KR100736702B1 - Composition comprising an extract of Cassia mimosoides var. nomame MAKINO for the prevention and treatment of ischemic diseases - Google Patents

Abstract

The present invention is a tea pool having improved cell viability under hypoxic conditions (Cassia mimosoidesvar. nomame The present invention relates to a composition containing MAKINO) extract, the tea extract of the present invention shows a strong inhibitory activity against apoptosis in ischemic disease animal model, the composition is a pharmaceutical composition for the prevention and treatment of ischemic disease and health It can be usefully used as a functional food.

Tea grass, apoptosis, ischemic disease, pharmaceutical composition, health functional food

Description

Composition for the prevention and treatment of ischemic diseases, including cha extract extract {Composition comprising an extract of Cassia mimosoides var. nomame MAKINO for the prevention and treatment of ischemic diseases}

Figure 1a is a graph showing the improvement of survival rate of HepG2 cells according to the addition of the extract of the green tea extract at different concentrations in the culture medium in hypoxic conditions,

Figure 1b is a graph showing the improvement of the survival rate of HepG2 cells according to the addition of the tea extract crude extract with different concentration in the culture medium under normal oxygen conditions,

Figure 2a is a graph showing the survival rate of HepG2 cells according to the addition of the crude extract extract to the culture medium in hypoxic conditions,

Figure 2b is a diagram showing the DNA segment of HepG2 cells in hypoxic conditions,

Figure 2c is a diagram showing the effect of inhibiting the DNA segmentation of HepG2 cells according to the addition of the crude extract extract in culture medium under hypoxic conditions,

Figure 3 is a MTT assay result showing the survival of HepG2 cells according to the addition of the chapul hexane, ethyl acetate, butanol soluble fraction and water soluble fraction in different concentrations in the culture medium in low oxygen conditions,

Figure 4a is a graph showing the infarct inhibitory effect of tea extract crude intraperitoneal injection in cerebral infarction animal model,

Figure 4b is a result of staining TTC (2,3,5-triphenyltetrazolium chloride) in the brain of the cerebral infarction animal model,

Figure 4c is the result of TTC staining of the brain of the cerebral infarct animal model intraperitoneally injected with chapul crude extract,

Figure 4d is a graph showing the infarction inhibitory effect of the concentration of Chapul ethyl acetate soluble fraction intraperitoneal injection in animal models of cerebral infarction,

5 is a diagram showing the effect of inhibiting the kidney damage of the extract of tea extract during intraperitoneal injection in ischemic acute renal failure animal model in blood creatinine concentration test.

The present invention relates to a composition for the prevention and treatment of ischemic diseases containing tea extract.

Cerebral infarction and myocardial infarction, which are typical ischemic diseases, are blocked due to hypertension, hyperlipidemia, diabetes, smoking, arteriosclerosis in which blood vessels are narrowed, and coronary arteries that supply blood to the brain by blood clots or coronary arteries that supply blood to the heart. It is caused by necrosis of tissue.

Cardiovascular disease accounts for 30% of all deaths worldwide and is the number one cause of death, 75% of which are ischemic diseases such as myocardial infarction and cerebral infarction. Myocardial infarction and cerebral infarction each account for the highest mortality rates with cancer. The methods of reducing the mortality caused by myocardial infarction and cerebral infarction include the treatment of hypertension and hyperlipidemia to prevent the blockage of blood vessels and the reduction of necrosis of surrounding tissues when the vessel is blocked.

Among these, the best way to reduce the necrotic site is to perforate the blood vessels by thrombolytics to block them, but the heart and the brain, which are energized by breathing, are exposed to tissues within 3-6 hours of blockage. Necrosis and subsequent reperfusion can not expect the therapeutic effect. However, it is difficult to get to the hospital within three to six hours of occlusion, which is the time required to increase the treatment effect, and to reperfusion after treatment, and the heart and brain are not regenerated once damaged. Therefore, if the cell viability in the ischemic state can be improved until reperfusion in the hospital, the therapeutic effect can be increased. At this time, one of the causes of tissue necrosis is apoptosis (Crow MT et al., Circ . Res., 95 (10), pp957-970, 2004; Friedlander RM, N. Engl. J. Med . , 348 (14) , pp1365-1375, 2003), by inhibiting this can improve cell viability.

In addition, kidney transplantation (Daemen MA et al., Transplantation , 73 (11), pp 1693-1700, 2002) or plastic surgery (Gastman BR et al., Plast. Reconstr . Surg . , 111, pp1481-1496, 2003). Caused by apoptosis.

In addition, in the case of surgery to stop the heart, if the amount of oxygen required more than the amount of oxygen supplied by the cardiopulmonary system, myocardial damage or brain damage due to hypotension may occur. Heart failure due to ischemia and myocardial damage and brain damage, for example, in cases where some blood is blocked, such as bypass graft surgery during coronary artery occlusion, aneurysm surgery in the cerebral artery or aorta. Side effects such as involuntary return may occur. Indeed, surgical or interventional treatment of aortic aneurysms results in adverse events such as myocardial ischemia, renal failure and paraplegia in 3-16% of patients. Therefore, if a drug that inhibits apoptosis in ischemic conditions is used for pretreatment, the side effects can be reduced.

The cause of neuronal suicide is not yet clear, but in the case of transient cerebral ischemia in the cerebrum, the supply of oxygen and glucose is blocked, resulting in the reduction of adenosine triphosphate (ATP) and edema in neurons. It is known to be induced. Excitatory neuronal suicide mechanism by which ischemia causes excessive glutamate to accumulate outside the cell by ischemia, and this glutamic acid enters the cell and eventually causes neuronal cell death due to excessive accumulation of intracellular calcium. Kang TC et al., J. Neurocytol . , 30 (12), pp945-955, 2001) and the increase of radicals in vivo due to sudden oxygen supply during ischemia-reperfusion damages DNA and cytoplasm, resulting in neuronal death. There is an oxidative neuronal cell death mechanism (Won MH et al., Brain Res. , 836 (1-2), pp70-78, 1999). Based on these mechanisms, many researches have been conducted to search for substances that effectively inhibit neuronal cell death caused by cerebral ischemia or to reveal the mechanism of the substance. However, few substances have been found to effectively inhibit neuronal cell suicide.

However, in the case of minocycline, a tetracycline antibiotic that inhibits apoptosis under ischemic conditions, cerebral infarction (Yrjanheikki J et al., Proc . Natl . Acad . Sci . USA , 96 (23), pp13496-13500, 1999), Myocardial infarction (Scarabelli TM et al., J. Am. Coll. Cardiol . , 43 (5), pp865-874, 2004) and ischemic acute renal failure (Wang J et al., J. Biol . Chem . , 279 (19) , pp19948-19954, 2004), is also known to be effective in the treatment of ischemic diseases. In addition, the present inventors have confirmed that such tetracycline-based antibiotics improve the survival of cells in ischemic conditions similar to this study (Korean Patent No. 0404134; US Patent No. 6716822, 6818625). Furthermore, aminoglycoside-based and quinolone-based antibiotics, as well as minocycline, improved cell survival under ischemic conditions. Among them, the aminoglycoside-based G418 (Genetisin) has been shown to treat myocardial infarction. (US Patent No. 6716822). In subsequent experiments, G418 suppressed apoptosis in ischemic conditions and showed a therapeutic effect on myocardial infarction as well as cerebral infarction. As a result, it has been expected that the samples showing the same cell survival improvement effect as G418 under ischemic condition will ultimately be effective in the treatment of ischemic diseases such as myocardial infarction. There is no disclosure about having.

Cassia mimosoides var. nomame MAKINO) is called Chapul because it is used for teasing at the beginning of the year of legumes, and young sprouts are also eaten as herbs. Outpost is mountain migraine ( Nomame Herba ) is also used as a herbal medicine. It is used for the treatment of jaundice caused by moist heat, soil erosion caused by food poisoning in summer, night blindness, and swelling. The main ingredients are emodin, physcion, myristic acid, Beta-sistosterol and the like (Andok-kyun, primary Korean book of primary color, 5, p174, 2002).

As a patent using cha grass, a patent was developed for foods that lower blood lipids (Korean Patent Registration No. 10-331064), and a patent using a mixture with other medicines as a blood circulation improving agent (China Patent Registration Nos. 1065793 and 1176814). And the literature on the effect of Chapulse on reducing blood lipids (Patil UK et al., J. Ethnopharmacol., 90 (2-3), pp249-252, 2004). There is no teaching or description anywhere in the literature regarding uses associated with diseases.

Accordingly, the present inventors have completed the present invention by recognizing the problems of the prior art in the treatment of ischemic diseases and confirming the improvement and therapeutic effect of chapul extract in ischemic disease animal model as an apoptosis inhibitor that improves cell viability in hypoxic conditions. It was.

It is an object of the present invention to provide a composition for preventing and treating ischemic diseases containing chapul extract and an extract that effectively inhibits apoptosis in ischemic conditions.

In order to accomplish the above object, the present invention provides a chapul ( Cassia mimosoides var. nomame MAKINO) Provides a pharmaceutical composition for the prevention and treatment of ischemic diseases containing crude extracts or purified fractions as an active ingredient and a pharmaceutically acceptable carrier or excipient.

The ischemic disease includes myocardial infarction, cerebral infarction, ischemic acute renal failure, ischemic acute liver failure, diabetic foot ulcer, diabetic nephropathy, or ischemic disease or organ tissue damage due to surgical side effects.

Ischemic diseases due to surgical side effects include ischemic heart failure, ischemic renal failure, ischemic liver failure or ischemic stroke.

The organ tissue damage is characterized in that it is due to long-term surgery or transplantation, recurrent traumatic limb rejoint with reperfusion after ischemia.

The organs include internal organs such as kidneys, liver, pancreas, lungs or heart.

The extract of the present invention includes a crude extract and chaff extract.

The crude extract of the present invention comprises water, ethanol, C ₁ to C ₄ lower alcohols such as methanol or mixed solvents thereof, preferably extracts soluble in methanol.

      In addition, the purified fraction of the present invention is a solvent selected from hexane, ethyl acetate or butanol after dissolving the crude extract of water in the water, preferably comprises a fraction available in ethyl acetate.

Hereinafter, the present invention will be described in detail.

Crude crude extract and purified fraction of the present invention can be obtained as follows.

The tea extract crude extract of the present invention is dried, and then, in a fine tea paste, a C ₁ to C ₄ lower alcohol such as about 1 to 15 times the volume, preferably about 5 to 10 times the amount of water, ethanol, methanol, etc. Or a mixed solvent having a mixing ratio of about 1: 0.1 to 1:10, preferably methanol, and at an extraction temperature of 0 to 100 ° C, preferably 20 to 100 ° C, for about 0.5 to 2 days, preferably 1 hour Cold extraction, hot water extraction, ultrasonic extraction, reflux cooling extraction, medicinal extractor, etc. for 1 to 1 days are extracted by extraction, preferably 1 to 12 times, preferably 2 to 4 times by filtration by ultrasonic extraction and then filtered The filtered extract was concentrated under reduced pressure at 20 to 100 ° C., preferably at 30 to 70 ° C., under reduced pressure, and the residue was dried with a vacuum freeze dryer to be soluble in water, lower alcohols, or a mixed solvent thereof. Can.

The non-polar solvent layer obtained by sequentially shaking the aqueous solution obtained by re-dissolving the crude extract of chapar immediately in the water with the same amount of hexane, ethyl acetate and butanol was concentrated under reduced pressure and lyophilized, respectively, to obtain the purified fractions of the present invention, hexane and ethyl. Acetate and butanol soluble fractions can be obtained, and the remaining aqueous layer can be concentrated under reduced pressure and lyophilized to yield a soluble fraction.

The present invention provides a pharmaceutical composition effective for the prevention and treatment of ischemic diseases containing the chapul extract of the present invention obtained as the active ingredient as an active ingredient.

Trypan blue pigment exclusion test using the human liver cancer cell line treated with crude extract of chapul according to the present invention was excellent in improving cell survival, DNA segment analysis test results using human liver cancer cell line treated with crude extract The suicide inhibiting activity was excellent, and MTT assay using human hepatocellular carcinoma cell line treated with Chapul ethyl acetate soluble fraction was effective in improving cell survival.

In addition, administration of the crude extract and ethyl acetate soluble fraction in the animal model that caused the ischemic disease significantly reduced tissue damage.

Tea grass extract of the present invention has little toxicity and side effects, so can be used with confidence even during prolonged use for prophylactic purposes.

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.

Pharmaceutical compositions comprising extracts according to the invention, in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterile injectable solutions, respectively, according to conventional methods. Can be formulated and used. Carriers, excipients and diluents that may be included in the composition comprising the 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, sucrose in the extract. Or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use may include various excipients, such as wetting agents, sweeteners, fragrances, preservatives, etc., in addition to water and liquid paraffin, which are commonly used to include suspensions, solutions, emulsions, and syrups. have. 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.

The compositions of the present invention can be administered via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular.

Preferred dosages of the tea extract of the present invention may 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, but the amount of 0.1 to 1000 mg / kg may be administered once to several times a day. Can be. Therefore, the above dosage does not limit the scope of the present invention in any aspect. Administration may be administered once a day or may be divided several times.

In addition, the present invention provides a health functional food comprising a tea extract crude extract or purified fraction and food acceptable food additives having an activity of preventing and improving ischemic disease.

As defined herein, "health functional food" means a food manufactured and processed using raw materials or ingredients having functional properties useful for the human body according to Act No. 6767 of the Health Functional Food Act, and "functional" means It means ingestion for the purpose of obtaining useful effects on health use such as nutrient control or physiological action on structure and function.

The dietary supplement for the prevention and improvement of the ischemic disease of the present invention, the chaff extract based on the total weight of the composition comprises 0.01 to 95%, preferably 1 to 80% by weight.

In addition, for the purpose of preventing and improving ischemic diseases, it is possible to manufacture and process as a health functional food in the form of pharmaceutical dosage forms such as powders, granules, tablets, capsules, pills, suspensions, emulsions, syrups, or health drinks. .

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

In addition to the above, the composition 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. The compositions of the present invention may also contain pulp 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 generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

In addition, the extract of the present invention may be added to food or beverage for the purpose of preventing the ischemic disease. 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.

Below, The invention is illustrated in detail by the following examples and experimental examples.

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

Example  One. Chapool Crude extract  Produce

After collecting domestically grown tea grass, wash it cleanly and dry it, and put 500 liters of tea grass sample into 5 liters of methanol, and extract it twice by using an ultrasonic extractor (sonicator, 8210R-DTH, Branson, USA) for 24 hours and filter it. , Concentrated under reduced pressure with a vacuum concentrator (NP-1, EYELA, Japan) to obtain 62 g of chapul methanol extract (hereinafter referred to as HY2207).

Example  2. Chapool Purified fraction  Produce

After collecting domestically grown grass, washed with water and dried, 100 g of the extracted by the method of Example 1 using 10 times the number of methanol and filtered with a filter paper (Whatman No. 3) 38 Concentration under reduced pressure at ° C yielded 16 g of crude methanol extract. 16 g of the crude crude methanol suspension was suspended in 200 ml of water, placed in a 1 L fraction filter, and 200 ml of hexane was added thereto. After 24 hours, the hexane layer was recovered, concentrated at 80 ° C., and repeated twice in the same manner. 2.9 g). After 200 hours of ethyl acetate solvent was added to the remaining water suspension in the remaining water suspension, the ethyl acetate layer was recovered and concentrated under reduced pressure at 38 ° C. The ethyl acetate fractions were repeated twice as described above (hereinafter referred to as CM-EA). Business card) 3.5 g was added to the remaining water suspension, and 200 ml of butanol solvent was added to the fractional filter, and after 24 hours, the butanol layer was recovered and concentrated under reduced pressure at 60 ° C .. The same procedure was repeated twice to obtain butanol fraction (hereinafter, CM- 2.6 g, and the remaining water suspension was concentrated under reduced pressure at 50 ° C. to obtain 6.8 g of a water fraction (hereinafter referred to as CM-WA).

Reference Example  1. Preparation of experimental animals

Experimental animals used Sprague-Dawley male rats (Hyochang Science Co., Ltd.) weighing 250-300 g in weight, and constant conditions (temperature: 21 ± 2 ℃, contrast: 12) in Daegu Catholic University medical school. In the time-contrast cycle, free intake of food and water was allowed, and sufficient water and food were provided before the start of the experiment, and the animals were pretreated for 10 minutes before the start of the experiment.

Experimental Example  One. Chapool Crude extract Cell viability  Improve activity measurement ( in vitro )

In order to investigate the effect of the extract of chapul crude extract on the survival of cells in hypoxic and normal oxygen conditions, after adding the medium to which the concentration of HY2207 prepared in Example 1 was added to the cells, the experiment as follows in hypoxic and normal oxygen conditions The degree to which cell survival is improved by performing the following methods is described in Trypan Blue, described in Sambrook J and Russel DW, Molecular Cloning 3rd ed., Vol. 3, A8.6-8.8, Cold Spring Harbor Laboratory Press, New York, 2001. It was measured by modifying trypan blue dye exclusion.

HepG2 (ATCC HB 8065, USA), a human hepatoma cell line, contains penicillin G sodium (100 Units / L, Invitrogen, USA) and streptomycin sulfate (100 mg / L). 12 well-plate using Invitrogen, USA) and Eagle's minimum essential midium (EMEM, Invitrogen, USA) added with 10% fetal bovine serum (Invitrogen. USA) as serum. ) Was incubated in 2 × 10 ⁵ cell number / 800 μl medium. After incubating the cells for 48 hours at 37 ° C. and 5% CO ₂ -95% atmosphere and replacing with the same fresh culture medium, HY2207 of Example 1 was dissolved in 50% ethanol to 100 μg / ml, 1000 μg / The experimental group added to the culture medium at the concentration of ㎖ and the negative control group (negative control) without any addition were incubated for 2 days at low oxygen (oxygen concentration 3%) and normal oxygen conditions using the following trypan blue staining exclusion method The cell number was measured. The medium was removed, washed once with phosphate buffered saline (PBS) and trypsin treatment. Centrifuge to collect the cells, add fresh medium to form a cell suspension, and then mix 1: 1 with 0.4% trypan blue solution (Invitrogen, USA) and dye the cells in blue light after 5 minutes. The cells which were not stained with the dead cells were regarded as living cells and the number of cells was measured by using a hemocytometer.

As shown in FIG. 1A, in the hypoxic condition, almost all cells die in the negative control group (Control) after one day of culture (Ratio = 0), whereas the HY2207 of the present invention survives cells in a concentration range of 100-1000 µg / ml. (Ratio> 0), as shown in Figure 1b, it was confirmed that under normal oxygen conditions HY2207 slightly inhibited the survival of cells in the concentration range of 100-1000 ㎍ / ㎖. Accordingly, it was confirmed that the HY2207 of the present invention effectively improves cell survival under hypoxic conditions, and does not inhibit cell growth under normal oxygen conditions and at the same time concentrations between 100 and 1000 ㎍ / ml It was confirmed that it should be administered.

Experimental Example  2. Chapool Crude extract Under hypoxic conditions  Apoptosis Inhibition Activity Measurement in vitro )

DNA fragmentation assay; Yoshida A et al., In Apoptosis : A practical approach (Studzinski GP (Ed.)), Pp47 to determine the mechanism by which HY2207 helps cell survival in hypoxic conditions. -48, Oxford University Press, New York, 1999).

HepG2 (ATCC HB 8065, USA), a human hepatoma cell line, contains penicillin G sodium (100 Units / L, Invitrogen, USA) and streptomycin sulfate (100 mg / L). Invitrogen, USA) and 1 × in a 60 mm dish using Eagle's minimum essential midium (EMEM, Invitrogen, USA) added with 10% fetal bovine serum (Invitrogen. USA) as serum. The cells were cultured in 10 ⁶ cell numbers / 4 ml medium. The cells were incubated for 48 hours at 37 ° C. and 5% CO ₂ -95% atmosphere, and then replaced with fresh culture medium. The HY2207 of Example 1 was dissolved in 50% ethanol and incubated at a concentration of 300 μg / ml. DNA fragmentation assay was performed by incubating the experimental group added to the medium and the negative control group without adding the medium for 2 days under hypoxic and normal oxygen conditions to investigate the appearance of the DNA ladder. As a result, the experimental group (HY2207) to which the HY2207 was added at 300 µg / ml improved the survival of cells as in Experimental Example 1 as compared to the control group (HY2207) without addition (see FIG. 2A), and also added HY2207. The experimental group (see FIG. 2C) delayed the appearance time of the DNA ladder than the control group (see FIG. 2B). Therefore, it was confirmed that HY2207 has an effect of suppressing apoptosis and improving cell survival.

Experimental Example  3. Chapool Purified fraction Cell viability  Improve activity measurement ( in vitro )

MTT assays described in the literature (Hoffman RM, In Cell Biology (Celis JE (Ed.)), Vol. 1, pp369-370, Academic Press, New York to confirm the activity of fractions purified from crude extracts of Chapul) , 1994) was added to the human liver cancer cell line HepG2 cell line was added to the medium containing fractions of varying concentrations, and the degree of improving cell viability was examined by performing the experiment as described below in hypoxic conditions.

HepG2 (ATCC HB 8065, USA), a human hepatoma cell line, contains penicillin G sodium (100 Units / L, Invitrogen, USA) and streptomycin sulfate (100 mg / L). Invitrogen, USA) and 12-well plate (12 well-) using Eagle's minimum essential midium (EMEM, Invitrogen, USA) added with 10% fetal bovine serum (Invitrogen. USA) as serum. plate) was incubated in 2 × 10 ⁵ cell number / 800 μl medium. After incubating the cells for 48 hours at 37 ° C. and 5% CO ₂ -95% atmosphere, the chapul purified fractions of Example 2 were purified using dimethyl sulfoxide (DMSO; for ethyl acetate fraction) or 50% ethanol (water Soluble fractions) were added to the culture medium at concentrations of 10, 100 and 1000 μg / ml, the negative control group to which nothing was added and G418 10 μg /, which was already found to improve cell viability. MTT assay was performed while the positive control (ml) was incubated for 2 days under hypoxic conditions.

As a result of the above experiment, as shown in FIG. 3, under hypoxia (hypoxia), HY2207 (WH) and CM-HX (HX) showed an effect of improving cell survival at a concentration of 1000 μg / ml, and CM-BU Did not improve cell survival even at a concentration of 1000 μg / ml, whereas in CM-EA (EA), the effect of improving cell survival was observed not only at 1000 μg / ml but also at 100 μg / ml. Therefore, it was confirmed that the active ingredient related to cell survival is selectively concentrated in CM-EA.

Experimental Example  4. Intraperitoneal injection, Chapool Crude extract  And Ethyl acetate soluble Fraction  Effect test on cerebral infarction ( in vivo )

In order to verify the therapeutic effect of HY2207 and CM-EA on cerebral infarction, the experiment described in (Han HS et al., J. Neurosci ., 22, pp3921-3928, 2002) was modified to rat cerebral infarction of Reference Example 1 above. Animal models were performed as shown below.

 4-1. Chapool Crude extract  Effect Test on Cerebral Infarction

Rats of Reference Example 1 were inhaled with enflurane (enflurane, Sino-Pharmaceutical Co., Korea), and then the blood pressure measurement and blood sampling paths were secured in the lower extremity. Ligation and 3-0 nylon sutures were inserted into the internal carotid artery to block the middle cerebral artery to create a cerebral infarction model.

HY2207 of Example 1 was injected into this cerebral infarction model to investigate the area of necrotic sites. At this time, in order to determine the appropriate dose which is a safe amount and a dose that does not exhibit toxicity, a method of raising the step amount by taking the minimum amount as a starting point based on the result of Experimental Example 1 was used. To this end, the dose increase was adopted by a fold increase method, starting with 50 mg / kg and increasing to 100 and 200 mg / kg.

HY2207 was intraperitoneally injected 200 mg / kg (1 ml) 20 hours and 1 hour before the middle cerebral artery was blocked to induce ischemia, and after 2 hours of ischemia induction, the suture was removed and the animals were recovered. . After 22 hours, the animals were euthanized, brain tissues were extracted, stained in TTC (2,3,5-triphenyltetrazolium chloride, Sigma, USA) solution, and the degree of damage to the hemispheres was analyzed by image analysis system (Qunatity One). 4.0, Biorad, USA) to calculate the ischemic index (ischemic index) in the same manner as in the following equation 1 to compare the effect of the drug.

Ischemic index (%) = A / B × 100

A: volume of the injury site in the hemisphere of the brain (mm ³ ),

B: total volume of brain hemispheres (mm ³ ).

As a result of the experiment, rats not administered HY2207 (8 rats, control group) had an ischemic index of 93%, whereas rats (8 rats, experimental group, HY2207) administered 62% had a volume of necrosis area. Was reduced by about 33% (p <0.05), indicating that HY2207 has a significant effect in the cerebral infarction model (see FIG. 4A). This phenomenon was confirmed as a result of the TTC staining, the tissue damage was significantly reduced in the experimental group (see Figure 4c) compared to the control (see Figure 4b).

 4-2. Chapool Ethyl acetate soluble Fraction  Effect Test on Cerebral Infarction

The area of the necrotic site was measured by injecting CM-EA, which was found to contain a large amount of the active substance in Example 3, to a cerebral infarction model made by the same method as Experimental Example 4-1.

CM-EA was intraperitoneally injected 5, 10 and 20 mg / kg (1 ml), respectively, 20 hours and 1 hour before the middle cerebral artery was blocked, and sutured for 2 hours after ischemia induction. Removed and the animals were recovered. After 22 hours, the animals were euthanized, brain tissues were extracted, TTC staining was performed, and the ischemic index was measured in the same manner as in Experimental Example 4-1.

As a result of the experiment, rats not injected with CM-EA (8 rats, control group, 0 mg / kg) had an ischemic index of 93%, compared with 5, 10 and 20 mg / kg of CM-EA, respectively. Rats (4, 8, 8; 5 mg / kg, 10 mg / kg, 20 mg / kg) had ischemic indices of 70.8, 61.4, and 50.8%, respectively, and necrotic areas were 23.9, 34.0, and 45.4% (p <0.05, respectively). ) Decreased (see FIG. 4D). From this, HY2207 showed a significant effect on the cerebral infarction model at a concentration of about 200 mg / kg, while CM-EA, an ethyl acetate soluble fraction of HY2207, was found to have a significant effect at a concentration of 10 mg / kg or more.

Experimental Example  5. Intraperitoneal injection, Chapool Crude extract Ischemic premature failure  For effect test ( in vivo )

In order to verify the therapeutic effect of Chapul crude crude extract on ischemic acute renal failure, Wang J et al., J. Biol . Chem . , 279 (19), pp19948-19954, 2004. The rat ischemic nephrotic insufficiency model was made of 1 rat and animal experiment was performed as follows.

Rats of Reference Example 1 were anesthetized with 50 mg / kg ketamine (ketamine, Yuhan, Korea) and 20 mg / kg xyazine (xylazine, Sigma, USA) 30 minutes before surgery, followed by an abdominal incision. . The renal artery and renal vein of the left kidney were clamped to block the flow of blood, and then the right kidney was extracted to create a model of ischemic renal failure.

Inject the left renal artery into the abdominal cavity with HY2207 400 mg / kg (1 ml) dissolved in 0.9% saline one hour before inducing ischemia, and leave the ischemic state for 45 minutes after ischemic induction. Recovered. After 24 hours, blood was collected from experimental animals, and the concentration of creatinine (mg / dl) in the blood was measured.

As a result of the experiment, the concentration of creatinine in rats (4 mice, control group) without injection of HY2207 was 3.9 mg / dl, whereas the concentration of injected rats (6 mice, experimental group, HY2207) was 1.5 mg / dl. The creatinine concentration was reduced by 62% (p <0.05), suggesting that HY2207 inhibits kidney damage, thereby facilitating the release of creatine through the kidneys. It was confirmed that the effect (see Fig. 5).

Experimental Example  6. Toxicity test

The change was observed by injecting HY2207 of Example 1 into 5 Sprague-Dawley male rats of 320 ± 20 g.

24 hours after HY2207 (500 mg / kg) was injected intraperitoneally, weight was not significant. There was also no change in behavior.

It describes an example of the formulation of the pharmaceutical composition containing the extract tea extract of the present invention, the present invention is not intended to limit this but only to explain in detail.

Formulation example  One. Powder  Produce

300 mg of HY2207 in Example 1 above

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 Tablets

50 mg of HY2207 of Example 1

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

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

Formulation example  3. Manufacture of capsule

50 mg of HY2207 of Example 1

Corn starch 100 mg

Lactose 100 mg

2 mg magnesium stearate

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

Formulation example  4. Preparation of Injectables

50 mg of HY2207 of Example 1

Appropriate sterile distilled water for injection

pH adjuster

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

Formulation example  5. Liquid  Produce

100 mg of HY2207 of Example 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 and dissolved in purified water, lemon flavor is added to the mixture, and then the above ingredients are mixed, purified water is added to adjust the total amount to 100 ml, and then filled in a brown bottle. The solution is prepared by sterilization.

Formulation example  6. Manufacture of healthy food

HY2207 1000 mg of Example 1

Vitamin Mixture

70 μg of Vitamin A Acetate

Vitamin E 1.0 mg

Vitamin B1 0.13 mg

Vitamin B2 0.15 mg

Vitamin B6 0.5 mg

0.2 μg of vitamin B12

Vitamin C 10 mg

10 μg biotin

Nicotinic Acid 1.7 mg

Folate 50 ㎍

Calcium Pantothenate 0.5mg

Mineral mixture

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 mixture is a composition which is relatively suitable for healthy foods in the preferred embodiment 1, the compounding ratio may be arbitrarily modified, and the above-mentioned ingredients may be mixed according to a conventional health food manufacturing method. Next, the granules may be prepared and used for preparing a health food composition according to a conventional method.

Formulation example  7. Manufacture of health drinks

HY2207 1000 mg of Example 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 a conventional healthy beverage production 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 then refrigerated and stored in the present invention For the preparation of healthy beverage compositions.

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

The tea extract of the present invention, when administered to an ischemic animal model, not only inhibits apoptosis and reduces infarcts of tissues, but also can be used in medicines and health functional foods for the treatment of ischemic diseases because there are no side effects even when taken for a long time. .

Claims (10)

Cassia mimosoides var. delete delete delete delete The pharmaceutical composition of claim 1, wherein the crude extract is extracted in water, ethanol, methanol, or a mixed solvent thereof. delete delete (Cassia mimosoides var. Nomame MAKINO) A dietary supplement for the prevention and improvement of cerebral infarction or ischemic acute renal failure containing crude extract or ethyl acetate soluble fraction of the crude extract as an active ingredient. delete

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