KR102025245B1 - Composition for improving inhibition activity against angiotensin converting enzyme - Google Patents

Abstract

The present invention relates to an angiotensin inhibitor having an inhibitory activity against angiotensin converting enzyme, and specifically, to a hydrolyzed protease, trypsin, bromelain and papain, which are hydrolysed, angiotensin converting enzyme inhibitory activity is markedly increased. The present invention relates to an angiotensin inhibitor which can be usefully used for the prevention or treatment of hypertension, kidney disease or complications thereof by containing a tree extract as an active ingredient.

Description

Composition for enhancing angiotensin converting enzyme inhibitory activity {COMPOSITION FOR IMPROVING INHIBITION ACTIVITY AGAINST ANGIOTENSIN CONVERTING ENZYME}

The present invention relates to a composition having an inhibitory activity against angiotensin converting enzyme, and more particularly to a composition having an angiotensin converting enzyme inhibiting activity by containing a sulfur-lacquered tree extract treated with a hydrolase as an active ingredient.

In recent years, nutritional status has been dramatically improved due to economic growth, improved education, and scientific development, and diseases caused by changes in various life patterns including dietary life and prolongation of life expectancy have also tended to diversify.

Cerebrovascular disease, which is the leading cause of death after cancer in Korea, is due to high onset of hyperlipidemia and hypertension. In particular, hypertension is a representative cause of complications such as cerebrovascular disease and cardiovascular disease, and it is increasing as the diet is westernized and aging. For the treatment of hypertension, diuretics, vasodilators, calcium blockers, etc. are mainly used, but there are problems such as headaches, dizziness, orthostatic hypotension, renal dysfunction, etc., which requires attention to use.

NO is known to relax blood vessel smooth muscles to expand blood vessels, thereby preventing blood pressure by controlling blood pressure, preventing blood clot formation that causes strokes and heart attacks, and softening 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 (hereinafter referred to as GABA) is a non-protein amino acid that is a neurotransmitter in mammals. It has been reported to prevent blood pressure by improving blood flow in the brain and acting on the vascular center, inhibiting the secretion of antidiuretic hormones, and expanding blood vessels to lower blood vessels.

On the other hand, patients with kidney disorders are also gradually increasing, which may include the increase of diabetic nephropathy due to changes in living environment, aging or increase in diabetic patients. The number of patients with renal failure due to renal failure or inadequate dialysis is increasing every year, and dialysis therapy causes side effects such as complications due to erythrocyte production disorders, microglubulin accumulation, and increased cardiovascular lesions. . In addition to coercive therapy, patients undergoing renal failure are administered with drugs that suppress the rise of electrolytes in the blood, and low-protein diets. In cases of renal anemia, erythropoietin is administered. It is not considered enough to stop. In kidney disease such as nephritis, diabetic nephropathy, renal failure, and the like, it is often accompanied by hypertension, and hypertension is considered to be one of the deteriorating factors of kidney disease. ought. Especially, the renin- angiotensin type inhibitor attracts especially attention.

The renin-angiotensin-aldosterone system is an endocrine pathway that regulates blood pressure and extracellular fluid volume. Lenin, activated by glomerular neighboring cells and secreted into the blood, converts angiotensinogen to angiotensin I, and angiotensin I is converted to angiotensin II by an angiotensin converting enzyme (ACE). ACE hydrolyzes the C-terminal dipeptide (his-Leu) of inactive angiotensin I and converts it to angiotensin II, which constricts vascular wall smooth muscle and releases aldosterone secretion in the adrenal cortex. Accelerating increases the resorption of sodium and water in the kidneys. This increases the amount of salt and water in the body, increasing the total amount of fluid and blood pressure. In addition, it may inactivate the bradykinin nanopeptide (nanopeptide) having a vascular relaxation action to inactivate it. ACE activity is a cause of hypertension, arteriosclerosis, heart failure, diabetes and various kidney diseases.

ACE inhibitors reduce the formation of angiotensin II by inhibiting the conversion of angiotensin I to angiotensin II by acting on the ACE enzyme. ACE inhibitors were first approved as treatments for the treatment of hypertension and later found to be effective in other cardiovascular and renal diseases, and have been widely applied to the treatment of various diseases such as acute myocardial infarction, heart failure, and diabetic nephropathy. . Specifically, ACE inhibitors suppress the secretion of aldosterone, an antidiuretic hormone in the adrenal cortex, and have a therapeutic effect on kidney diseases such as renal failure, lowering blood vessel resistance in the kidneys and inhibiting reabsorption of sodium to prevent sodium excretion through urine. It is known that by increasing diuretic can help. ACE inhibitors are thought to have renal protective effects that are not suppressive. For example, studies show that ilbesartan's effect on inhibiting the progression of diabetic nephropathy was superior to control of blood pressure by calcium antagonists. have. For this reason, especially in diabetic nephropathy, ACE inhibitors are widely used even in the normal range of blood pressure.

In addition, ACE inhibitors may inhibit the degradation of bradykinin to exhibit vasodilation effects, inhibit sympathetic nerves, and inhibit β 1 receptor activity, thereby exhibiting therapeutic effects on coronary artery diseases such as heart failure. In addition, by suppressing the decomposition of prostaglandin can reduce the elevated intraocular pressure due to glaucoma, it can be used to prevent vision loss. Representative ACE inhibitory drug formulations are known as Captopril.

Synthetic ACE inhibitors generally have side effects that are considered unsafe, and the development of ACE inhibitors from natural products with few side effects increases the demand for the development of pharmaceuticals and food materials. Known natural ACE inhibitors include kazenine hydrolysates (Kohmura, M, et al., Agric. Biol. Chem., 54: 835 (1990)), Murariryl produced by Nocardia orientales. (muraryl) peptide (Caren B. et al., The Journal of Antibiotics, 37: 330 (1984)), peptides obtained from porcine serum (Tadahiko, H. and Ryoichi. K., Biochem. Biophys. Com., 139: 52 (1986)), peptides of tuna gut autodigestion (see Nobuyasu, M. and Toshio, S., Biophys.Res.Com., 57: 695 (1993)), sardine meat part Hydrolysates obtained by treatment with alkaline proteolytic enzymes of Bacillus rickeniformis (see Matsui, T. et al., Biosci. Biotec. Biochem., 57: 922 (1933)), peptides in anchovy salt (see : Kim, Sun-Bong et al., Journal of the Korean Fisheries Society, 26: 321 (1993)), Peptides Isolated from Cultures of Steptomyces species (Uasuji, K. et al., The Journal of Antibiotics) , 36: 1295 (1983)), peptides in vinegar (Wakako T. et al., Nippon Shokuhin Kogyo Gakkaishi, 39: 188 (1002)) prepared from undegreased rice, poly-derived from organic solvent extracts of green tea Phenols (Ref .: Contrast and Choi Cheong, Korean Journal of Food Science and Technology, 25: 238 (1993)), Hot Water Extract of Tangerine Peel (Ref .: Yoshijara, M. et al., Agric. Biol. Chem., 49: 909 (1985)) ) And Cheongju and its by-products (cf. Seiji, B.S., et al., 51: 463 (1993)) have been reported. Such an ACE inhibitor may be used as a functional food or drug such as lowering blood pressure.

Crude drugs are medicines that use natural or naturally produced natural products as raw materials or processed. Unlike medicines made of single-component compounds, many kinds of ingredients are mixed, and the effects are known to be complex and have relatively few side effects. Yellow Chilli ( Dendropanax) morbifera Lev . ) Is an evergreen broad-leaved arboreous tree native to the southern coast of Korea and Jeju Island. It is a species that does not fall in winter. Hwangchil has been used as a rare paint that emits the golden color of emperor's armor, helmets, and other metal ornaments since the Three Kingdoms. It is a Korean envoy, written in the Goryeo Dynasty, Guilin History, Guilin, and Haedong History. It is recorded and remains a specialty of Baekje, the old book of Tang Dynasty History Book. The main wood of Lee Si-Jin has been recorded that Hwangchil-Tak is effective in removing heat, treating eye and burns, and leprosy and is harmless.

KR 100204112 B1

 Manjusri, D. and Richard, L. S., J. Biol. Chem., 250: 6162 (1975); Davis, J. O., fed. Proc., 36: 1753 (1977); Hall, J. E. et al., J. Hypertens., 4: 387 (1986)  N Engl J Med 2001 Sep 20; 345 (12): 851-60

An object of the present invention is to provide an angiotensin converting enzyme inhibitor comprising a hydrolyzed Hwangchil wood extract as an active ingredient.

According to an aspect of the present invention, the hydrolyzed by the protease, trypsin, bromelain and papain ( Dendropanax Provided is an angiotensin converting enzyme (ACE) inhibitor for the prevention or treatment of hypertension, kidney disease, or complications thereof, comprising an extract of Morbifera ) as an active ingredient.

In addition, the hydrolysis treatment is a first step of immersing the raw wood raw material in water and treating protease and trypsin; And it may be carried out in a second step of treating bromelain and papain in the sulfuric acid dipping solution prepared in the first step.

In addition, the hwangchil wood extract may be a hot water extract.

In addition, the hydrolyzed Hwangchil wood extract may be increased L-arginine and Gaba content.

In addition, the complications include arteriosclerosis, coronary artery disease, myocardial infarction, heart failure, peripheral blood circulation disorder, vascular stenosis, stroke, cerebral infarction, cerebral hemorrhage, ischemic brain disease, renal failure, diabetes, diabetic nephropathy, diabetic retinopathy, proteinuria, Edema, vision disorders due to vascular narrowing of the eye, and glaucoma.

Angiotensin converting enzyme inhibitor according to an aspect of the present invention can exhibit a markedly increased angiotensin converting enzyme inhibitory activity by using the hydrolyzed Hilchi chinensis extract containing as an active ingredient. Through this, the renin- angiotensin- aldosterone system can be usefully used as a composition for the prevention, improvement or treatment of hypertension, kidney disease or complications thereof.

In addition, L-arginine and Gaba components are markedly increased, which can exhibit an excellent vasorelaxant effect on the contracted vessels. Through this, it can be used as a pharmaceutical composition for the purpose of preventing or treating hypertension.

Figure 1 shows the angiotensin converting enzyme inhibitory activity of the Hwangchil-tree extract and the hydrolyzed Hwangchil-tree extract,
Figures 2 and 3 confirm the vascular relaxation effect of the Hwangchil-tree extract and the hydrolyzed Hwangchil-tree extract by basal tension test.

The present invention relates to an angiotensin converting enzyme inhibitor containing as an active ingredient the extracts of Dendropanax Morbifera hydrolyzed with protease, trypsin, bromelain and papain.

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

Angiotensin converting enzyme inhibitor of the present invention includes a hydrolyzed Hwangchil-tree extract as an active ingredient.

The part which becomes the raw material of the hwangchil tree which can separate the hwangchil tree extract 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 or branches of the yellow lacquer tree are selected. More preferably, the leaves or young branches of the yellow lacquer tree are selected. The young branch means a branch of 1 year or less. It can be used by cutting, drying and pulverizing by a conventional method. The said drying method is not specifically limited, A conventional method can be used. For example, natural drying, hot air drying or freeze drying may be used.

The hydrolyzate may be prepared by treating the hydrolysing enzyme with the raw material of the yellow lacquer tree and extracting the solvent with water as a solvent. In the preparation of the hydrolyzed Hwangchil wood extract of the present invention, the order of the preferred embodiment may consist of immersing the raw material in water and dividing it into two steps, followed by hydrolysis and heating to extract the enzyme inactivation at the same time. have. The hydrolysis treatment is a first step of immersing the raw material of Hwangchil wood in water and treating protease and trypsin for the first hydrolysis; And it can be carried out in the second step of treating bromelain and papain for hydrolysis in the sulfuric acid dipping solution prepared in the first step. In the present invention, the hydrolysis treatment of the raw materials is carried out in two steps as described above to allow enzymes having different reaction temperature conditions to react at the optimum temperature, while the optimum reaction temperatures are similar and do not induce reverse reactions with each other. Enzyme groups can be treated together in the same step to shorten the reaction time, thereby shortening the time performed for the entire process, thereby increasing productivity. The enzyme to be added during the hydrolysis may be treated in the form of an enzyme solution dissolved in a solvent.

In the first step of the hydrolysis treatment, first, the raw material may be immersed in water, and the first hydrolysis treatment may be performed by adding a protease for the first hydrolysis treatment. Specifically, the first step of the hydrolysis treatment may be performed by adding protease and trypsin to the hwangchil immersion liquid which is the water of the hwangchil wood. The water is an immersion solvent and forms a reaction pool for the enzymatic reaction, and may be used in 7 to 15 times the weight of the sulfur wood raw material. The protease may be added to increase the amount of protein eluted from the raw wood lacquer, it is preferable that the protease derived from Bacillus subtilis ( Bacillus subtilis ) is selected. The trypsin may be added to decompose the hwangchil-derived protein into polypeptides and a few amino acids. The protease and trypsin treated in the first step may be treated with 0.1 to 1% by weight, based on 100% by weight of the total raw wood lacquer. The first step may be performed by treating at 40 to 50 ° C. for 5 to 15 hours. Preferably, the treatment is carried out at 42 to 47 ° C. for 6 to 10 hours.

Next, a second step of hydrolysis treatment can be performed. The second step may be carried out by treating the aminopeptidase to the raw material treated with the proteolytic enzyme in the sulfur lacquer tree in the previous step. Specifically, it may be carried out by adding bromelain and papain to the protease and trypsin-treated hwangchil immersion liquid prepared in the first step. The bromelain and papain treated in the second step may be treated in an amount of 0.1 to 1% by weight based on 100% by weight of the raw Hwangchiranmu raw material. The first step may be performed by treatment at 50 to 60 ° C. for 12 to 20 hours. Preferably, the treatment is performed at 53 to 57 ° C. for 14 to 18 hours. In the second step, bromelain and papain may be prepared and treated with an enzyme solution dissolved in a solvent, and the solvent for preparing the enzyme solution may be used as 0.5 to 2 times the weight of the raw material of yellow chile, but is not limited thereto.

Next, hot water extraction may be performed on the hydrolyzed Hwangchil wood dipping solution by the method comprising the first and second steps to inactivate the treated enzyme and to separate the Hwangchil wood extract which is a useful component. . Through this, the production of the Hwangchil wood extract and the inactivation of the enzyme is made in one process can increase productivity by reducing the overall manufacturing time and effort while improving the convenience of the production process. The treatment temperature at the time of hot water extraction may be 80 to 120 ° C, preferably 100 to 120 ° C. Extraction for 1 to 72 hours within the above temperature range can be carried out separation and enzyme inactivation of the Hwangchil wood extract. Preferably it can be extracted for 2 to 12 hours, more preferably for 3 to 7 hours. If necessary, hot water extraction may be performed by further adding 8-12 times water based on the weight of the yellow lacquer tree to increase the extraction yield. Hydrolyzed Hwangchil-tree extract prepared by hot water extraction may be provided in the form of concentrated liquid or solid powder by adjusting the concentration by removing the solvent through filtration, concentration or drying.

Hydrolyzed Hwangchil-tree extract of the present invention has excellent angiotensin converting enzyme inhibitory activity. In detail, the angiotensin converting enzyme inhibitory activity of the yellow lacquer tree raw material can be enhanced by hydrolyzing the raw lacquer tree raw material. Through this, by inhibiting the conversion of angiotensin I to angiotensin II, it is excellent in reducing the increase in the amount of salt in the body, the increase in the total body fluid and the increase in blood pressure caused by the action of angiotensin II, preventing the prevention of hypertension, kidney disease or their complications It can be used as a composition for improving, or treating. For example, it may be used as a diuretic for promoting the discharge of body fluids for the improvement or treatment of hypertension, kidney disease or complications thereof. The complications include, for example, arteriosclerosis, coronary artery disease, myocardial infarction, heart failure, peripheral blood circulation disorder, vascular stenosis, stroke, cerebral infarction, cerebral hemorrhage, ischemic brain disease, renal failure, diabetes, diabetic nephropathy, diabetic retinopathy , Proteinuria, edema, visual impairment due to vascular narrowing of the eye, and glaucoma. In addition, the hydrolyzed Hwangchil-tree extract of the present invention can significantly amplify the content of L-arginine and GABA, which are useful components of Hwangchil-tree raw material, through hydrolysis, thereby releasing better blood vessels. Can be effective. Since the hydrolyzed Hwangchil tree extract of the present invention has excellent angiotensin converting enzyme inhibitory activity and increased L-arginine and GABA content, it is possible to prevent, ameliorate or treat vascular disorders associated with vasoconstriction. It can be used as a composition of high quality and high efficiency. The vascular disorders include, for example, hypertension, arteriosclerosis, coronary artery disease, myocardial infarction, heart failure, (peripheral) blood circulation disorders, vascular stenosis, stroke, cerebral infarction, cerebral hemorrhage or ischemic brain disease.

Angiotensin converting enzyme inhibitor containing the hydrolyzed Hwangchil-tree extract of the present invention as an active ingredient may be prepared as a pharmaceutical preparation for application to the prevention or treatment of hypertension, kidney disease or complications thereof. The pharmaceutical formulation of the present invention uses at least one selected from the group consisting of pharmaceutically acceptable carriers, diluents and excipients, according to methods which can be easily carried out by those skilled in the art. Can be prepared in unit dose form or incorporated into a multi-dose container. The formulations can then be in the form of solutions, suspensions or emulsions in oils or aqueous media or in the form of extracts, powders, granules, tablets or capsules. The formulation may further comprise a dispersant or stabilizer.

If desired, the pharmaceutical formulation may further comprise a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier is conventionally used in the formulation, 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 oils.

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, but is not limited to, at least one selected from the group consisting of lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, and preservatives.

The pharmaceutical preparation of the present invention may be administered orally or parenterally, and in the case of 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 may be controlled by factors such as the formulation method, mode of administration, age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion and response to the patient. In general, the skilled practitioner can readily determine and prescribe a dosage effective for the desired treatment or prophylaxis. 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 dose of the pharmaceutical composition of the present invention can be adjusted by a skilled physician.

Angiotensin converting enzyme inhibitor containing a hydrolyzed sulfuric acid extract of the present invention as an active ingredient may be prepared as a functional food. The functional food is a food prepared by adding the hydrolyzed sulfuric acid extract to a food material, or a food prepared from capsules, powders, suspensions, and the like, and when ingested, has a health-specific meaning of health. Contains food. Examples of the foods include drinks, meat, sausages, breads, biscuits, rice cakes, chocolates, candy, snacks, sweets, pizza, noodles, gum, ice cream, soups, beverages, tea, and vitamin complexes. It is not limited to this.

If necessary, the functional food of the present invention may further include ingredients that are commonly added during food production. For example, nutritional supplements, vitamins, minerals (electrolytes), flavors, colorants, neutralizers, pectic acids, peptates, alginic acids, alginates, organic acids, protective colloidal thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohols , Carbonizing agent, pulp, etc. may be further included, but is not limited thereto.

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

Hereinafter, the present invention will be described in more detail with reference to the following examples in order to help understanding of the present invention. However, these examples are merely illustrative of the present invention and are not intended to limit the appended claims, it will be apparent to those skilled in the art that various changes and modifications to the embodiments are possible within the scope and spirit of the present invention. Naturally, such modifications and variations fall within the scope of the appended claims.

Example

<Preparation of Hwangchil Tree Extract>

Hot air drying was carried out by cutting the leaves up to 50cm within the greenery area of the base of the Hwangchil-tree to 2cm as a raw material. 20 times of distilled water was added based on the weight of the raw material of Hilchi chile, followed by hot water extraction at 100 ° C. for 4 hours, concentrated to a concentration of 30 brix or more, and lyophilized to obtain a hot water extract in powder form. Using 2.5 kg of raw material, 521.55 g of extract was obtained, and the yield was found to be 20.9% (w / w).

<Preparation of Hydrolyzed Hwangchil Tree Extracts>

1 kg of dried fine yellow lacquer leaves was used as a raw material, and 9 parts of distilled water was added to the raw material to be immersed. 1 g of protease and 1 g of trypsin were dissolved and treated in distilled water, and immersion extraction and primary hydrolysis of the raw materials were simultaneously performed at 45 ° C. for 8 hours. After completion of the first hydrolysis, 1 g of bromelain and 1 g of papain were dissolved in distilled water in separate multiples based on the weight of the raw material, prepared as an enzyme solution, treated in an immersion solution, and subjected to secondary hydrolysis at 55 ° C. for 16 hours. . After completion of the second hydrolysis, additional distilled water of 10 times by weight of raw material is added to extract distilled water of Hwangchil wood in distilled water of 20 times by weight of raw material. Has gone through the process of performing. After concentration to 30brix or more concentration and lyophilized to obtain a hot water extract in the form of a powder.

<Determination of L-Arginine and Gava Contents of Yellow Chili Extract>

The contents of L-arginine and Gabba were analyzed for H. Chil-chil extract and hydrolyzed Hwang-chil-tree extract. As a comparative example, Hwangchil-tree extract was used as an example of the hydrolyzed Hwangchil-tree extract of the present invention. For analysis of L-arginine and Gaba content, 6410A triple quadrupole mass spectroscopy (Agilent, USA) combined with 1200 series LC (Agilent, USA) was used. Samples are ionized with a positive electro-spray ionization (+ ESI) device and analyzed in multiple reaction monitoring (MRM) mode. The nebulizer pressure, N ₂ gas flow rate, and temperature are 40 psi, 10 L / min, and 310 ° C, respectively. The capillary voltage was maintained at 4kV. Gemini-NX C18 (4.6 × 150 mm, 3 μm, Phenomenex) was used as a column. The sample injection amount was 5 μl and the column oven was maintained at 35 ° C. 95% (v / v) starting at initial 0% (v / v) B using water (A) and methanol (B) mixed with 5 mM ammonium acetate and 0.1% (v / v) formic acid as mobile phase After sequentially changing to B and lowering to 0% (v / v) B again for 30 minutes, the flow rate was maintained at 0.5 ml / min. The analysis for each sample was measured twice and the results are shown in Table 1 below. In Table 1 below, the L-arginine and Gava content units are mg / g.

division Measure
repeat L-arginine
(Mg / g) Gava
(Mg / g) Comparative example

1 time 3.45 19.975 Episode 2 2.903 18.017 Average 3.177 18.996 Example

1 time 16.840 25.176 Episode 2 18.730 30.450 Average 17.770 27.813

Referring to Table 1, it can be seen that both the L-arginine and Gaba content of the hydrolyzed Hwangchil wood extract compared to the Hwangchil wood extract. Based on the comparative example, the average amplification rate of the L-arginine content of the Example was 559% (w / w), and the average amplification rate of the Gabba content was 146% (w / w).

<Checking ACE Inhibitory Activity>

Angiotensin converting enzyme (ACE) inhibitory activity was determined from spectrophotometric assay and properties of the angiotensin-I converting enzyme of rabbit lung, described by Cushman and Cheung (1971), Biochem. Pharmacol., 20, 1637-1648. Some deformation was measured. Hippuric acid-Histtidine-Leucine (Hip-His-Leu, Sigma Chemical Co., St. Louis, USA) was used as a substrate for measuring ACE inhibitory activity. It was analyzed through the principle of measuring. Extract of Comparative Example or Example in concentrations 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) for analysis 100 μL of a 5 mM Hip-His-Leu substrate solution dissolved in 0.4 M sodium borate buffer containing 50 μL and 0.3 M sodium chloride (NaCl) was mixed and reacted at 37 ° C. for 30 minutes, followed by 100 mU / mL angiotensin conversion. 50 μL of the enzyme solution was added and reacted at 37 ° C. for 1 hour. After the reaction was completed, 100 μL of 1 M HCl was added to stop the reaction, and 1 mL of ethylacetate (ethylacetate) was mixed to extract hypofuric acid, and the mixture was sufficiently mixed with a vortex mixer. After centrifugation at 3000 × g for 15 minutes, 500 μL of the supernatant was completely dried in a dryer at 65 ℃ and dissolved by adding 1 mL of 1 M sodium chloride and the 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 ACE inhibitory activity was expressed as IC ₅₀ value. Quantitative analysis was carried out by measuring the ACE inhibitory activity of each Comparative Example and Example, the inhibitory effect is shown in Figure 1 and Table 2 in terms of% compared to the untreated group. The untreated group was prepared by adding distilled water instead of the extract of Comparative Example or Example. In FIG. 1, (A) is a measurement result of a comparative example and (B) is a measurement result of an Example. The measurement was performed four times each, and captopril, known as an ACE inhibitor, was used as a reference for a comparison of ACE inhibitory activity. As a control, IC ₅₀ , an inhibitory effect of captopril, was 9.47 μM.

Condition density
(μg / ml) Repeat measurement Inhibitory effect
(%) Repeat measurement Inhibitory effect
(%) Repeat measurement Inhibitory effect
(%) Repeat measurement Inhibitory effect
(%) Comparative example 0.03 1st round -3.00 2nd round 0.00 3rd round 6.00 4th round 8.00 0.1 1st round -4.00 2nd round 1.25 3rd round 7.25 4th round 2.00 0.3 1st round -0.86 2nd round -0.86 3rd round 1.29 4th round 2.14 One 1st round 0.69 2nd round 0.95 3rd round 0.95 4th round 1.00 3 1st round 4.00 2nd round 4.75 3rd round 7.75 4th round 7.75 10 1st round 10.75 2nd round 13.00 3rd round 7.00 4th round 11.00 30 1st round 10.75 2nd round 13.00 3rd round 11.00 4th round 13.00 100 1st round 22.00 2nd round 25.00 3rd round 31.00 4th round 30.00 300 1st round 44.00 2nd round 46.00 3rd round 46.00 4th round 52.00 Example 0.03 1st round 3.95 2nd round 4.25 3rd round 4.40 4th round 4.40 0.1 1st round 8.00 2nd round 8.20 3rd round 9.20 4th round 9.30 0.3 1st round 11.38 2nd round 11.75 3rd round 12.38 4th round 14.25 One 1st round 12.38 2nd round 13.50 3rd round 14.25 4th round 13.88 3 1st round 15.32 2nd round 15.00 3rd round 14.68 4th round 14.53 10 1st round 25.75 2nd round 25.00 3rd round 25.00 4th round 23.75 30 1st round 32.33 2nd round 32.00 3rd round 31.33 4th round 30.00 100 1st round 37.00 2nd round 38.00 3rd round 40.00 4th round 45.00 300 1st round 63.33 2nd round 73.33 3rd round 75.00 4th round 75.00

1 and Table 2, the comparative example, when treated with a concentration of 300μg / ml was only 47% of the inhibitory effect on average compared to the untreated group, in the case of the same concentration in the case of the same treatment, the inhibitory effect compared to the untreated group 71.7%. Through this, it can be seen that the hydrolyzed Hwangchil-tree extract of the present invention exhibits significantly increased ACE inhibitory activity compared to the non-hydrolyzed general Hwangchil-tree extract. The hydrolyzed Hwangchil-tree extract of the present invention has excellent ACE inhibitory activity and can easily inhibit the conversion of angiotensin I to angiotensin II, thereby applying to high blood pressure, kidney disease or complications thereof to prevent, improve or It can be usefully used as a composition for treatment.

<Evaluation of Blood Pressure Reduction by Thoracic Aortic Vascular Relaxation>

Rat blood vessel sections were prepared for the experiment. Sprague-Dawley rats (Sampaco, Korea) of about 160 to 200g are used for experiments after adapting to the environment of feeding room with sufficient supply of solid feed (LabDiet 5L79, Orient Bio, Korea) and water. It was. The rats were stunned immediately after bleeding, and then opened along the abdominal midline, the descending thoracic aorta was isolated and cut into rings of approximately 2 mm in length. Oxygen-saturated crab solutions at 4 ° C (Krebs) buffer (mM / L); 118.4 NaCl, 25 NaHCO ₃ , 11.66 glucose, 4.7 KCl, 1.18 MgSO ₄ .7H ₂ O, 2.5 CaCl ₂ · 2H ₂ O, 1.19 KH ₂ PO ₄ , pH7.4). While continuously supplying oxygen, the surrounding adipose tissue and connective tissue were removed cleanly, and then cut horizontally to a length of about 2 mm to form a ring segment.

The functional activity of rat vascular aortic vascular tissues was first measured in order to analyze the effects of normal and wild hydrolyzed yellow chile extracts on the relaxation ability of the thoracic aortic vascular tissues. The solution in the organic chamber was ventilated with 95% oxygen (O ₂ ) -5% carbon dioxide (CO ₂ ) mixed gas at 37 ° C and pH 7.4. Blood vessel tension is measured by a polygraph system (PowerLab 8/35, ADInstruments, Dunedin, New Zealand) connected to an isometric transducer (MLT0201, Panlab, Cornella, Spain) and a computer analyzer (LabChart). Pro V8-MLS260 / 8, AD Instruments, Dunedin, New Zealand). Before drug testing, thoracic aortic blood vessels were loaded with a basal tone by applying one gram of force to the vascular slices by hanging one part of the vascular tissue on a ring installed at the bottom of the chamber and the other part hanging on a force-displacement transducer. after holding the equilibrium constant when the baseline is maintained in vessel can festival phenyl LES principal (Phenylephrine) 10 ^-5 after administration of the M, acetylcholinesterase (acetylcholine) in the contracted aorta ^{10 - 5 M, 10 - 4} M, 10 - ^{It was} determined that complete relaxation was added 3M. In addition, when contracted with Phenylephrine 10 ^-5 M, when exposed to the craps solution, it was confirmed that the reaction is stable to determine the functional activity. This is shown in Fig. 2A.

After confirming the functional activity of the rat thoracic aortic vascular tissue, the experiment was carried out using the Comparative Examples and Examples. Phenyl LES principal (Phenylephrine) 10 ^{- 5} administered in M concentration by the after-induced vasoconstriction, comparative example general hwangchil wood extracts 0.1, 0.3, 1, the relaxation degree of the contracted blood vessel and administered in 3, 10μg / ml It confirmed and the result was shown to FIG. 2 (B). In addition, in order to analyze the effect of the hydrolyzed Hwangchil-tree extract as an example on the thoracic aortic relaxation of rats, the same concentration as that of the comparative example was 0.1, 0.3, 1, 3, and 10 μg / ml after contracting the aorta. The relaxation reaction was confirmed at, and the results are shown in (C) of FIG. 2. The results of comparing the relaxation effects for each treatment concentration of Comparative Examples and Examples are shown in FIG. 3.

2 and 3, the EC ₅₀ value of the comparative example was found to be 6.61 ± 0.06 μg / ml, and the EC ₅₀ value of the example was 3.50 ± 0.07 μg / ml. Compared with the comparative example, it can be seen that the vascular relaxation effect of the example shows an excellent effect twice. Comparing the IC ₅₀ value, the comparative example is 6.61 μg / ml and the example is 3.50 μg / ml, and the result is obtained by taking an ACE inhibitor containing the hydrolyzed sulfur-lactose extract of the present invention to see the vasorelaxant effect. In this case, it can be seen that the equivalent effect can be obtained even when the intake amount corresponding to half is used as compared to the case of using the extract of Hilchi chinensis obtained by the simple extraction by the general method.

The ACE inhibitor of the present invention is enhanced by the hydrolase treatment, and the ACE inhibitory activity and L-arginine and Gabba are simultaneously amplified by containing the extract of Hilchi chinensis as an active ingredient, the blood vessel relaxation effect is further amplified, preventing or treating hypertension It can be usefully applied as a high efficiency therapeutic agent.

Claims (5)

A first step of immersing the raw material of Hwangchil-chi in water and treating trypsin and Bacillus subtilis-derived protease with an amount of 0.1 to 1% by weight based on 100% by weight of the total material;
A second step of treating bromline and papain in the content of 0.1 to 1% by weight, based on 100% by weight of the raw material, to the sulfuric acid dipping solution prepared in the first step; And
A third step of extracting hot water; Containing the extract of Hwangchil wood ( Dendropanax Morbifera ) prepared by a manufacturing method comprising an active ingredient,
Angiotensin converting enzyme (ACE) inhibitory activity, a pharmaceutical composition for preventing or treating hypertension or complications thereof.
According to claim 1, wherein the hydrolyzed Hwangchil wood extract is characterized in that the L-arginine and Gaba content is increased,
Pharmaceutical compositions for the prevention or treatment of high blood pressure or complications thereof.
According to claim 1, wherein the complications are arteriosclerosis, coronary artery disease, myocardial infarction, heart failure, peripheral blood circulation disorders, vascular stenosis, stroke, cerebral infarction, cerebral hemorrhage, ischemic brain disease, renal failure, diabetes, diabetic nephropathy, diabetic retinal Characterized in that it is a symptom or disease selected from the group consisting of pathology, proteinuria, edema, vision impairment due to vascular narrowing of the eye, and glaucoma,
Pharmaceutical compositions for the prevention or treatment of high blood pressure or complications thereof.
A first step of immersing the raw wood material into water and treating trypsin and Bacillus subtilis-derived protease;
A second step of treating bromelain and papain to the sulfur-clad immersion liquid prepared in the first step; And
A third step of extracting hot water; Containing the extract of Hwangchil wood ( Dendropanax Morbifera ) prepared by a manufacturing method comprising an active ingredient,
Angiotensin converting enzyme (ACE) inhibitory activity, a pharmaceutical composition for the prevention or treatment of kidney disease or its complications.
The method of claim 4, wherein the first step is characterized in that carried out for 5 to 15 hours at 40 to 50 ℃,
Pharmaceutical composition for the prevention or treatment of kidney disease or complications thereof.

Images