KR20060080874A - Composition comprising an extract of paeoniae radix alba for the prevention and treatment of ischemic diseases - Google Patents

Abstract

The present invention relates to a composition containing an extract of Paeoniae Radix Alba having a cell viability improving activity under hypoxic conditions, and the extract of the present invention exhibits potent inhibitory activity against apoptosis in ischemia models. , The composition may be usefully used as a pharmaceutical composition and health functional food for the prevention and treatment of ischemic diseases.

Earl pill, apoptosis, ischemic disease, pharmaceutical composition, dietary supplement              

Description

Composition comprising an extract of Paeoniae Radix Alba 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 crude extract of different concentrations in culture medium under hypoxic conditions,

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

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

3 is an MTT assay showing the survival of HepG2 cells according to the addition of hexane soluble fraction, ethyl acetate soluble fraction, butanol soluble fraction, and water soluble fraction of Baekjak at different concentrations in culture medium under hypoxic conditions,

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

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

4C is a result of TTC staining of the myocardium of the myocardial infarction model intraperitoneally injected with the crude extract extract,

5 is a graph showing the infarct inhibitory effect of the crude extract extract orally administered to myocardial infarction animal model,

Figure 6a is a diagram comparing the infarct inhibitory effect of the crude extract extract orally administered to cerebral infarction animal model,

Figure 6b is the result of TTC staining by extracting the brain of the cerebral infarction model,

Figure 6c is the result of TTC staining by extracting the brain of the cerebral infarction model orally administered the crude extract crude extract,

Figure 7a is a diagram showing the renal damage inhibitory effect of the crude extract extract intraperitoneal injection in ischemic acute renal failure animal model in blood creatinine concentration test,

Figure 7b is a diagram showing the effect of inhibiting the kidney damage of the ethanol Ethyl acetate soluble fraction in the peritoneal injection in the 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 the extract.

Cerebral infarction and myocardial infarction, which are typical ischemic diseases, are blocked due to hypertension, hyperlipidemia, diabetes, smoking, and atherosclerosis, in which blood vessels are narrowed. 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 repermit the 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 a decrease in adenosine triphosphate (ATP) and edema in neurons. It is known to cause suicide. Excitatory neuronal suicide mechanism by which ischemia causes excessive glutamate to accumulate outside the cell due to 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 cell survival under 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, aminoglycoside-based G418 (Genetisin) has been shown to have a myocardial infarction effect. (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 which show 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.

Peony (Paeoniaceae) is a perennial plant of the genus Amphitaceae , Peony , Paeonia, Paeoniae Radix Alba and Paeoniae Radix Rubra. Count and red peony are determined by the presence or absence of a peel, and the one with the peel is called the peony and the peeled peel is called peony ( Altern . Med . Rev., 6 (5), pp 495-499, 2001). In addition to antipyretics, earl pill has the effects of contraction inhibitory effect on gastric and intestinal smooth muscle and uterine smooth muscle, prevention of atherosclerosis, blood pressure lowering and blood flow improvement for vascular disease. These effects of peony extracts were found to have antioxidant effects (Ohsugi M et al., J. Ethnopharmacol . , 67, pp 111-119, 1999), platelet aggregation inhibitory effects (Lin HC et al., Planta Med , 65, pp 595-599, 1999). ), Antithrombotic effect (Ishida H et al., Chem . Pharm . Bull , 35 (2), pp849-852, 1987), antihyperlipidemia (Yang HO et al., Fitoterapia , 75 (1), pp45-49, 2004) and vascular endothelial protection from hyperlipidemia (Goto H et al., Phytother . Res , 13 (6), pp526-528, 1999). In addition, glycoside, which is a component of the peony, has been reported to be effective in the treatment of cerebral infarction (Yang J et al., Zhong Yao Cai , 23 (2), pp95-97, 2000), which appears to be due to the protective effect of peoniflorin, a constituent of the peony, on neurons (Zhang GQ et al., Acta Pharmacol . Sin. , 24 (12), pp 1248-1252, 2003).

Peony patents include 8 kinds of herbal medicine patents, including antipyretics for use in preventing angina by reducing hyperlipidemia (US Pat. No. 6413554). Peonyflorin, the main ingredient of peony, is used to treat arteriosclerosis and myocardial infarction. Patents (Japanese Patent No. 63030415) and the like for a technology for producing an antioxidant for the following. However, there is no teaching or description in any of the above documents regarding the use of the currant extract of the present invention as a main ingredient for the treatment and prevention of ischemic diseases.

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

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

In order to accomplish the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of ischemic diseases containing Paeoniae Radix Alba crude extract or tablet fraction 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 organ is characterized in that it comprises internal organs such as kidney, liver, pancreas, lung or heart.

The extract of the present invention includes the extract extracted from the leaf portion, flower portion, stem portion or root portion, preferably the root portion, and the purified purified fraction of the white peony.

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

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

Crude crude extract of the present invention, after drying, C ₁ to C ₄ lower alcohol, such as water, methanol, ethanol, or the like, about 1 to 15 times the volume, preferably about 5 to 10 times the weight of the finely divided Root root, or A mixed solvent having a mixing ratio of about 1: 0.1 to 1:10, preferably water, is added at an extraction temperature of 20 to 100 ° C, preferably 50 to 100 ° C, for about 0.5 to 2 days, preferably 1 hour to Cold extraction, hot water extraction, ultrasonic extraction, reflux cooling extraction, extractor bath extraction for 1 day is an extraction method, preferably extracting by repeated extraction 1 to 12 times, preferably 3 to 4 times by a bath extractor and then filtered. The filtered extract was concentrated under reduced pressure at 20 to 100 ° C., preferably at 40 to 70 ° C. with a vacuum rotary concentrator, and the extracted residue was dried with a vacuum freeze dryer to be used in water, lower alcohols or mixed solvents thereof. Drug crude extract can be obtained.

In the case of the crude extract, the water extract immediately before the concentration step, the concentrated liquid obtained by depressurization to 1/5 to 1/20 volume, preferably 1/10 volume, or concentrated in the case of methanol extract completely concentrated under reduced pressure and then again in water The solvent layer obtained by successively shaking the dissolved aqueous solution with the same amount of hexane, ethyl acetate and butanol was concentrated under reduced pressure to obtain hexanoic acid, ethyl acetate and butanol soluble fraction, and the remaining aqueous layer was concentrated under reduced pressure and lyophilized. Yong fractions can be obtained.

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

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

Crude crude extract and ethyl acetate soluble fraction were also administered to animal models that caused ischemic disease, resulting in an excellent reduction in tissue damage.

Earl extract of the present invention has little toxicity and side effects, so can be used with confidence even for long-term use for the purpose of prevention.

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 and sucrose in the extract. Or lactose, gelatin, carboxymethyl cellulose 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 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 dietary supplement comprising a crude extract or tablet fraction and a food acceptable food supplement additive having a prophylactic and ameliorating activity for 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.

Health functional foods for the prevention and improvement of ischemic diseases of the present invention, the total weight of the composition comprises the extract of Baekjak 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 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 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 proportion of 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 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, 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 beverages 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.

The present invention will be described in more detail based on the following examples and experimental examples, but the present invention is not limited thereto.

Example  1. Earl Crude extract  Produce

Wash the roots with freshly harvested Baekjak as a sample, and add 2 liters of water to Baekak 200g according to the conventional Botanical Extraction Method, and extract it over 2 hours in an electric bath (Daewoongdeaktang DWP-2000, Daewoong) for 2 hours. It was filtered with a filter paper (Whatman No. 3). Two liters of the extract obtained by filtration were lyophilized to obtain 30 g of the white water extract (hereinafter referred to as HY6081).

Example  2. Earl Purified fraction  Produce

1.4 ㎏ of Root of Peony purchased in the market was washed with water, dried, leached with methanol for 24 hours, extracted twice, filtered with filter paper (Whatman No. 3), and the crude extract was concentrated under reduced pressure at 38 ° C. using a vacuum condenser. 240 g of methanol was obtained. 210 g of the crude crude methanol suspension was suspended in 1 L of water, added to the 3 L fraction filter, and 1 L of hexane was added thereto. After 24 hours, the hexane layer was recovered and concentrated at 80 ° C .. The hexane fraction was repeated twice in the same manner. 10 mg was obtained (hereinafter named PRA-He). Ethyl acetate solvent was added to the fractional filter in the remaining water suspension, and after 24 hours, the ethyl acetate layer was recovered and concentrated under reduced pressure at 38 ° C. The ethyl acetate fraction was repeated twice as described above (hereinafter referred to as PRA-EA). ) 42 g of ethanol was added to the remaining water suspension and 1 liter of butanol solvent was added to the fractional filter. 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 referred to as PRA-BU). 65 g was obtained, and the remaining water suspension was concentrated under reduced pressure at 50 ° C. to obtain 91 g of water fraction (hereinafter referred to as PRA-WA).

Reference Example  1. Preparation of experimental animals

The experimental animals were Sprague-Dawley male rats (Hyochang Science Co., Ltd.) weighing 250-300 g, and were subjected to constant conditions (temperature: 21 ± 2 ° C, 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  1. Earl Crude extract Cell viability  Improve activity measurement ( in vitro )

In order to investigate the effect of the crude extract extract on the survival of cells in hypoxic and normal oxygen conditions, the medium was added to the cells by the concentration of HY6081 prepared in Example 1, and then experimented in the hypoxic and normal oxygen conditions as follows The degree to which cell viability is improved by performing the following methods is described in Trypan Blue as 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, HY6081 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 HY6081 of the present invention survives cells in a concentration range of 100-1000 µg / ml. (Ratio> 0), and as shown in FIG. 1B, it was confirmed that under normal oxygen conditions, HY6081 slightly inhibited cell survival at a concentration of 100 μg / ml or more. Accordingly, it was confirmed that the HY6081 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 administered at a concentration of about 100 ㎍ / ml in order to show the effect under hypoxic conditions. It should be confirmed.

Experimental Example  2. Earl 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 HY6081 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 serum 1 × in a 60 mm dish using Eagle's minimum essential midium (EMEM, Invitrogen, USA) added with 10% fetal bovine serum (Invitrogen. USA). 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. Then, HY6081 of Example 1 was dissolved in 50% ethanol and incubated at a concentration of 400 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 survival of cells was improved as in Experimental Example 1 as compared to the control group (HY6081) to which HY6081 was added (see FIG. 2A), and also to the experimental group to which HY6081 was added (see FIG. 2C). ) Delayed the time of appearance of the DNA ladder than the control (see Figure 2b). Therefore, it was confirmed that HY6081 has an effect of inhibiting apoptosis and improving cell survival.

Experimental Example  3. Earl 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, 1994 to confirm the activity of fractions purified from HY6081. ) Was added to the human hepatocarcinoma cell line HepG2 cell line, and the medium containing the fractions of different concentrations was examined. The degree of improvement of cell viability was investigated by performing the experiment as described below under 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 Earl tablet fractions of Example 2 were treated with dimethyl sulfoxide (DMSO; for ethyl acetate fraction) or 50% ethanol (water 10 g of G418, which was dissolved in the soluble fractions) and added to the culture medium at concentrations of 100 μg / ml and 1000 μg / ml, negative control with nothing added and already improved cell viability. MTT assay was performed while the positive control added with / ml was incubated for 2 days under hypoxic and normal oxygen conditions.

As a result of performing the experiment, as shown in Figure 3, the effect of improving the survival of cells in the concentration range of 10-1000 ㎍ / ㎖ PRA-WA and PRA-BU of Example 2 under hypoxia (hypoxia) is not observed Did. On the other hand, PRA-He was observed to improve the survival of cells at a concentration of 1000 μg / ml. However, in the case of PRA-EA, the effect of improving cell viability was observed not only at 1000 ㎍ / ml but also at 100 ㎍ / ml. Therefore, it was confirmed that cell-survival-related active ingredients were selectively concentrated in PRA-EA.

Experimental Example  4. Abdominal injection, Count Crude extract  Effect Test on Myocardial Infarction in vivo )

In order to verify the therapeutic effect of HY6081 on myocardial infarction, the experiment described in (Haisong J et al., Circulation , 97, pp892-899, 1998) was modified and the myocardial infarction model was used as the rat of Reference Example 1 as follows. The effect of HY6081 intraperitoneal injection was measured.

Rats of Reference Example 1 were administered with 10 mg / kg ketamine (ketamine, Yuhan, Korea) and 5 mg / kg xyazine (xylazine, Sigma, USA) to induce anesthesia, intubate the airway, and perform general anesthesia. . Then, the third and fourth ribs were excised to open the chest and draw the heart from the inside of the intercostal space. Myocardial infarction was created by tying the left coronary artery with 5-0 prolene sutures, relocating the heart into the chest, and suturing the subcutaneous tissue and skin.

The HY6081 of Example 1 was injected into the myocardial infarction model to investigate the area of necrosis. 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.

One hour prior to tying the left coronary artery to induce ischemia, 1 ml of 400 mg / kg HY6081 was injected into the abdominal cavity, allowed to stand in ischemic state for 3 days, and then the myocardium was extracted and TTC (2,3,5- triphenyltetrazolium chloride, Sigma, USA) and staining the solution, the degree of damage was measured by the image analysis system (Qunatity One 4.0, Biorad, USA) to calculate the ischemic index (Ischemic index) in the same manner as in the following formula (1) The effect of was compared.

Ischemic index (%) = A / B × 100

A: volume of injury site of the heart (mm3),

B: total volume of the heart (mm 3).

As a result of the experiment, rats not injected with HY6081 (6 rats, control group) had an ischemic index of 4.9%, whereas rats injected with rats (7 rats, experimental group, HY6081) were only 2.2%. The necrotic site volume was reduced by 55% (p <0.01), indicating that HY6081 had an excellent effect in the myocardial infarction model (see FIG. 4A). This phenomenon was also confirmed by TTC staining significantly reduced tissue damage in the experimental group (see Figure 4c) compared to the control (see Figure 4b).

Experimental Example  5. When administered orally, Count Crude extract  Effect Test on Myocardial Infarction in vivo )

In order to verify the therapeutic effect of HY6081 on myocardial infarction, the experiment described in Lee BH et al., Drug Develop. Res. 54, pp182-190, 2001 was modified to create a myocardial infarction model as in Experimental Example 4. Upon oral administration of HY6081, the effect was measured.

For 3 days, rats of Reference Example 1 were fed with 400 mg / kg of HY6081 per day in the form of powder, and then fed the left coronary artery to induce ischemia, and left for 30 minutes in ischemic state. The tied artery was released and left to reperfusion state. Subsequently, 1% Evans Blue dye (Sigma, USA) was administered intravenously, myocardium was extracted, and measured by an image analysis system to calculate the ratio of the non-blooded area to the entire left ventricle to calculate the AAR (area at risk). Thereafter, the myocardium was immediately stained in a TTC solution and measured by an image analysis system to calculate an infarcted area (IA) by calculating the ratio of the damaged area to the entire left ventricle. Using this method, the ischemic index was calculated in the same manner as in Equation 2 to compare the effects of drugs.

Ischemic index (%) = IA / AAR × 100

IA:% of injured area relative to the total left ventricle

AAR: Percentage of non-blood supply to total left ventricle

As a result of the experiment, rats not administered HY6081 (4 rats, control group) had an ischemic index of 53.1%, whereas rats (5 rats, experimental group, HY6081) received only 30.9%. The necrotic site volume was reduced by 42% (p <0.05), indicating that HY6081 had an excellent effect on myocardial infarction (see FIG. 5).

Experimental Example  6. When administered orally, Earl Count Crude extract  Effect test on cerebral infarction ( in vivo )

In order to verify the therapeutic effect of HY6081 on cerebral infarction (Han HS et al., J. Neurosci ., 22, pp3921-3928, 2002) by modifying the experiment described in the rat of the reference example 1 to make an infarction model Animal experiments were performed as follows.

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. HY6081 of Example 1 was orally administered every day for 7 days for 7 days before surgery to block the middle cerebral artery and 24 hours before surgery, and left to ischemic state for 2 hours after ischemia induction. The sutures were then removed and the animals were restored. After 22 hours, the animals were euthanized, brain tissues were extracted, stained in TTC solution, and the degree of damage to the hemispheres was measured by an image analysis system. Calculation was compared to compare the effects of the drugs.

Ischemic index (%) = A / B × 100

A: the volume of the injury site in the hemisphere of the brain (mm3),

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

As a result of the experiment, rats that did not receive HY6081 (12 rats, control group) had an ischemic index of 93%, whereas rats (4 rats, experimental group, HY6081) who received rats had 32% volume of necrotic sites. Was reduced by about 65% (p <0.05), indicating that HY6081 has a significant effect in the cerebral infarction model (see FIG. 6A). This phenomenon was confirmed as a result of the TTC staining significantly reduced tissue damage in the experimental group (see Figure 6c) compared to the control (see Figure 6b).

Experimental Example  7. Abdominal injection, Count Crude extract  And Ethyl acetate soluble Fraction Ischemic premature failure  For effect test ( in vivo )

In order to verify the therapeutic effect of HY6081 and PRA-EA on ischemic aplastic nephropathy, the experiment described in Wang J et al., J. Biol . Chem . , 279 (19), pp19948-19954, 2004 was modified. Rats of Reference Example 1 were made with ischemic nephrotic insufficiency model and animal experiments were performed as follows.

7-1. Earl Crude extract Ischemic premature failure  For effect test

Rats of Reference Example 1 were anesthetized with 50 mg / kg ketamine and 20 mg / kg xyazine 30 minutes before surgery, and then the abdominal cavity was dissected. 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.

One hour before the left renal artery was picked up to induce ischemia, HY6081 400 mg / kg (1 ml) dissolved in 0.9% saline was injected into the abdominal cavity. 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 HY6081 was 3.5 mg / dl, whereas the concentration of injected rats (6 mice, HY6081) was 1.9 mg / dl. The creatinine concentration was reduced by 44% (p <0.05), suggesting that HY6081 inhibits kidney damage, thereby facilitating the release of creatine through the kidneys. It was confirmed that the effect (see Fig. 7a).

7-2. Earl Ethyl acetate soluble Fraction Ischemic premature failure  For effect test

Ethyl acetate soluble fraction (PRA-EA) of Baekjak, which was found to contain a large amount of the active substance in Experimental Example 3, was injected into an ischemic stellar renal failure model made in the same manner as in Experimental Example 7-1 to measure blood creatinine concentration.

One hour before the left renal artery was picked up to induce ischemia, 100 mg / kg (1 ml) of PRA-EA dissolved in 0.3% carboxymethylcellulose (CMC) was injected into the abdominal cavity and left for 45 minutes. The clamps were then released to recover the animals. 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, rats not injected with PRA-EA (23 rats, control group) had a creatinine concentration of 4.2 mg / dL, whereas the rats (52 rats, experimental group, PRA-EA) were injected. 1.9 mg / dL decreased creatinine concentration by 55% (p <0.05), indicating that PRA-EA inhibits kidney damage, thereby facilitating the release of creatine through the kidneys. -EA also showed a significant effect in the ischemic nephropathy model (see Figure 7b).

Experimental Example  8. Toxicity Test

HY6081 of Example 1 was injected into 300 ± 20 g of Sprague-Dawley male rats to observe changes.

24 hours after HY6081 (2 g / kg) was injected into the abdominal cavity, the weight was not significant. There was also no change in behavioral behavior and no revenge when incised.

The preparation example of the pharmaceutical composition containing Baekjak extract of the present invention will be described, but the present invention is not intended to limit this, but is intended to explain in detail only.

Formulation example  One. Powder  Produce

300 mg of HY6081 of Example 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 Tablets

50 mg of HY6081 of Example 1, above

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. Manufacture of capsule

50 mg of HY6081 of Example 1, above

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 HY6081 of Example 1, above

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 HY6081 of Example 1

10 g of isomerized sugar

5 g of mannitol

Purified water

After dissolving each component in purified water according to the usual method of preparing a liquid solution, adding lemon flavor appropriately, mixing the above components, adding purified water, adjusting the whole to 100 ml by adding purified water, and then filling into a brown bottle. The solution is prepared by sterilization.

Formulation example  6. Manufacture of healthy food

HY6081 1000 mg of Example 1

Vitamin mixture proper amount

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 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. Manufacture of health drinks

HY6081 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 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 since there are no side effects even when taken for a long time. .

Claims (11)

Paeoniae Radix Alba 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 pharmaceutical composition according to claim 1, wherein the ischemic disease is myocardial infarction, cerebral infarction, ischemic acute renal failure, ischemic acute liver failure, diabetic foot ulcer, diabetic nephropathy, ischemic disease or organ tissue damage due to surgical side effects. The pharmaceutical composition according to claim 2, wherein the ischemic disease caused by the surgical side effect is ischemic heart failure, ischemic renal failure, ischemic liver failure or ischemic stroke pharmaceutical composition. 3. The pharmaceutical composition according to claim 2, wherein the organ tissue damage is due to long-term surgery or transplantation with reperfusion after ischemia, and reattachment of the traumatic amputated limb. The pharmaceutical composition according to claim 4, wherein the organ is an internal organ of the kidney, liver, pancreas, lung, or heart. The pharmaceutical composition according to claim 1, wherein the crude extract and the purified fraction are extracted or fractionated from the leaf portion, flower portion, stem portion or root portion of the white peony. The pharmaceutical composition of claim 1, wherein the crude extract is extracted from C ₁ to C ₄ lower alcohols such as water, methanol, and ethanol, or a mixed solvent thereof. 2. The pharmaceutical composition according to claim 1, wherein the purified fraction is sequentially dissolved in hexane, ethyl acetate or butanol after dissolving the crude extract from water. The pharmaceutical composition according to claim 8, wherein the purified fraction is an ethyl acetate soluble fraction. A dietary supplement comprising the earl extract or tablet fraction of claim 1, which has a prophylactic and ameliorating activity for ischemic diseases, and a food acceptable additive as an active ingredient. The health functional food according to claim 10, which is in the form of powder, granule, tablet, pill, capsule, liquid or health drink.

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