KR101016996B1 - A composition comprising ginsenosides for treating and preventing heart disease - Google Patents

Abstract

PURPOSE: A composition containing ginsenoside extracted from leaves, stems, or buds of ginseng plant is provided to reduce or enhance the contraction of heart chamber muscle and to treat cardiac insufficiency, myocardial infarction, and hypertension. CONSTITUTION: A composition for preventing or treating hypertension or angina pectoris contains ginsenoside F1 of chemical formula 1 or notoginsenoside R1 isolated from leaves, stems, or buds of ginseng plant. A health food for preventing or treating hypertension or angina pectoris contains ginsenosdie F1 or notoginsenoside R1. The health food is a dairy product including drinks, meats, sausage, bread, candy, snack, noodle, and ice cream and a supplementary product including beverage, alcoholic beverage, and vitamin. The used ginseng is Panax ginseng, Panax quinquefolia, Panax notoginseng, Panax japonicas, Panax vietnamensis, Panax elegatior, Panax wangianus, or Panax bipinratifidus.

Description

A composition comprising ginsenosides for treating and preventing heart disease

The present invention relates to a composition for the prevention and treatment of cardiovascular diseases containing ginsenosides extracted from the leaves, stems or buds of ginseng.

More specifically, the present invention relates to a composition for the prevention and treatment of cardiovascular diseases containing ginsenosides that are effective in increasing or decreasing myocardial (ventricular muscle) contraction to treat heart failure, myocardial infarction, hypertension and the like. .

According to clinical practice in traditional Chinese medicine and combinations of medicinal herbs and ginseng, ginseng and its preparations improve myocardial contractility, coronary artery enlargement, increased coronary blood flow, myocardial oxygen consumption, protection of myocardium from reperfusion injury, inhibition of platelet aggregation, It has been confirmed to have pharmacological activity for acting as an antithrombin and to be used clinically for the treatment of chest injury, heart pain (coronary artery disease), arteriosclerosis and the like.

Research in modern science has shown that the main bioactive component of ginseng is ginsenoside, a type of saponin. Ginsenosides are known to exist in the roots of ginseng, as well as in the leaves, stems, and buds of ginseng. The most known ginsenosides in terms of effect and quantity include ginsenosides such as ginsenosides Rg2, Rg3, Rh1 and Rh2 which are contained in processed ginseng. The ginsenosides are secondary ginsenosides produced by hydrolysis of ginsenosides during the processing procedures of ginseng (eg, steam treatment and drying steps). These secondary ginsenosides have a number of novel activities, for example ginsenosides Rh1, Rh2 and Rg3 have significant anticancer activity and will induce regeneration of cancer cells (ie, induce cancer cells to re-differentiate into healthy cells). While other ginsenosides do not have this effect or their effects are relatively weak. Ginsenoside Rg3 shows a relatively strong inhibitory effect on platelet aggregation induced by collagen and the like. Ginsenoside Rhl has a significant inhibitory effect on the conversion of fibrinogen to fibrin induced by thrombin. In vivo metabolism of ginsenosides has also been studied through serum pharmacology.

Ginsenoside F1 (ginsenoside F1) used in the present invention is known as a secondary ginsenoside mainly produced by the metabolism of intestinal bacteria, and is contained in a high content even in the leaves, stems, or buds of ginseng. Isolated in large quantities from the leaves of ginseng. Notoginsenoside R1 (notoginsenoside) is known as the major ginsenoside of Panax notoginsengs , a type of ginseng, but was isolated from the leaves of ginseng in this experiment. Floringinsenoside Kc (floralginsenoside Kc) is a novel ginsenoside identified in the present invention and is a compound isolated from the buds of ginseng.

The present invention aims to treat cardiovascular diseases by controlling the contraction of myocardium (ventricular muscle) using the composition containing the ginsenosides. Increased myocardial contraction can prevent and treat diseases such as heart failure, myocardial infarction, myocardial ischemia, myocardial contraction disorder, ischemic heart disease, cardiac hypertrophy, cardiomyopathy, congestive heart failure, rhythm disorder, and heart attack. May lower the blood pressure of blood released from the ventricles to treat hypertension or angina.

Myocardial infarction is when the coronary artery to the heart is acutely blocked by thrombosis or rapid contraction (spasm) of blood vessels. Refers to the situation of death (necrosis). Blood clots don't necrosis due to blood clots, but pain in the chest due to poor blood flow in the blood vessels.

Heart failure is a heart condition in which the body cannot supply enough blood. It is the final and deadly form of various heart diseases such as cardiac hypertrophy, coronary atherosclerosis, myocardial infarction, heart valve disease, or cardiomyopathy. Heart failure manifests itself initially in the form of reduced motor performance, but as symptoms progress, the heart's ability to supply decreases rapidly, resulting in a lack of sufficient blood in normal conditions and causing a fatal condition such as a heart attack.

Hypertension refers to a systolic blood pressure of 140 mm Hg or more or a diastolic blood pressure of 90 mm Hg or more in adults 18 years of age or older. Hypertension can be broadly classified into two types. The cause of the disease is known and hypertension is caused by the occurrence of secondary hypertension, and the cause of the disease is not found is called primary (hypertension) hypertension. About 95% of all hypertensive patients are essential hypertension. The underlying reason for the development of essential hypertension is not clear, but is due to an increase in cardiac output (a volume of blood ejected from the heart for 1 minute) or an increase in peripheral vascular resistance, which leads to a decrease in myocardial contractility. Hypertension can be treated.

An object of the present invention to provide a composition for the prevention and treatment of cardiovascular diseases containing ginsenosides extracted from the leaves, stems or buds of ginseng.

Another object of the present invention is to prepare a health functional food for the prevention and improvement of cardiovascular diseases containing ginsenosides extracted from the leaves, stems or buds of ginseng.

The present invention is preferably ginsenoside represented by the following formula 1 of a plurality of ginsenosides obtained by purely separating and purifying ginsenosides isolated from the leaves, stems or buds of ginseng and confirming their structure through chemical structure and physicochemical characterization. The present invention was completed after confirming that the side regulates myocardial contraction.

[Formula 1]

Ginseng as defined in the present invention is a perennial plant belonging to the genus Panax , Panax ginseng , Panax quinquefolia , Panax ginseng , Panax notoginseng , Panax japonicus, Vietnamese ginseng , Panax vietnamensis ), Panax elegatior , Panax wangianus , or Panax bipinratifidus , preferably Panax ginseng .

In addition, as an extraction solvent for extracting ginsenoside from the leaves, stems or buds of ginseng, water, an alcohol, an organic solvent or a mixed solvent of the alcohol or the organic solvent and water may be used.

Ginsenosides isolated from the leaves, stems or buds of ginseng according to the present invention have high stability and can be used as raw materials or additives for foods and pharmaceuticals.

The pharmaceutical compositions comprising ginsenosides according to the present invention may be prepared in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, external preparations, suppositories, and sterile injectable solutions, respectively. It can be formulated and used in the form. Examples of carriers, excipients and diluents that can be included in the composition containing the extract include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations include at least one excipient such as starch, calcium carbonate, sucrose or lactose, gelatin, and the like. Mix is prepared. 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. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.

The dosage of the active ingredient will vary depending on the age, sex and weight of the subject to be treated, the particular disease or pathology to be treated, the severity of the disease or pathology, the route of administration and the judgment of the prescriber. Dosage determination based on these factors is within the level of skill in the art and generally dosages range from 0.01 mg / kg / day to approximately 2000 mg / kg / day. More preferred dosage is 0.1 mg / kg / day to 500 mg / kg / day. Administration may be once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect. The extract of the present invention can be administered to mammals such as mice, livestock, humans, etc. by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injections. Since the extract of the present invention has little toxicity and side effects, it is a drug that can be used safely even when taken for long periods of time.

Ginsenosides isolated from the leaves, stems or buds of ginseng according to the present invention was found to be able to control the contraction of ventricular muscles, thereby preventing and treating cardiovascular diseases. Increasing myocardial contraction may treat diseases such as heart failure, myocardial infarction, myocardial ischemia, angina pectoris, myocardial contraction disorder, ischemic heart disease, cardiac hypertrophy, cardiomyopathy, congestive heart failure, rhythm disorder, and heart attack. Is known to be able to treat high blood pressure ginsenoside derived from the leaves, stems or buds of the ginseng of the present invention can be usefully used and applied in high-efficiency prevention and treatment for cardiovascular diseases.

1 is a representative graph showing that the intensity of muscle contraction is reduced when ginsenoside F1 is treated to red ventricular muscle cells in comparison to a control.
FIG. 2 is a graph showing statistical reduction of muscle contractile force when ginsenoside F1 is treated to multiple red ventricular muscle cells in comparison with the control group.
3 is a representative graph showing that the intensity of muscle contraction decreases when notoginsenoside R1 is treated to red ventricular muscle cells as compared to the control group.
Figure 4 is a graph showing the statistical treatment that muscle contractility decreases when treated with red ventricular muscle cells notoginsenoside R1 compared to the control.
FIG. 5 is a graph showing an increase in the strength of muscle contraction when floral ginsenoside Kc was treated to red ventricular muscle cells compared to the control group.
FIG. 6 is a graph showing statistically the increase in muscle contractility when treated with floral ginsenoside Kc in red ventricular muscle cells compared to the control group.

Hereinafter, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, it is provided to ensure that the contents introduced herein are thorough and complete, and that the spirit of the present invention to those skilled in the art can fully convey.

< Example  1. the leaves of ginseng or From buds Ginsenoside  How to Extract>

1-1. Extract from Ginseng Leaves

2.3 kg of dried Korean ginseng leaves were extracted three times with methanol (6 L, 50 ° C.) and vacuum dried. 630 g of methanol extract was suspended in 4 L of water and then extracted three times with 4 L of n-hexane and 4 L of dichloromethane to extract 45 g of n-hexane fraction and 20 g of dichloromethane fraction, respectively. The remaining water layer was eluted with a concentration gradient of MeOH-H ₂ O (25, 50, 75, 100% MeOH, v / v) on a DIION HP-20 column to obtain four fractions (fr.1.1-fr). .1.4). Of these, fr.1.3 (48.0g) was fractionated in a silica gel column under a concentration gradient of CH ₂ Cl ₂ -MeOH (20: 1-1: 1) to obtain five fractions (fr.3.1-fr.3.5 ). Thereafter, fr.3.2 (3.0 g) was obtained in four fractions under conditions of CHCl ₃ -MeOH-H ₂ O (6: 1: 0.1) on a silica gel column (fr.4.1-fr.4.4), and fr.4.2 (450 mg) was fractionated in a reverse phase chromatography column under conditions of MeOH-H ₂ O (2: 1) to obtain ginsenoside F1 (150 mg), and fr.4.4 (600 mg) was subjected to reverse phase chromatography. Fractionation was performed under a MeOH-H ₂ O (4: 3) condition on a chromatography column to obtain notoginsenoside R1 (5.0 mg).

1-2. Extract from Ginseng Buds

1.2 kg of dried ginseng buds were extracted three times with methanol (3 L, 50 ° C.) and vacuum dried. 204 g of methanol extract was suspended in 2.5 L of water and then extracted three times with 2.5 L of n-hexane and 2.5 L of dichloromethane to extract 29.4 g of n-hexane fraction and 15.2 g of dichloromethane fraction, respectively. The remaining water layer was eluted with a concentration gradient of MeOH-H ₂ O (25, 50, 75, 100% MeOH, v / v) on a DIION HP-20 column to obtain four fractions (fr.1.1-fr). .1.4). Among them, Fr.1.4 (20.0g) was fractionated in a silica gel column under a concentration gradient of CH ₂ Cl ₂ -MeOH (20: 1-1: 1) to obtain eleven fractions (fr.3.1-fr. 3.11). Thereafter, floral ginsenoside Kc (Floralginsenoside Kc, 5.8 mg) was obtained by fractionation under MeOH-H ₂ O (9: 5) under a reversed phase chromatography column at fr.3.11.

< Example  2. Isolated from the leaves or buds of ginseng Ginsenoside  Physical and chemical characterization- ^One H NMR  Data>

2-1. Notoginsenoside R1

white powder; [α] + 10 ( c 0.1, MeOH); ESI-MS: m / z 967 [M + H] ⁺ . ¹ H-NMR (CD ₃ OD, 400 MHz): δ 0.90, 0.91, 0.94, 1.05, 1.30, 1.58, 1.60, 1.64 (3H each, all s, H ₃ -30, 19, 18, 29, 26, 21 , 27, 28), 3.34 (1H, dd, J = 12.0, 5.2 Hz, H-3), 3.72 (1H, m, H-12), 4.08 (1H, m, H-6), 4.35 (1H, d, J = 8.0 Hz, H-1 "), 4.37 (1H, d, J = 8.0 Hz, H-1 '''), 4.60 (1H, d, J = 8.0 Hz, H-1'), 5.10 (1H, broad singlet, J = 6.4 Hz, H-24);

2-2. Ginsenoside F1

white powder; [α] +36 ( c 0.6, MeOH); ESI-MS: m / z 639 [M + H] ⁺ . ¹ H-NMR (CD ₃ OD, 400 MHz): δ 0.90, 0.92, 1.04, 1.25, 1.30, 1.58, 1.60, 1.64 (3H each, all s, H ₃ -30, 19, 18, 29, 26, 21 , 27, 28), 3.58 (1H, dd, J = 12.0, 5.2 Hz, H-3), 3.98 (1H, m, H-12), 4.55 (1H, d, J = 8.0 Hz, H-1 ' ), 4.85 (1H, m, H-6), 5.06 (1H, br t, J = 6.4 Hz, H-24)

2-3. Floralginsenoside Kc

white amorphous powder; [α] +11 ^o ( c 0.10, MeOH); IR (KBr) ν _max 3452, 2918, 1743, 1262, and 1058 cm ¹ negative ion mode (ESI-MS): m / z 917 [MH ₂ OH], 755 [MH ₂ OC ₆ H ₁₀ O ₅ -H] HR-ESI-TOF- Negative ion mode (MS): m / z 917.4734 [(MH ₂ O) -H] (calcd for C ₄₅ H ₇₃ O ₁₉ , 917.4746); IR spectrum: ν _max 3452, 1743, and 1058. ¹ H (C ₅ D ₅ N, 500 MHz): δ _H 4.07 (1H, dd, J = 7.0, 14.0 Hz, H-23)

The floral ginsenoside is a novel compound [Tung NH, Song GY, Kim JA, Hyun JH., Kang HK, Kim YH. Dammarane-Type Saponins from the Flower Buds of Panax ginseng and Their Effects on Human Leukemia Cells. Bioorg Med Chem Lett 2010, 20: 309-314.] Details are known.

< Example  3. Preparation of Cardiac Muscle Cells and Measurement of Cell Contractility>

Rat ventricular myocytes for determining myocardial contractility were extracted from the male SD (Sprague-Dawley) red [Lee et al., 2008]. Red was anesthetized with pentobarbital sodium (150 mg / kg, i.p.), and the heart was removed by opening the chest by a surgical method following the animal ethical guidelines of Chungnam National University.

The aorta of the extracted heart was connected to a Langendorf perfusion device and passed through the aorta for the first 3 minutes. Ca ^{2 +} -free Tyrode solution: 137 mM NaCl, 5.4 mM KCl, 10 mM HEPES , 1 mM MgCl 2, 10 mM glucose, pH 7.3), and then calcium-free containing collagenase (collagenase, 1.4 mg / ml, Type 1, Roche) and protease (protease, 0.14 mg / ml, TypeXIV, Sigma). The thyroid solution was perfused for 12 minutes. Finally, the enzymes were removed by perfusion for 8 minutes with a 0.2mM CaCl2-containing thyroid solution, and the cells were harvested by taking a ventricle and cutting it into small pieces.

The isolated ventricular muscle cells were allowed to attach to the bottom of the cell chamber placed on the inverted microscope, and continuously flowed a thyroid solution containing 2 mM Ca ²⁺ . Two platinum wires were placed on both sides of the chamber to induce cardiomyocyte contraction and 1 Hz stimulation was applied to the cells with an electrical stimulator. The change in cell length during contraction of cardiomyocytes was measured with a video edge-detector (model VED-105, Crescent Electronics), and a device for converting analog signals to digital signals (Digidata1 322, Molecular device, USA) After going through, it was recorded in a PC computer program (PC computer program, pClamp 9.0, Molecular device, USA). Shrinkage strength and data analysis were performed using a computer program (Clampfit, 9.0, Molecular device USA).

Each ginsenoside was prepared in high concentration in dimethyl sulfoxide (DMSO), diluted with extracellular fluid (Tyroid solution containing 2mM Ca ²⁺ ) to 1μM, and then flowed into cardiomyocytes. The ginsenoside treatment solution was treated until none.

As shown in FIGS. 1 to 4, ginsenoside F1 and notoginsenoside R1 markedly decreased the contractile force of cardiomyocytes, and floral ginsenoside Kc increased the contractile force of cardiomyocytes as shown in FIGS. 5 and 6. It was confirmed. In addition, the results showed similar results in ginsenoside F1, notoginsenoside R1 or floral ginsenoside Kc obtained by extracting leaves and buds of ginseng.

In addition to the ginsenoside, the ginseng extract itself using methanol or water was used to confirm the contractile force of the cardiomyocytes. The extract mainly containing the ginsenoside F1 and the notoginenoside R1 has a contractile force of the cardiomyocytes at a concentration of 1 μg / ml. It was confirmed that the remarkable decrease, the extract containing mainly the floral ginsenoside Kc significantly increased the contractile force of the cardiomyocytes at a concentration of 1 μg / ㎖.

< Example  4. Toxicity Test>

4-1. Acute Toxicity

To investigate the toxicity of ginsenoside F1, notoginsenoside R1, and floral ginsenoside Kc isolated from the leaves or buds of ginseng in acute (within 24 hours) in animals in a short time. This experiment was conducted to determine mortality. Twenty ICR mouse strains, which are common mice, were assigned to 10 control groups and 10 experimental groups. In the control group, nothing was administered, and the experimental group contained 2.0 g / kg of ginsenoside F1, notoginsenoside R1, and floral ginsenoside Kc isolated from the leaves or buds of ginseng (50 of the amount used in general animal experiments). Oral administration). After 24 hours of administration, the mortality was examined, and all the groups treated with ginsenoside F1, notozinenoside R1, and floral ginsenoside Kc isolated from the control group and leaves or flowers of 2.0 g / kg ginseng survived. It was.

4-2. Long-term and tissue toxicity experiments in experimental and control groups

Long-term toxicity studies were conducted to examine the effects of ginsenoside F1, notozinenoside R1, and floral ginsenoside Kc isolated from the leaves or flowers of ginseng on C57BL / 6J mice. Blood was collected after 8 weeks from the animals in the experimental group and the control group administered with the solvent, and the blood concentrations of glutamate-pyruvate transferase (GPT) and blood urea nitrogen (BUN) were measured using a Select E (Vital Scientific NV, Netherland) device. It measured using. As a result, in the case of GPT, which is known to be related to hepatotoxicity, and BUN, which is known to be related to nephrotoxicity, there was no difference in the experimental group compared to the control. In addition, histological observations were performed on the liver and kidney from each animal, and the histological observation was performed by optical microscopy through a conventional tissue fabrication process. No abnormalities were observed.

< Example  1: Pharmaceutical Formulation example >

1-1. Manufacture of tablets

200 g of ginsenoside F1 isolated in Example 1 according to the present invention was mixed with 175.9 g of lactose, 180 g of potato starch, and 32 g of colloidal silicic acid. 10% gelatin solution was added to the mixture, which was then ground and passed through a 14 mesh sieve. It was dried and the mixture obtained by adding 160 g of potato starch, 50 g of talc and 5 g of magnesium stearate was made into a tablet.

1-2. Preparation of Injection

1 g of ginsenoside F1, 0.6 g of sodium chloride and 0.1 g of ascorbic acid isolated in Example 1 according to the present invention were dissolved in distilled water to make 100 ml. The solution was bottled and sterilized by heating at 20 ° C. for 30 minutes.

< Example  2: food Production Example >

2-1. Preparation of Cooking Seasonings

Ginsenoside F1 separated in Example 1 according to the present invention to 0.2 to 10% by weight to prepare a cooking seasoning for health promotion.

2-2. Manufacture of flour food products

Ginsenoside F1 isolated in Example 1 according to the present invention is added to flour at 0.1 to 5.0% by weight, and bread, cake, cookies, crackers and noodles are prepared using this mixture to prepare foods for health promotion. It was.

2-3. soup  And juicy ( gravies Manufacturing

Ginsenoside F1 isolated in Example 1 according to the present invention was added to the soup and gravy by 0.1 to 1.0% by weight to prepare meat products for health promotion, soup and gravy of noodles.

2-4. dairy product( dairy products Manufacturing

Ginsenoside F1 isolated in Example 1 according to the present invention was added to milk at 0.1 to 1.0% by weight, and various dairy products such as butter and ice cream were prepared using the milk.

< Example  3: drink Production Example >

3-1. Vegetable Juice  Produce

0.5 g of ginsenoside F1 isolated in Example 1 according to the present invention was added to 1,000 ml of tomato or carrot juice to prepare vegetable juice for health promotion.

3-2. Fruit juice  Produce

0.1 g of ginsenoside F1 isolated in Example 1 according to the present invention was added to 1,000 ml of apple or grape juice to prepare a fruit juice for health promotion.

Claims (8)

Prevention of hypertension or angina characterized in that it comprises at least one of ginsenoside F1 and notoginsenoside R1 of formula 1 isolated from leaves, stems or buds of ginseng Therapeutic composition.
[Formula 1]

delete delete Prevention of hypertension or angina characterized in that it comprises at least one of ginsenoside F1 and notoginsenoside R1 of formula 1 isolated from leaves, stems or buds of ginseng Health functional food for improvement.
[Formula 1]

The method of claim 4, wherein
The health functional food is selected from the group consisting of nutritional products including drinks, meat, sausages, bread, candy, snacks, noodles, dairy products including ice cream, soups, beverages including ionic drinks, alcoholic beverages and vitamin complexes Health functional food for the prevention or improvement of hypertension or angina characterized in that it is. delete delete delete

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