KR20050094081A - Pharmaceutical composition comprising the viscos substances extracted from chongkukjang for fibrinolysis - Google Patents

Abstract

The present invention relates to a thrombolytic composition containing soybean jeomjilmul extract was separated from the soybean, a soybean jeomjilmul extract Specifically beans of the present invention will then fermented with strains of Bacillus natto (Bacillus natto) producing a soybean, soybean About 2 to 12 times of distilled water was added to the mixture, followed by slow shaking and extraction at about 25 ° C. for about 1 to 6 hours, and the extract obtained by filtration was filtered through a 30 to 100 mesh filter network and about 10 to about 10 to 1500 g. It is obtained by taking the supernatant by centrifugation for 30 minutes, and exhibits excellent thrombolytic ability, so that it can be used as a pharmaceutical composition for thrombolytic medicine and health functional food.

Description

Pharmaceutical composition comprising the viscos substances extracted from Chongkukjang for fibrinolysis}

The present invention relates to a composition for thrombolysis, which contains the extract of Chungkookjang Viscose from the Cheonggukjang.

Fibrin is a major protein component of blood coagulants formed by the action of thrombin from fibrinogen. In vivo, blood is always in balance with coagulation and lysis. Thrombus formation and thrombolysis in the body is complex and finely regulated, and thrombus generated during normal purification is immediately degraded and does not affect the human body. However, if various factors cause imbalance of this regulation and accumulate blood clots that accumulate in the blood vessels, they block the blood vessels and cause thrombosis, which interrupts the blood circulation and stops the supply of nutrients and oxygen to the tissues. , Myocardial infarction or other cardiovascular disease (Cerinic MM., Et al., Semin.Arthritis Rheum ., 32 (5) , pp285-95, 2003).

Medicines that have typically been used to treat thrombosis to date include urokinase, streptokinase, and tissue-type plasminogen activator (tPA). However, these are expensive, have a short half-life, side effects of systemic bleeding, and have the disadvantages that oral administration is impossible except for urokinase.

Therefore, new methods for treating thrombosis are currently being sought because of the limitation of such thrombosis therapeutics.

Cheonggukjang and natto are fermented soybean products from Korea and Japan, respectively, and have a history of hundreds of years. Salted fish and shiokara are also widely used as fermented seasonings in Korea and Japan, respectively. Therefore, the separation of thrombolytic substances from these foods could lead to the development of inexpensive and safe treatment for thrombosis.

In particular, Cheonggukjang is a traditional soybean fermented food of Korea and is known as an excellent food containing bioactive substances such as protein and dietary fiber, isoflavones and phenols (David, JA, et al., Am. J. Clin. Nutr. , 34 , pp 362-366, 1981; Adlercreutz, H., et al., W. Ann. Med. , 29 , pp95-120, 1997). In addition, Cheonggukjang has the potential to produce new bioactive substances as well as various bioactive substances in soybeans and is expected to help health (Kim, OH, et al., Food) . Industry and Nutrition , 4 , pp 40-46 1999). On the other hand, Cheonggukjang is decomposed soy protein by the enzyme action produced by Bacillus natto and Bacillus subtilis during fermentation ripening process, and has its own unique taste and smell. ( Journal of Korean Food Science Society , 3 , p64, 1971; Kim Kyung-ja et al., Korean Food Science Society , 14 , p301, 1982).

In the research performed on the soybean has a characteristic such as the study of the nitrogen component change during changes and soy protein in the chemical composition of the process Soybean Meju fermentation (bakgyein, Korea thickening Journal, 15, p93, 1972; bakgyein, Korea thickening Journal , 15, p111, 1972) and a study on the holding component (yisukhui etc., Journal of the Korea Food, 15, p93, 1972), soybean produced using rice straw (gimgyeongja etc., Korea Food Science, 14, p301, 1982), strain Studies on the Production of Cheonggukjang with Different Diets (Hyeonja Lee et al . , 14 , p97, 1981; Seo Jeong-sook et al., Korean Food Science Society , 14 , p309, 1982; Seo Jeong-sook et al . , 15 , p385, 1983 ). In addition, techniques related to Cheonggukjang include Korean Patent Publication Nos. 2001-2980, 1999-70171, 1999-60113, 1993-11894, 1993-1812, 1991-7446, and 1988-5879. Nos. 2001-18095, 1999-72468, 2001-25273, 2001-18094, 1997-64430, 1999-65689, 1990-12548 and 1998-8040 Patent Publication Nos. 1994-10744, 1992-2675, 1993-2356 and 1996-16567. However, these technologies are all focused on eliminating off-flavor and improving physical properties.

On the other hand, Korean Patent Publication No. 2002-21178 relates to functional Cheonggukjang and its manufacturing method, and describes anti-cancer functional Cheonggukjang, and Korean Patent Publication No. 2003-59985 fermented milk for preventing adult diseases containing soybean fermented extract and its manufacture The present invention discloses a method for reducing serum cholesterol and lowering blood pressure thereof, and Korean Patent Laid-Open Publication No. 2002-79719 discloses Bacillus megaterium jabi-42 strain having excellent thrombolytic ability isolated from Chunggukjang. Disclosed are thrombolytic enzymes.

However, none of the above literature teaches or discloses a thrombolytic composition containing Cheonggukjang viscous extract produced during fermentation.

In this regard, the present inventors searched the thrombolytic ability of Cheonggukjang mucus extract isolated from Cheonggukjang, and confirmed the effect to complete the present invention.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for thrombolysis, which contains the extract of Cheonggukjang slime from the Cheonggukjang.

In order to achieve the above object, the present invention provides a pharmaceutical composition for thrombolysis containing Cheonggukjang viscous extract isolated from Cheonggukjang.

Hereinafter, the present invention will be described in detail.

The cheonggukjang viscous extract is (1) fermented soybean to prepare the chunggukjang, (2) mixed with about 2 to 12 times the distilled water in Cheonggukjang, then slowly shaken for about 1 to 6 hours at about 25 ℃ Step 2 to extract, (3) the extract is filtered through a 30 to 100 mesh screen, centrifuged at about 1500 to 3000 g for about 10 to 30 minutes to take the supernatant, additionally (4) spray the supernatant Characterized in that obtained by drying, hot air drying or lyophilization.

Specifically, in the first step, after washing the beans, soaked in water three to five times the volume for about 12 to 24 hours, preferably 18 hours, soaked and then drained, steam cooker, pressurization The mixture is heated at 100 to 150 캜, preferably at 121 캜 for 10 minutes to 2 hours, preferably 20 minutes, using a porcelain or the like, and then cooled to a temperature of 50 to 70 캜.

Thereafter, Bacillus natto or Bacillus subtilis strain, preferably Bacillus natto , incubated in steamed soybeans, inoculated at 1 to 3% by weight, preferably 2% by weight of the raw soybeans. Fermented at 40-42 ° C. for 40 hours to 80 hours, preferably 72 hours, and additionally freeze-dried to prepare Cheonggukjang, and then store the prepared Cheonggukjang in a storage container such as vinyl to prevent the flow of Cheonggukjang. .

In the second step, about 2 to 12 times, preferably about 6 times, distilled water is added to the Cheonggukjang prepared in the first step, followed by mixing, and then 1 to 6 hours at room temperature, preferably about 25 ° C., preferably The extract is obtained once to three times, preferably three times with slow shaking for 1 hour.

In the third step, the extract obtained in the second step is filtered through a 30 to 100 mesh, preferably 80 mesh filter, about 1500 to 3000 g, preferably 10000 g for about 10 to 30 minutes, preferably Centrifuge for 20 minutes to take the supernatant to obtain the Chunggukjang slime extract.

Additionally, in the fourth step, the Chungkookjang viscous extract obtained in the third step is cooled to 40 ° C. or lower, and then spray-dried at 75 ° C. or lower to dry to 8% or less of moisture, or a hot air dryer having a temperature of about 65 to 75 ° C. After drying to 8% or less of water content, it is powdered to 80 mesh or more to obtain Cheonggukjang viscous extract powder.

Cheonggukjang viscous extract powder obtained through the manufacturing process can be confirmed that there is no odor, and has a unique flavor of light yellowish Cheonggukjang.

The present invention provides a pharmaceutical composition for thrombolysis, which contains the extract of Cheonggukjang viscous material obtained by the above method as an active ingredient.

In addition, the chungkukjang viscous extract has been used as a food for a long time, the extract of the present invention extracted therefrom also has no problems such as toxicity and side effects.

The thrombolytic ability of the Cheonggukjang viscous extract obtained by the above method was searched, and the excellent thrombolytic effect was confirmed.

The thrombolytic pharmaceutical composition of the present invention comprises the extract in an amount of 0.02 to 50% by weight based on the total weight of the composition.

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

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

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

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

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

In addition, the present invention provides a health functional food comprising the Cheonggukjang viscous extract and food acceptable food supplement additives exhibiting a thrombolytic effect. Examples of the food to which the extract can be added include various foods, beverages, gums, teas, vitamin complexes, and health functional foods.

It may also be added to foods or beverages for the purpose of thrombolytic effects. At this time, the amount of the extract in the food or beverage may be added in 0.01 to 15% by weight of the total food weight, the health beverage composition may be added in a ratio of 0.02 to 5 g, preferably 0.3 to 1g based on 100 ml. have.

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

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

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

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

Reference example. Cheonggukjang Ingredient Analysis

General components of Chungkookjang were analyzed by AOAC method. Moisture, crude protein, crude fat, crude ash and total dietary fiber content were measured. Moisture was analyzed by atmospheric heating, crude fat by Soxhlet extraction, crude protein by semi-micro Kjeldhal method, and crude ash by incineration (Shin Hyo-sun. ), Shinkwang Publishing Co., pp. 69-87, 1992). The total dietary fiber content was measured by AOAC method developed and modified by Prosky et al. (Prosky, L., et al., Journal-Association of Official Analytical Chemists , 71 , pp1017-1023, 1988).

Example 1. Preparation of Cheonggukjang

The soybean used in the experiment was 2 kg of US soybean, washed, soaked in water for 18 hours, soaked, drained and steamed at 121 ℃ for 20 minutes using a steam cooker, and then the temperature was 60 ℃. It was cooled to be. Bacillus natto (Korea Food Research Institute) was inoculated with steamed beans in 2% of the weight of the raw soybeans, fermented at 37 ° C. for 72 hours to produce 500 g of Cheonggukjang, and then prepared Was stored in a storage container such as vinyl to prevent the flow of cheonggukjang viscous material, and the contents were analyzed according to the method of the reference example. In addition, it was additionally lyophilized and powdered to determine its content.

The general contents of Cheonggukjang were 55.7% of moisture, 19.7% of protein, 8.3% of crude fat, 2.4% of crude ash, and 7.0% of total fiber. The water content of lyophilized Chunggukjang was 8.6%, protein 44.5%, fat 18.7%, ash 5.4%, total dietary fiber 15.8% and carbohydrate 7.0%.

Example 2. Preparation of Cheonggukjang Viscous Extract

2-1. Isolation of Cheonggukjang Viscous Extract

After mixing and adding 3 L of distilled water to 500 g of Cheonggukjang prepared in Example 1, the mixture was extracted three times by slowly shaking the extract at about 25 ° C. for 1 hour, and then the obtained extract was 80 mesh. The filtrate was filtered through, and centrifuged at 1,000 g for 20 minutes, and then the supernatant was taken to obtain 24 g of Cheonggukjang viscous extract (see FIG. 1).

2-2. Preparation of Cheonggukjang Viscous Extract Powder

2-2-1. Spray Drying of Cheonggukjang Viscous Extract

24 g of the Chungkookjang viscous extract obtained in Example 2-1 was cooled to 40 ° C. or lower, spray-dried at 75 ° C. or lower, and dried to 8% or less of water to obtain a powder of Chungkookjang viscous.

2-2-2. Hot air drying of Cheonggukjang viscous extract

24 g of the Cheonggukjang viscous extract obtained in Example 2-1 was dried to a moisture content of 8% or less with a hot air dryer (HY-D01, Hanyoung STE) at about 65 to 75 ° C., and then powdered to 80 mesh or more and powdered. Cheonggukjang viscous extract of was obtained.

2-2-3. Lyophilization of Cheonggukjang Viscous Extract

Cheonggukjang slime extract 24g obtained in Example 2-1 was lyophilized in a lyophilizer to obtain a chungkukjang slime extract in powder form.

Experimental Example 1. Experiment of Determination of Thrombolytic Activity of Cheonggukjang Viscous Extract

In order to measure the fibrinolytic activity of the Cheonggukjang viscous extract obtained in Example 2-1, the following experiment was performed.

First, 0.2 ml thrombin (20 NIH / mL) solution was mixed well with 6 mL of 0.1% 15% (w / v) fibrinogen solution and placed on a petri dish and left for 30 minutes to allow fibrin plate medium (fibrin). plate). 10 μl of the Chungkookjang viscous extract solution of Example 2-1 at 2 mg / ml was added dropwise to the plate medium, and the fibrin lysed zones were formed after leaving at 37 ° C. for 5 hours to compare the diameters of the fibrin lysed zones. The thrombolytic activity of the extracts of Chungkookjang was measured. Meanwhile, the standard plasmin solution was prepared at 0.075, 0.15, 0.30, 0.45 unit / ml, and the diameter of the dissolved region formed by dropping 10 μl dropwise into the fibrin plate medium was measured and compared with the activity of the Chungkookjang viscous extract. . At this time, a calibration curve was prepared as shown in FIG. 2 using a plasmin standard (3.3 unit / mg protein) in order to measure the thrombolytic ability of the extract of Cheonggukjang slime.

As a result, it was confirmed that the dissolution region of the Chungkookjang viscous extract of 2 mg / ml concentration was large. In addition, after the heat treatment of the chungkukjang viscous extract for 5 minutes at 60, 70, 80, 90, 100 ℃ for 5 minutes, the dissolution activity was measured. Activity remained.

In addition, the thrombolytic ability after heat treatment at 100 ° C. for 5, 10, 20, and 30 minutes decreased enzymatic activity with increasing heat treatment time at 100 ° C., but compared with the diameter of the dissolution zone which was not heat treated even after 30 minutes of heat treatment. In all, enzyme activity of about 45% was maintained.

Hereinafter, an example of the preparation of the chungkukjang viscous extract will be described, but this is not intended to limit the present invention but merely to explain in detail.

Formulation Example 1 Preparation of Powder

20 mg of Chungkookjang viscous extract of Example 2-1

Lactose 100 mg

Talc 10 mg

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

Formulation Example 2 Preparation of Tablet

Cheonggukjang viscous extract 10 mg of Example 2-1

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

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

Formulation Example 3 Preparation of Hard Capsule

Cheonggukjang viscous extract 10 mg of Example 2-1

3 mg of crystalline cellulose

Lactose 14.8 mg

Magnesium Stearate 0.2 mg

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

Formulation Example 4 Preparation of Soft Capsule

Cheonggukjang viscous extract 10 mg of Example 2-1

Lecithin 0.5 mg

Di-alpha-tocopherol 0.5 mg

Soybean oil 8 mg

According to the conventional method for producing a soft capsule, a soft capsule is prepared by mixing the above components.

Formulation Example 5 Preparation of Injection

Cheonggukjang viscous extract 10 mg of Example 2-1

Mannitol 180 mg

Sterile distilled water for injection 2974 mg

Na ₂ HPO _4, 12H ₂ O 26 mg

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

Formulation Example 6 Preparation of Liquid

20 mg of Chungkookjang viscous extract of Example 2-1

10 g of isomerized sugar

5 g of mannitol

Purified water

According to the conventional method of preparing a liquid solution, each component is added to the purified water to dissolve it, the lemon flavor is added appropriately, the above components are mixed, purified water is added, the whole is adjusted to 100 ml by the addition of purified water, and then filled in a brown bottle. The solution is prepared by sterilization.

Formulation Example 7 Preparation of Health Functional Food

1000 mg of Cheonggukjang viscous extract of Example 2-1

20 g of vitamin mixture

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B ₁ 0.13 mg

Vitamin B ₂ 0.15 mg

Vitamin B ₆ 0.5 mg

0.2 μg of vitamin B ₁₂

Vitamin C 10 mg

10 μg biotin

Nicotinamide 1.7 mg

Folate 50 ㎍

Calcium Pantothenate 0.5mg

Inorganic Mixtures

Ferrous Sulfate 1.75 mg

Zinc Oxide 0.82 mg

Magnesium carbonate 25.3 mg

Potassium monophosphate 15 mg

Dibasic calcium phosphate 55 mg

Potassium Citrate 90 mg

Calcium Carbonate 100 mg

Magnesium chloride 24.8 mg

Although the composition ratio of the above-mentioned vitamin and mineral mixtures is mixed with a component suitable for a health food in a preferred embodiment, the compounding ratio may be arbitrarily modified, and the above ingredients are mixed according to a conventional health food manufacturing method. The granules may be prepared and used for preparing a health food composition according to a conventional method.

Formulation Example 7 Preparation of Healthy Drink

1000 mg of Cheonggukjang viscous extract of Example 2-1

Citric acid 1000 mg

100 g oligosaccharides

Plum concentrate 2 g

1 g of taurine

Add 900 ml of purified water

After mixing the above components in accordance with the conventional healthy beverage manufacturing method, and stirred and heated at 85 ℃ for about 1 hour, the resulting solution is filtered and obtained in a sterilized 2 L container, sealed sterilization and refrigerated Used to prepare the healthy beverage composition of the invention.

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

As mentioned above, the pharmaceutical composition containing the cheonggukjang viscous extract of the present invention as an active ingredient exhibits excellent thrombolytic ability and can be usefully used for thrombolytic drugs and health functional foods.

1 is a view of the production method of the Chungkukjang viscous extract,

Figure 2 is a diagram of the calibration curve prepared for the comparative measurement of the thrombolytic ability of the extract of Chungkookjang viscous.

Claims (6)

A thrombolytic pharmaceutical composition containing the extract of Cheonggukjang isolated from Cheonggukjang. The extract of claim 1, wherein the extract of Cheonggukjang viscous is obtained by adding distilled water of about 2 to 12 times to Cheonggukjang prepared by fermenting soybean, and then slowly shaking and extracting at about 25 ° C. for about 1 to 6 hours. To obtain a supernatant by filtration through a filter network of 30 to 100 mesh, centrifugation at about 1500 to 3000 g for about 10 to 30 minutes and additionally obtained by spray drying, hot air drying or lyophilization of the supernatant. . The pharmaceutical composition according to claim 2, wherein the soybean is fermented with Bacillus natto strain when fermented soybean is prepared. The pharmaceutical composition of claim 1, comprising 0.02-50% by weight of the Chungkookjang viscous extract, based on the total weight of the composition. Health functional food comprising the Chungkookjang viscous extract of claim 1 and a food acceptable food supplement additive exhibiting a thrombolytic effect. The health functional food of Claim 5 which is a health drink.