KR100643191B1 - Compositions comprising Rhei Rhizoma, Scutellaria baicalensis, and Coptis chinensis for inhibition of reendothelialization and prevention and treatment of cardio vascular diseases - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for inhibiting novel vascular restenosis and preventing and treating cardiovascular diseases from natural substances, and more particularly, to inhibit vascular restenosis containing methanol extract of rhubarb, gold and rhubarb as an active ingredient. Pharmaceutical composition for the prevention or treatment of cardiovascular diseases or pharmaceutical composition for the prevention of vascular restenosis and prevention or treatment of cardiovascular diseases containing Baicalein, Berberine or Emodin as an active ingredient will be.

The composition of the present invention exhibits an inhibitory effect on intimal thickening in the accelerated atherosclerosis model, and inhibits proliferation and migration activity of vascular smooth muscle cells. It may be usefully used as a pharmaceutical and health functional food for inhibiting vascular restenosis and preventing or treating cardiovascular diseases.

Rhubarb, gold, barberry, vascular restenosis inhibitor, cardiovascular disease preventive

Description

Compositions comprising Rhei Rhizoma, Scutellaria baicalensis, and Coptis chinensis for inhibition of reendothelialization and prevention and treatment of cardio vascular diseases}             

1 is a graph showing the body weight change ( A ) and food intake ( B ) of animals after administration of the control group, simvastatin (SV), rhubarb, gold and barberry methanol extract (SST),

Figure 2a is a graph of the neointimal thickening inhibitory ability through the control group, simvastatin (SV), rhubarb, gold and rhubarb methanol extract (SST) after the intima area ( A ), intima / media area ( B ) and stenosis ratio ( C ) ego,

Figure 2b is an arterial angiogram of Figure 2,

Figure 3 shows the vascular smooth muscle cells of simvastatin (SV; ◆), rhubarb, gold and rhubarb methanol extract (SSV; ■), Baikal (Bai; ＊), berberine (Ber; ▲), emodine (Emo; A7r5 cells) is a graph showing the inhibition of proliferation,

Figure 4 is a graph measuring the cell cycle inhibition of vascular smooth muscle cells (A7r5 cells) of rhubarb, gold and rhubarb methanol extract by flow cytometry,

5 is a Western blot photograph showing the inhibitory effect of cyclin B1 and β-actin protein expression on vascular smooth muscle cells (A7r5 cells) of rhubarb, golden and rhubarb methanol extracts and baikal, berberine, and emodin,

Figure 6a is a graph showing the inhibition rate of vascular smooth muscle cells (A7r5 cells) of rhubarb, gold and rhubarb methanol extract by the Transwell Chamber method,

FIG. 6B is a micrograph of FIG. 6A;

Figure 7 is a RT-PCR result showing the inhibition of MMP-2 and MMP-9 gene expression of vascular smooth muscle cells of rhubarb, gold and rhubarb methanol extract and Baikallein, berberine, emodin.

The present invention relates to a pharmaceutical composition for inhibiting vascular restenosis and preventing or treating cardiovascular diseases containing rhubarb, golden and rhubarb extract or Baicalein, Berberine or Emodin as an active ingredient. .

Cardiovascular disease is a disease including heart disease, hypertensive heart disease, arrhythmia, congenital heart disease, myocardial infarction, angina, and other vascular diseases such as stroke and peripheral vascular disease. These diseases occur at various age groups and can lead to death with serious sequelae if not treated in a timely manner. Due to westernized dietary and lifestyle changes, the incidence and mortality rate of cardiovascular diseases continues to increase in recent years, but there are no effective treatments for treating them.

Current treatments for coronary artery disease include pharmacotherapy, gene therapy, percutaneous transluminal coronary angioplasty and stenting (PTCA), and reperfusion therapy (CABG) of coronary artery bypass graft (CABG). revascularization therapy is underway. However, even after reperfusion therapy, the drug should be continued and there is a possibility that coronary artery disease may return.

In particular, percutaneous coronary angioplasty (PTCA) has been widely used since 1977 for the treatment of patients with coronary atherosclerosis (Gurentzig, Lancet, 4, 263, 1978). There is a problem that restenosis occurs in about 40% of patients after 6 months (Ryan et al., J. Am. Coll. Cardiol., 22, 2033-2054, 1993). The mechanism of the development of restenosis after PTCA is to promote the migration and proliferation of vascular smooth muscle cells by producing and secreting various cytokines from balloon-damaged vascular endothelial cells, activated platelets or macrophages. Therefore, it is known that the production and secretion of extracellular matrix (ECM) is promoted, leading to thickening of the endocardium (intima) and developing into restenosis (Godfried et al, Am. Heart J., 129, 203-210 , 1995).

To date, attempts have been made to prevent restenosis using antiplatelet agents, anticoagulants, cholesterol biosynthesis inhibitors, and antiallergic agents (Lefkovits et al., Progr. Cardiovas. Dis., 40, 141-158, 1997). Fish oil (Faggin et al., Arterioscler. Thromb. Vasc. Biol. , 20, 152-163, 2000), vitamin C or vitamin E (Nunes et al., Circulation , 88, I-372, 1993 Research has been conducted on the prevention of restenosis of natural products-derived components. However, these drugs have not yet obtained sufficient results in clinical practice.

Taxol (Herdeg et al ., Zeischrift fur Kardiologie , 89, 390-397, 1999), heparin (Cairns et al., Circulation , 94, 1553-1560, 1996), EPA (Leaf et al., Circulation , 90 , 2248-2257, 1994), estrogen (O'Keefe et al., J. Am. Coll. Cardiol ., 29, 1-5, 1997) Although the back is under research and development as a vascular stenosis inhibitor, the study of the herbal medicine on the inhibition of vascular stenosis has been conducted only by the present inventors.

Golden Gate Rhubarb or Genusgrass, the origin of Rhei Rhizoma , is a perennial plant of the dicotyledonous genus Mardiaceae, which is 6-7 years old by removing the shells and burdock-shaped roots of the roots. Whole sliced and dried is called rhubarb. In oriental medicine, it has been used as an anti-inflammatory laxative since bc, and it is used in combination with various prescriptions. Main components are anthraquinone derivatives, glycosides and tannins thereof. Ingestion in small doses is effective for constipation as a laxative but in large cases (Jung Bo-seop and Shin Min-kyo); Medicinal metabolism, Yeonglimsa, pp327-329, 1998), anti-inflammatory action (Mian et al., J. Pharma. Pharmacol) ., 39, 845-847, 1987, Nonaka et al., Chem. Pharm. Bull., 29, 874-878, 1981), cytostatic action (Fujitsuka et al., Jpn. J. Nephrology, 39, 693 -700, 1997) and ACE inhibitory activity (Inokuchi et al., Chem. Pharm. Bull. , 32, 3615-3619, 1984).

Golden ( Scutellaria baicalensis ) is a perennial herb used to dry golden roots. The roots of gold contain baicalein, baicalin, wogonin, and neobaicalein. Physiological activity and pharmacological action of gold, antipyretic, anti-inflammatory, anti-allergic action, and expands blood vessels, and further lowers blood pressure and inhibits atherosclerosis. In particular, Baikalin contained in gold is sedating and hemostasis (止血) because it reduces the permeability of capillaries. In addition, by strengthening the mast cell membrane to suppress the release of chemical transport material, it has anti-allergic effect, in particular to improve hepatitis caused by allergies and inhibit vascular permeability (Jung Bo-seop and Shin, Min-Kyo; Hyangjedae, Yeonglimsa, pp864- 865, 1998), anti-inflammatory and hyperlipidemic effects (Uchiyama et al., J. Med. Pharm. Soc. Wakan-Yaku, 6, 158-164, 1989; Kubo et al., Chem. Pharm. Bull., 32, 2724-2729, 1984).

Coptis chinensis is a perennial herbaceous plant belonging to the family Ranunculus, and its roots are dried in the sun. The roots of nasturtium contain alkaloids such as berberine, coptisine, warenine, and palmatine as active ingredients. Lactobacillus has antimicrobial and antipathogenic effects, in particular berberine enhances the action of acetylcholine, relaxes vascular smooth muscle (Jung Bo-seop and Shin, Min-Kyo; Pharmacological Dictionary, Yeonglimsa, pp490-493, 1998), anti-inflammatory Action (Ko et al., Eur. J. Pharmacol., 399, 187-196, 2000), antioxidant activity (Yokozawa et al., J. Pharm. Pharmacol., 56, 547-556, 2004) and blood pressure lowering effects.

Accordingly, the present inventors have searched for herbal medicines to prevent restenosis after PTCA, and as part of a study to improve usability as a medicine, the new inner lining thickening of the methanol extract of Samhwangsasimtang, which is the herbal medicine containing rhubarb, gold and rhubarb, is excellent. The present invention was confirmed to have an inhibitory and preventive effect on cardiovascular diseases, and thus, the present invention was completed.

It is an object of the present invention to inhibit vascular restenosis and cardiovascular disease containing methanol extract containing rhubarb, gold and rhubarb or its active ingredient, baicalein, berberine or emodin, as an active ingredient. It provides a pharmaceutical composition and health functional food for the prevention or treatment of.

In order to achieve the above object, the present invention comprises an extract containing rhubarb, gold and rhubarb as an active ingredient, inhibiting vascular restenosis and preventing or treating cardiovascular diseases, including a pharmaceutically acceptable carrier, excipient or diluent It provides a pharmaceutical composition.

The extract includes a polar solvent selected from water, a lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, preferably an extract available in water.

In addition, the present invention includes the prevention of vascular restenosis and prevention of cardiovascular diseases, including baicalein, berberine or emodin as an active ingredient and a pharmaceutically acceptable carrier, excipient or diluent Or it provides a pharmaceutical composition for treatment.

Hereinafter, the present invention will be described in more detail.

The extract is characterized in that it is included in the form of a pulverized product, extract or powder extract thereof. At this time, the composition according to the present invention includes a mixed extract comprising a single extract of each herbal medicine, or a complex extract prepared by extracting a plant containing two or more of each herbal medicine.

The extract according to the present invention may be a crude extract prepared by applying a solvent extraction method using a conventional water extract, alcohol, etc. as a solvent, and may also be a fraction extract purified using column chromatography. Extraction method according to the present invention can be applied to all extraction methods known to those of ordinary skill in the art, for example, extraction with water, alcohol or mixed solvent, extraction method with bath or room temperature, etc. Including but not limited to.

In a preferred embodiment of the present invention, dried herbal medicines may be washed to remove foreign substances, etc. present on the surface of the material, and then dried in a dryer, mixed in an appropriate blending ratio, and ground to obtain the herbal powders. In addition, 1 to 20 times volume / weight of distilled water, lower alcohol having 1 to 4 carbon atoms or a mixed solvent thereof, preferably about 1 to 20 times, and most preferably about 3 to 10 times Of methanol at 20 to 100 ° C., preferably at 50 to 70 ° C. for about 1 to 10 days, preferably about 2 to 5 hours, at about 50 to 70 ° C. extraction temperature, hot water extraction, ultrasonic extraction, reflux cooling extraction, preferably The extract obtained using a reflux cooling extraction method is filtered and concentrated under reduced pressure to obtain a methanol extract.

Extracts for inhibiting vascular restenosis and preventing or treating cardiovascular diseases of the present invention are not limited thereto, but are based on the dry weight of each herbal medicine of rhubarb, gold, and yellow rye 1.5 to 2.5: 0.5 to 1.5: 1.5 to 2.5 It is preferable to contain in ratio. The herbal extracts have been used as food for a long time as the extract of the present invention extracted therefrom also has no problems such as toxicity and side effects.

In the experiment of preparing and evaluating a balloon injury model using a white rat by the present inventors, the rhubarb, gold, and rhubarb methanol extracts of the present invention exhibited an inhibition of neointimal formation of 54.5%. The Intima / Media ratio, which is a measure, also shows an inhibition rate of 55.5%. In addition, rhubarb, gold and rhubarb methanol extracts and the major components from these constituent herbal drugs, Baikallein, berberine and emodine, concentration-dependently inhibit the proliferation of vascular smooth muscle cells. Specifically, the proliferation of vascular smooth muscle cells of rhubarb, gold and rhubarb methanol extracts and their main components, bikallein, berberine, and emodin, correlated with the protein expression of cyclin B1, which is a G2 / M progression regulator In order to confirm the Western blot method, the concentration-dependent inhibition of Cyclin B1 protein expression suppresses the proliferation of vascular smooth muscle cells by inhibiting cell cycle progression to G ₂ / M phase.

In addition, rhubarb, golden and rhubarb methanol extracts and their main components, Baikallein, berberine, and emodine, were confirmed by transwell chamber method of vascular smooth muscle cell migration. It inhibits activity in a concentration-dependent manner, and these inhibitory effects also inhibit MMP-2 and MMP-9 gene expression.

Therefore, the present invention measures the effect of rhubarb, golden and rhubarb methanol extract on the proliferation and suppression of vascular smooth muscle cells, and atherosclerosis of rhubarb, gold and rhubarb methanol extract and its major components Baikallein, berberine and emodine Provide research methods. In other words, the bioassay of rhubarb, gold and rhubarb methanol extract and its major components Baikal, berberine and emodine provided by the present invention observes the proliferation and pluripotency of vascular smooth muscle cells, an important mechanism of vascular restenosis. It can be useful to

The present invention provides a pharmaceutical composition for inhibiting vascular restenosis and preventing or treating cardiovascular diseases, comprising the rhubarb, golden and rhubarb extract as an active ingredient and a pharmaceutically acceptable carrier, excipient or diluent. In addition, the present invention provides a pharmaceutical composition for inhibiting vascular restenosis and preventing or treating cardiovascular diseases, including a baccalin, berberine or emodine as an active ingredient of the extract and including a pharmaceutically acceptable carrier, excipient or diluent. to provide.

The cardiovascular diseases are diseases including atherosclerosis, heart failure, hypertensive heart disease, arrhythmia, congenital heart disease, myocardial infarction, angina and other vascular diseases such as stroke and peripheral vascular disease. And most preferably is atherosclerosis. Accordingly, the present invention can provide a method for preventing or treating atherosclerosis by administering an anti-arteriosclerosis composition comprising an effective amount of rhubarb, gold and yellow lotus methanol extracts or bikallein, berberine, and emodine.

The pharmaceutical composition for inhibiting vascular restenosis and preventing or treating cardiovascular diseases includes 0.01 to 50% by weight of the extract or compound based on the total weight of the composition.

Pharmaceutical dosage forms of the extracts of the present invention may be used in the form of their pharmaceutically acceptable salts, or 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 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. 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 oils such as olive oil, injectable esters 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 or compounds 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 or compound of the present invention is preferably administered at 10 to 2000 mg / kg, preferably at 100 to 1000 mg / kg. Administration may be administered once a day or may be divided several times. The pharmaceutical composition of the present invention can be administered to various mammals such as rats, mice, livestock, humans, and the like. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections.

Complex herbal extract of the present invention is a drug that can be used with confidence even when taken for a long time for the purpose of prevention because there is little toxicity and side effects.

The present invention provides a dietary supplement comprising a methanol extract of rhubarb, gold, and yellow lotus as an active ingredient exhibiting inhibitory effect on vascular restenosis and preventing cardiovascular diseases, and a food supplement acceptable additive. In addition, the present invention provides a health functional food comprising an active ingredient of Baikallein, berberine or emodine, and a food supplement, which is an active ingredient of the extract, which exhibits an effect of inhibiting vascular restenosis and preventing cardiovascular diseases.

In addition, a composition containing methanol extract of rhubarb, golden and rhubarb or its active ingredient Baikallein, berberine or emodine may be added to foods or beverages for the purpose of preventing atherosclerosis or cardiovascular diseases, and the food type is particularly It is not limited, For example, various foods, such as confectionery, bread, noodles, etc., Drinks, such as water, a soft drink, fruit drink, gum, tea, a vitamin complex, seasonings, a health functional food, etc. are mentioned. At this time, the amount of the extract or compound in the food or beverage, in general, in the case of the health functional food composition of the present invention can be added to 0.01 to 15% by weight, preferably 0.1 to 5% by weight of the total food weight, health drink The composition may be added at a ratio of 0.01 to 30 g, preferably 0.1 to 1 g based on 100 ml. Since the health functional food of this invention obtained in this way contains the composition of this invention, it is a food which can fully utilize the health prevention effect and health treatment effect which the compound of this invention has.

The health beverage composition of the present invention has no particular limitation on the liquid component except for containing the extract or compound as essential ingredients in the indicated proportions, and may contain various flavors or natural carbohydrates, etc. as additional ingredients, like ordinary 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 (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 1-20g, preferably about 5-12g per 100ml of the composition of the present invention.

The food of this invention is easily obtained by adding the process of adding the composition of this invention mentioned above to the manufacturing process of the base food, or by adding the process of adding the composition of this invention mentioned above after manufacture of the foodstuff which is a base material. Can be. At this time, a taste and odor correction agent may be added as needed.

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.

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

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

Example 1.Preparation of Rhubarb, Golden and Rhubarb Methanol Extracts

After selecting and cutting rhubarb, gold and rhubarb purchased from Gyeongdong market, add 180 g of each of golden ginseng, rhubarb and rhubarb to 2 l of 100% methanol and reflux cooler (B㎺CHI Rotavator R-114, B㎺CHI LABORTECHNIK AG) was extracted three times for 3 hours in a water bath. The extract was filtered on a filter paper and concentrated under reduced pressure to obtain 48.63 g (yield 27.0%) of methanol extract, which was used as a sample.

Experimental Example 1. Evaluation of vascular stenosis and stenosis

1-1. Manufacture of Balloon injury model

The left iliac artery of the 13-week-old rat (350 g) was incised and the iliac artery was cut one third, then a balloon catheter (2F size, Baxter Co.) was inserted and advanced about 15-20 cm upwards. Insert into the right carotid artery. About 0.02 ml of saline was injected to inflate the balloon of the catheter, and then the catheter was retracted by 1 to 2 cm to endothelial denudation. After repeating the above procedure 2-3 times, the catheter was removed and the left artery was cut to induce vascular narrowing to prepare a balloon wound model.

1-2. Evaluation of Vascular Stenosis

Rhubarb, golden and rhubarb methanol extract (194 mg / kg) (SST) prepared in Example 1 was orally administered to the balloon wound model prepared in Example 2-1 once daily. On the other hand, simvastatin (0.83 mg / kg) (SV), which is being used as an antihyperlipidemic agent, was administered for the same method and period (p <0.05) (see FIG. 1 ). After 2 weeks of administration, the syringe was inserted into the left ventricle, and PBS was injected at a dose of 10 ml per minute using a 100 ml vacuum pump to perfusion the components in the blood. Thereafter, 100 ml of a 10% formalin solution was injected to reflux the arterial tissue, and then the artery was extracted and deposited. The isolated artery was constructed into 3 μm sections, and then stained with a hematoxylin-eosin solution, followed by intima using the NIH image analysis program (1.62 ver.). After analyzing media and lumen areas, the degree of stenosis was evaluated by calculating the Intima / Media ratio. The results are shown in Figures 2a and 2b.

Endothelial Inhibition Rate (%) Intima / Medium Film Ratio Control 100.0 ± 7.4 1.00 ± 0.09 Simvastatin-treated group (SV)  69.3 ± 6.7 0.70 ± 0.05 Methanol extract administration group of the present invention (SST)  54.5 ± 6.2 0.56 ± 0.04

As a result, the extracts of gold, sulfur and rhubarb methanol showed an inhibition of neonatal formation of 54.5%, which showed an inhibition rate of 55.5% even in the ratio of intima / medium, which is a measure of stenosis (Table 1). In other words, the gold, sulfur and rhubarb methanol extract of the present invention was confirmed to have excellent vascular restenosis inhibiting ability.

Experimental Example 2 Evaluation of Inhibitory Effect of Vascular Smooth Muscle Cell Proliferation in Vitro

Inhibition of vascular smooth muscle cell proliferation of rhubarb, gold and rhubarb methanol extract prepared in Example 1 and Baikallein, berberine, emodin (Sigma) was examined.

2-1. Evaluation of Efficacy of Vascular Smooth Muscle Cell Proliferation of Rhubarb, Golden and Rhubarb Methanol Extracts

Vascular smooth muscle cells, A7r5 cells (prepared from ATCC), were suspended at 10x FBS-DMEM at 1x10 ⁵ cells / ml. The cell suspension was added 200 μl per well of a 96-well culture plate and incubated at 37 ° C., 5% CO ₂ for 24 hours to attach cells to the culture plate. Washed twice with PBS and then passaged with fresh medium, then treated with golden, sulfur and rhubarb methanol extracts of various concentrations (200-800 μg / ml) for 24, 48, 72 hours at 37 ° C., 5% CO ₂ . Incubated.

After 72 hours of incubation, the supernatant was collected, and 180 µl of the culture solution was added to each well in a 96-well culture plate, and the MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) reagent was added to 20 ml. After incubation for 4 hours by ㎕, discard the broth and dry for a while in the air. Thereafter, 200 μl of DMSO was completely dissolved in formazan attached to the bottom of the well, and then absorbance was measured at 570 nm. The inhibition rate (%) of each sample was calculated based on the difference between the absorbances generated in the group treated with DMSO (dimethyl sulfoxide) and the group not treated (see FIG. 3).

As a result, rhubarb, the gold and goldthread methanol extract 1000 ㎍ / ㎖ processed with vascular smooth muscle cell proliferation was inhibited 56.3%, whereas IC ₅₀ of the simvastatin is the 5.53 ㎍ / ㎖, rhubarb, the gold and goldthread methanol extract 490.5 ㎍ / Ml, showing an excellent cell proliferation inhibitory activity.

2-2. Evaluation of Efficacy of Vascular Smooth Muscle Cell Proliferation of Baikallein, Berberine and Emodine

As described in Experiment 2-1, the effect of inhibiting cell proliferation was measured in A7r5 cells, which are vascular smooth muscle cells, and cultured by treating various concentrations (2.5, 5, 10, and 20 µg / ml) of Baikallein, berberine, and emodine. , Absorbance was measured. The inhibition rate (%) of each sample was calculated based on the difference in absorbance generated in the group treated with DMSO and the group not treated (see FIG. 3).

As a result, the effect of inhibiting vascular smooth muscle cell proliferation was confirmed depending on the increase in the treatment concentration of Baikallein, berberine, and emodine. The IC ₅₀ of simvastatin is 5.53 μg / ml, whereas the baccalin is 12.26 μg / ml, the berberine is 5.90 μg / ml, and the emodine is 15.08 μg / ml. I could confirm that.

Experimental Example 3. Study on the cell cycle of vascular smooth muscle cells in vitro

The cell cycle of the rhubarb, gold and rhubarb methanol extracts prepared in Example 1, and vascular smooth muscle cells of Baikallin, berberine, and emodin were measured.

Specifically, 1 × 10 ⁶ cells / ml of vascular smooth muscle cells (A7r5 cells) were dispensed into 10 mm dishes treated with 10% FBS-DMEM, and then cultured at 37 ° C., 5% CO ₂ for 24 hours. After 24 hours of incubation in Free DMEM medium, 24, 48 in 10% FBS-DMEM containing 800 μg / ml of rhubarb, golden and rhubarb methanol extracts or 20 μg / ml of bicalin, berberine, and emodine, respectively Incubate again for hours. Cells were then removed with trypsin-EDTA, centrifuged with PBS (1500 rpm, 5 min) and cells were fixed for 2 hours at 4 ° C. with cold 70% ethanol. After the fixed cells were centrifuged again with PBS (1500 rpm, 5 minutes), 0.25 mg / mL RNase solution was added and left at 37 ° C for 30 minutes, followed by 50 ㎍ / mL PI solution at 4 ° C for 30 minutes. Stained. DNA content of the stained cells was measured by flow cytometry, and the cell cycle was analyzed (see FIG. 4).

As a result, it was confirmed that the treatment of rhubarb, gold and rhubarb methanol extract or baikal, berberine, and emodine inhibited the progression of G2 / M phase of vascular smooth muscle cells.

Experimental Example 4. Measurement of Cyclin B1 Protein Inhibition Effect in Vitro

The cell cycle of the rhubarb, gold and rhubarb methanol extracts prepared in Example 1, and vascular smooth muscle cells of Baikallin, berberine, and emodin were measured.

Specifically, A7r5 cells were 1 × 10 ⁶ / ml Dogs were suspended in DMEM medium containing 10% FBS and then attached to a 100 mm culture dish and incubated at 37 ° C., 5% CO ₂ for 24 hours. After incubation, washed twice with PBS and incubated for 24 hours in DMEM containing no FBS. DMEM medium containing 10% FBS was further treated with 800 μg / ml of golden, rhubarb, rhubarb methanol extract or 20 μg / ml of simvastatin, bikallin, berberine, and emodine, respectively, for 24 and 48 hours. The cells are then washed three times with PBS and boiling lysis buffer (250 mM Tris-HCl buffer, pH 6.8), 4% SDS. 10% glycerol. 0.006% bromophenol blue. 2% beta-mercaptoethanol ) Was added to each culture dish to lyse the cells, and the cells were denatured by heating for 5 minutes. Protein amount was quantified by the BCA method, and 30 µg of the protein was electrophoresed for 2 hours using a 12% acrylamide gel to separate the protein. The separated protein was transferred to a PVDF membrane (Polyvinylidenedifluoride membrane, Millipore) for 1 hour 20 minutes, and then the membrane was washed twice with methanol and PBST (PBS + Tween), followed by blocking buffer (5% non-fat dry milk). in 0.1% Tween 20-PBS) was incubated at room temperature for 1 hour and then washed three times with PBST for 5 minutes. Primary antibody after washing (rabbit polyclonal cyclin B1 antibody diluted to 1/1000 (Santa Crutz Biotechnology) and mouse monoclonal β-actin antibody diluted to 1/2000 (Sigma-Aldrich)) Was added to a PBS solution containing 3% non-fat dry milk, and incubated for 1 hour and 30 minutes at room temperature. After incubation, the membrane was washed three times with PBST for 5 minutes and the secondary antibody (Horceradish peroxidase (HRP) -conjugated goat anti-rabbit-IgG diluted to 1/1500 cyclin B1, HRP-conjugated beta-actin) Goat anti-mouse-IgG, Santa Crutz Biotechnology Inc.) was incubated for 1 hour at room temperature. Then, washed three times with PBST for 5 minutes and the protein expression inhibitory effect was detected using ECL (Enhanced chemiluminescence detection system, Pharmacia Biotech, USA) (see Figure 5).

As a result, the expression of cyclin B1 protein was significantly decreased when rhubarb, gold and rhubarb methanol extracts were treated, and the expression of protein was almost suppressed at 48 hours. Berberine also reduced the expression of cyclin B1 protein.

Experimental Example 5. Evaluation of vascular smooth muscle cell migration inhibition effect in vitro

Transwell Chamber method (Bilato et al., J. Clinical to observe whether rhubarb, gold and rhubarb methanol extracts prepared in Example 1 and Baikallein, berberine, emodine inhibit vascular smooth muscle cell migration Invest., 96, 1905-191, 1995).

5-1. Evaluation of Efficacy of Vascular Smooth Muscle Cells Inhibition of Rhubarb, Golden and Rhubarb Methanol Extracts

The lower part of the chamber was coated with collagen as an extracellular matrix in a polycarbonate filter (8 μm pore size), and A7r5 cells, the vascular smooth muscle cells, were topped with 1x10 ⁵ cells / ml, 0.2 ml in DMEM medium containing 0.1% BSA. It was. The lower chamber was treated with 10% FBS-DMEM in various concentrations (200-800 μg / ml) of rhubarb, golden and yellow lotus methanol extracts and incubated at 37 ° C., 5% CO ₂ for 4 hours. After incubation, the non-host cells of the upper filter were removed with a cotton swab, and then the filter was fixed with methanol solution. The filter was stained with hematoxylin-eosin and then dehydrated in xylene solution. After the filter was sealed with a Euokitt solution and covered with a cover glass, the number of cells residing with the filter was counted under a microscope. Inhibition rate (%) of each sample was measured at 100% difference between the control group and the test group (see FIGS. 6A and 6B).

As a result, when the methanol extract of the present invention 800 ㎍ / ㎖ treated vascular smooth muscle cell induction activity 73.8% inhibited, it was confirmed that the inhibition of vascular smooth muscle cells in the concentration-dependent treatment of rhubarb, gold and rhubarb methanol extract .

5-2. Evaluation of Efficacy of Vascular Smooth Muscle Cell Inhibition of Baikallein, Berberine and Emodine

As shown in Experiment 5-1, the effect of inhibiting cell migration in A7r5 cells, which are vascular smooth muscle cells, was measured and treated with various concentrations (2.5, 5, 10, and 20 ㎍ / ml) of Baikallein, berberine, and emodine. Inhibition of lactose was measured. Inhibition rate (%) of each sample was calculated by making the difference between the cell numbers of the control group and the test group 100% (see FIGS. 6A and 6B).

As a result, it was confirmed that all three substances significantly inhibited vascular smooth muscle cell migration since barcalane, berberine, and emodine concentrations were proportionally dependent.

Experimental Example 6. Measurement of the Inhibitory Effect of MMP-2 and MMP-9 Gene Expression in Vitro

The inhibitory effects of the expression of MMP-2 and MMP-9 genes in vitro of rhubarb, gold and rhubarb methanol extracts prepared in Example 1 and Baikallin, berberine and emodine were examined.

Specifically, A7r5 cells (1 × 10 ⁶ / ml) were suspended in DMEM medium containing 10% FBS and then attached to a 100 mm culture dish and incubated at 37 ° C., 5% CO ₂ for 24 hours. After incubation, washed twice with PBS and incubated for 24 hours in DMEM containing no FBS. DMEM medium containing 10% FBS was further treated with 800 μg / ml of golden, rhubarb and rhubarb methanol extracts or 20 μg / ml of simvastatin, bacalin, berberine, and emodine, respectively, for 6, 24, and 48 hours. . After incubation, the cells were collected in each dish, and then 1 ml of TRI reagent (Sigma) was added to lyse the cells, RNA was extracted using chloroform and isopropanol, and the absorbance was measured at 260 and 280 nm. Concentration and purity were determined. RNA was reverse transcribed into cDNA using RT system (Promega), and then PCR was performed using Taq DNA polymerase (Taq PCR master mix kit, Qiagen). After completion of the PCR reaction, the amplified DNA was electrophoresed at 80 V for 1 hour with 2% agarose gel, and then stained with 10,000-fold diluted SYBR Gold staining solution for 1 hour to 1 hour 30 minutes. The stained DNA was then observed using an Alpha Imager ™ (Alpha Innotech Corporation) (see FIG. 7).

As a result, the expression of MMP-2 was significantly decreased after 24 hours of rhubarb, gold and rhubarb methanol extract, and the expression of the gene was almost suppressed after 48 hours. In addition, the gene expression of MMP-9 was reduced after 6 hours of rhubarb, gold and rhubarb methanol extract, and the expression of the gene was inhibited after 24 and 48 hours of treatment. That is, it was confirmed that the rhubarb, gold and rhubarb methanol extracts of the present invention and the compounds from the constituent herbal medicines inhibit vascular smooth muscle cell proliferation and diurnal activity, thereby effectively inhibiting the neointimal thickening and can be used as anti-arteriosclerosis agents.

Experimental Example 7. Toxicity Test

In order to investigate the presence or absence of toxicity by oral administration to rats of each of the methanol dry extract of Example 1, Baikallein, berberine, and emodine, 1 g / kg and 2 g of male and female rats of the SD system, respectively, were examined. Five doses of / kg and 3 g / kg were administered once orally, and 14 deaths, general symptoms, weight change and autopsy findings were observed. The result of this test is as follows.

(1) There were no deaths observed during the test period in all the administration groups of the compositions of the present invention during the test period, and there was no change in general symptoms specifically observed compared to the control group.

(2) Body weight change showed significant weight loss on day 3 after administration in 5 g / kg dose group, but was not different from control group in all other administration groups.

As a result, no clinical symptoms were observed in all the test substance-treated groups compared to the death and control groups, and there were almost no specific symptoms in the anatomical changes at the time of body weight and autopsy. In addition, since the compositions did not show a clear toxicity at doses of 5 g / kg or less, the LD ₅₀ (lethal dose) in rats by oral administration was determined to be 5 g / kg or more.

Hereinafter, the formulation examples of the pharmaceutical preparations will be described, which are intended to explain in detail only, not to limit the present invention.

Formulation Example 1-1. Preparation of injectables (1)

Baikallein .............................................. 1.0mg

Sodium metabisulfite ........ 3.0mg

Methylparaben ............... 0.8 mg

Propylparaben ...................... 0.1 mg

Sterile distilled water for injection ..............

The above ingredients are mixed and made into 2 ml by a conventional method, and then filled into 2 ml ampoules and sterilized to prepare an injection.

Formulation Example 1-2. Preparation of injectables (2)

Berberine ............... 1.0mg

Sodium metabisulfite ........ 3.0mg

Methylparaben ............... 0.8 mg

Propylparaben ...................... 0.1 mg

Sterile distilled water for injection ..............

The above ingredients are mixed and made into 2 ml by a conventional method, and then filled into 2 ml ampoules and sterilized to prepare an injection.

Formulation Example 1-3. Preparation of injectables (3)

Emodine ............... 1.0 mg

Sodium metabisulfite ........ 3.0mg

Methylparaben ............... 0.8 mg

Propylparaben ...................... 0.1 mg

Sterile distilled water for injection ..............

The above ingredients are mixed and made into 2 ml by a conventional method, and then filled into 2 ml ampoules and sterilized to prepare an injection.

Formulation Example 2 Preparation of Tablet

Methanol dry extract of Example 1. 20 mg

Lactose 100 mg

Starch .............................. 100 mg

Magnesium Stearate ...............

The above components are mixed and tableted according to a conventional method for producing tablets to produce tablets.

Formulation Example 3 Preparation of Capsule

Methanol dry extract of Example 1. 10 mg

Lactose 50 mg

Starch ........................................ 50 mg

Talc ........................................ 2mg

Magnesium Stearate ...............

Capsules are prepared by mixing the above ingredients and filling into gelatin capsules according to a conventional method for preparing capsules.

Formulation Example 4 Preparation of Liquid

Methanol dry extract of Example 1 ............ 100 mg

Sugar .................................. 20g

Isomerized sugar ......................................... 20 g

Lemon Flavor ......................

Purified water is added to the whole ........... 100ml

The above components are mixed according to a conventional method for preparing a liquid, filled into a 100 ml brown bottle, and sterilized to prepare a liquid.

Formulation Example 5 Preparation of Healthy Food

Methanol dry extract of Example 1 ........ 1000 mg

Vitamin Mixture ......................

Vitamin A Acetate ......... 70 μg

Vitamin E ................................... 1.0 mg

Vitamin B1 ................................. 0.13 mg

Vitamin B2 ..................... 0.15 mg

Vitamin B6 .................. 0.5 mg

Vitamin B12 ................................. 0.2 μg

Vitamin C ......................................... 10 mg

Biotin ......................................... 10 μg

Nicotinamide ........................................ 1.7 mg

Folic acid ......................................... 50 ㎍

Calcium Pantothenate ......................................... 0.5 mg

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 6 Preparation of Healthy Drink

Methanol dry extract of Example 1 ... 1000 mg

Citric Acid .................................... 1000 mg

Oligosaccharide ........................... 100 g

Plum concentrate ........................... 2 g

Taurine ......................................... 1 g

Purified water is added .............. 900 ml total

After mixing the above components according to a conventional healthy beverage production method, and then 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 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.

Rhubarb, golden and rhubarb methanol extracts of the present invention and baicalein, berberin and emodin alleviate neointimal thickening by inhibiting the proliferation and migration activity of vascular smooth muscle cells And inhibiting vascular restenosis, the pharmaceutical composition for inhibiting arteriosclerosis may be usefully used as a pharmaceutical composition and health functional food for preventing or treating cardiovascular diseases.

Claims (10)

Prevention of heart failure, hypertensive heart disease, arrhythmia, congenital heart disease, myocardial infarction, angina pectoris, stroke and atherosclerosis, including rhubarb, golden and rhubarb methanol extracts as active ingredients and a pharmaceutically acceptable carrier, excipient or diluent And therapeutic pharmaceutical compositions. delete The pharmaceutical composition according to claim 1, wherein the extract contains rhubarb, gold, and yellow lotus in a ratio of 1.5 to 2.5: 0.5 to 1.5: 1.5 to 2.5 based on the dry weight of each herbal medicine. delete delete delete Prevention and improvement of heart failure, hypertensive heart disease, arrhythmia, congenital heart disease, myocardial infarction, angina pectoris, stroke and arteriosclerosis, including rhubarb, golden and rhubarb methanol extracts as active ingredients and food acceptable additives Dietary supplements. The health functional food according to claim 7, wherein the health functional food is a health beverage. delete delete

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