angiotensin inhibitory activity>

An angiotensin I-Converting Enzyme (ACE) activity assay is described in Cushman and Cheung (1971) as'Spectrophotometric assay and properties of the angiotensin-I converting enzyme of rabbit lung', Biochem. Pharmacol. , 20, 1637-1648). Hippuric acid- Histidine- Leucine (Hip-His-Leu, Sigma Chemical Co., St. Louis, USA) was used as a substrate for measuring angiotensin inhibitory activity, and hippuric acid produced by the action of angiotensin converting enzyme (hippuric acid). For analysis, 50 μL and 0.3 M of test extracts of 0.03, 0.1, 0.3, 1, 3, and 10 μg/mL in 100 μL (pH8.3) of 0.4 M sodium borate buffer containing 0.3 M sodium chloride (NaCl) 100 μL of 5 mM Hip-His-Leu substrate solution dissolved in 0.4 M sodium borate buffer containing sodium chloride (NaCl) was mixed and reacted at 37° C. for 30 minutes, followed by addition of 50 μL of 100 mU/mL angiotensin converting enzyme solution 37 The reaction was carried out for 1 hour at ℃. After the reaction was completed, 100 μL of 1 M HCl was added to stop the reaction, and 1 mL of ethylacetate was mixed with vortex mixer to extract hypuric acid, and then centrifuged at 3000×g for 15 minutes. After centrifugation, 500 μL of the supernatant was completely dried in a dryer at 65° C., dissolved by adding 1 mL of 1 M sodium chloride, and absorbance was measured at a wavelength of 228 nm with a microplate spectrophotometer. At this time, the sample extract content required to represent 50% of the inhibitory activity of angiotensin was expressed as an IC ₅₀ value.

For comparison of the inhibitory effect of enzyme activity, Captopril, an inhibitor of angiotensin converting enzyme, was used as a standard, and IC ₅₀ , an inhibitory effect of Captopril as a control, was about 9.47 μM. Angiotensin inhibitory activity was measured for a complex extract containing a hydrolyzed hwangchil tree extract and an immature and bokbunja extract at a mixing ratio of 2:1 (1:0.5), and quantitative analysis was performed for 0.03, 0.1, 0.3 1, 3, It is measured in 10, 30, 100μg / ml is shown in Figure 4.

Referring to FIG. 4, the hydrolyzed hwangchil tree extract showed IC ₅₀ 95.3 μg/ml, and the immature and bokbunja extract showed IC ₅₀ 93.4 μg/ml, and the complex extract according to the present invention IC ₅₀ =44.9 μg/ml As can be seen, it can be seen that it exhibits a significantly superior angiotensin inhibitory effect compared to each single extract. The complex extract according to the present invention is useful to be applied as a composition that prevents the action of angiotensin I in angiotensin II to lower blood pressure and relax blood vessels, and thus has a low concentration and excellent effect on cardiovascular diseases related to hypertension It can be utilized as a composition.

<Verifying the vasodilating effect of hydrolyzed hwangchil tree extract and immature and bokbunja extract>

In order to confirm the vasodilating effect of the herbal extract, a vascular tissue fragment of a rat was prepared. Sprague-Dawley rats (Samtaco, Korea) of 200~300g or so are used for experiment after adapting to the breeding room environment while supplying solid feed (LabDiet 5L79, Orient Bio, Korea) and water Did. After stunning the mouse, immediately bled, then opened along the abdominal center line, separated the thoracic aorta, cut into a ring shape of about 2-3 mm in length, and oxygen-saturated 4°C crabs The solution was immersed in a solution (Krebs buffer (mM/L); 118.4 NaCl, 25 NaHCO3, 11.66 glucose, 4.75 KCl, 1.18 MgSO4, 7H2O, 2.5 CaCl2, 2H2O, 1.19 KH2PO4, 0.02 EDTA, pH7.4). While continuously supplying oxygen, the surrounding adipose and connective tissues were removed cleanly, and then cut transversely to a length of about 2 to 3 mm to form ring segments.

To analyze the effect of herbal extracts on the relaxation capacity of the vascular tissue of the rat thoracic aorta, the functional activity of the vascular tissue of the rat vascular aorta was first measured. The solution in the organ chamber was maintained at 37°C, pH 7.4 with a mixed gas of 95% oxygen (O2)-5% carbon dioxide (CO2). The blood vessel tension includes a polygraph system (RPS212, Grass, RI, USA) and a computer analyzer (Power Analyzer) connected to a force-displacement transducer (FT03, Grass, Fhode Island, USA). Lab 400, MacLab system, Castle Hill, Australia)).

Before the drug experiment, the thoracic aortic vessel was loaded with a basal tension by applying 1.5 g of force to the vascular segment by hanging one part of the vascular tissue on a ring installed in the lower part of the muscle chamber and the other part hanging on a force-displacement transducer. After maintaining the equilibrium for a minute, if a constant baseline is maintained, phenylephrine (Phenylephrine) 10-5M is administered, and then acetylcholine 10-5M,10-4M,10-3M is injected into the constricted aorta. It was determined that the relaxation was complete upon addition. In addition, after contraction with Phenylephrine 10-5M, when exposed with a craps solution, it was confirmed that the reaction was stable, and thus it was possible to determine functional activity. This is shown in Fig. 5A.

Next, the effect of the hydrolyzed hwangchil tree extract of Preparation Example 2 on the thoracic aortic vasodilator response in rats was analyzed. After contracting the aorta, the samples were treated at concentrations of 0.1, 0.3, 1, 3, and 10 μg/ml, and the measurement results are shown in FIG. 5(B).

5 (B), as a result of confirming the relaxation reaction, it can be seen that the hydrolyzed hwangchil tree extract exhibits a vasodilating effect, and IC ₅₀ appears to be about 0.35±0.05 μg/ml.

Next, the immature and bokbunja extract was used as a sample to analyze the effect on the vascular relaxation response of the rat aorta. After contracting the aorta, the samples were treated at concentrations of 0.1, 0.3, 1, 3, and 10 μg/ml, and the measurement results are shown in FIG. 5(C).

5 (C), as a result of confirming the relaxation reaction, it can be confirmed that the immature and bokbunja extracts exhibit vasodilating effects, and the IC ₅₀ appears to be about 0.99±0.07 μg/ml.

<Confirmation of vasodilator effect of the complex extract>

Two complex extracts containing hydrolyzed hwangchil tree extract and immature and bokbunja extracts were prepared 1:1 and 2:1 (1:0.5), respectively, to analyze the effect on the vascular relaxation response of the thoracic aorta in rats. After contracting the aorta, relaxation reactions were confirmed at 0.1, 0.3, 1, 3, and 10 μg/ml, and the results are shown in FIG. 6.

Referring to FIG. 6, it can be confirmed that the IC ₅₀ of the complex extract exhibits a vasodilating effect of about 2.78±0.07 μg/ml in a sample with a 1:1 mixing ratio and 0.25±0.05 μg/ml in a sample with a 2:1 mixing ratio.

It was found that in both the composite extract samples, the concentration-dependent vasodilation effect was increased, and the vasodilation effect of the sample in which the hydrolyzed hwangchil tree and immature and bokbunja extracts were mixed in a weight ratio of 2:1 was remarkably excellent You can confirm that. In the case of the complex extract of the present invention in which the hydrolyzed hwangchil tree and the immature and bokbunja extracts are mixed in a weight ratio of 2:1, even when taking a small amount, it can exhibit excellent vasodilating effects.

The complex extract according to the present invention can be usefully utilized as a high-efficiency hypertensive treatment agent that further enhances the vasodilatory effect by using a hydrophilic enzyme-treated hwangchil tree extract in which L-arginine and gaba are simultaneously exposed and an immature and bokbunja extract.

Claims (8)

In the composition for preventing, improving or treating vascular diseases, including a complex extract of hwangchil tree ( Dendropanax Morbifera ) extract and immature and bokbunja ( Rubus coreanus ) extract,
The hwangchil tree extract is subjected to primary hydrolysis treatment with proteolytic enzymes and secondary hydrolysis treatment with proteolytic enzymes including bromelain and papain,
The composite extract comprises the hwangchil tree extract and the immature and bokbunja extract in a mixed weight ratio of 1:0.4 to 0.6, vascular disease prevention, improvement or treatment composition.
According to claim 1,
The protease contains a protease, vascular disease prevention, improvement or treatment composition.
According to claim 2,
The protease comprises a trypsin, a composition for preventing, improving or treating vascular disease.
delete According to claim 1,
The vascular disease is at least one selected from the group consisting of hypertension, arteriosclerosis, peripheral blood circulation disorder, vascular stenosis, cerebral infarction, angina, myocardial infarction and ischemic brain disease, a composition for preventing, improving or treating vascular disease.
According to claim 1,
The complex extract is a composition for preventing, improving or treating vascular diseases, which has a blood vessel relaxing function.
A pharmaceutical formulation for preventing or treating vascular disease, comprising the composition of any one of claims 1 to 3, 5 and 6.
Functional food for preventing or improving vascular disease, comprising the composition of any one of claims 1 to 3, 5 and 6.

Images