KR101126276B1 - Pharmaceutical composition for preventing and treating cardiovascular diseases comprising triterpenoid saponins - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention and treatment of cardiovascular diseases, comprising a triterpenoid saponin-based compound or a pharmaceutically acceptable salt thereof as an active ingredient. The triterpenoid saponin-based compounds according to the present invention can induce vascular endothelial cell activation by relaxing blood vessels, lowering blood pressure, increasing endothelial nitric oxide synthase (eNOS) expression, and inducing NAD (P) H. Of P47 ^phox , a component of oxidase There is an activity that can suppress the generation of reactive oxygen by NAD (P) H oxidase by reducing the expression. Therefore, the triterpenoid saponin-based compound or a pharmaceutically acceptable salt thereof may be usefully used for preventing or treating cardiovascular diseases.

Triterpenoids, saponins, cardiovascular system, diseases

Description

Pharmaceutical composition for preventing and treating cardiovascular diseases comprising triterpenoid saponins}

The present invention relates to a pharmaceutical composition for the prevention and treatment of cardiovascular diseases, comprising a triterpenoid saponin-based compound or a pharmaceutically acceptable salt thereof as an active ingredient.

Saponin is present in plant roots, stems, leaves, bark, seeds, etc., and has been used as an herbal medicine since it has a strong effect as a cardiac medicine or diuretic. In addition, saponins are known to have surfactant activity, cholesterol and complex formation, hemolytic action, antibacterial action, contraception, growth inhibition, expectoration, anti-inflammatory, analgesic, antiviral, enzyme inhibition and anticancer activity (Hostettmann K et al., Plant Biochem , 7: 435-471, 1991; Lacaille-Dubois et al., Phytomedicine , 2: 363-386, 1996; Price KR et al., CRC Crit Rev Food Sci Nutr , 26-27-135, 1987).

The saponin is a glycoside compound in a form in which aglycone (non-sugar portion) is structurally bound to one or more sugars and has a molecular weight of 600 to 2,000 Daltons or more. The sugar part of saponin is well known D-glucose, D-galactose, L-arabinose and aglycone is composed of triterpenoids or steroids. Saponins are roughly classified into triterpenoid saponins and steroid saponins according to the kind of aglycone.

Cardiovascular disease is a disorder of the heart and vascular system, including heart disease, diseases of the central and lower organs, such as the aorta, and peripheral blood vessels of tissues. According to the World Health Organization (WHO) statistics, the number of deaths from cardiovascular diseases is increasing every year, especially in Asia, including Japan and Korea.

There are various causes of cardiovascular disease, but it is now known that cardiovascular diseases such as atherosclerosis and hypertension are caused by increased free radical species in the blood vessels caused by NAD (P) H oxidase. Babior, Blood 93: 1464-1476, 1999, Griendling et al., Cir.Res. 86: 494-501, 2000). In addition, endothelial cells of blood vessels normally release NO to relax blood vessels, regulate blood flow, and protect against thrombosis of blood vessel walls. When pathologically abnormal function of endothelial cells is released, endothelial cells secrete NO. Is known to cause several cardiovascular dysfunctions (Albrecht et. Al., J. Pathol . 199: 817, 2003). Due to the above causes, diseases such as arteriosclerosis, hypertension, angina pectoris, hyperlipidemia, myocardial infarction, stroke and heart failure have been found in patients with cardiovascular diseases.

In order to treat such cardiovascular diseases, various compounds are currently used according to the site of action and the mechanism of action. For example, various antihypertensive agents, such as diuretics, antiadrenergic drugs, vasodilators, and angiotensin converting enzyme inhibitors, have been used as antihypertensive agents.

However, the triterpenoid saponin-based compounds derived from the elm are not known to have a preventive and therapeutic effect on cardiovascular diseases by blocking the cause of the above cardiovascular diseases.

Therefore, the present inventors have studied for many years to find a natural-derived compound having a therapeutic activity of cardiovascular diseases. The invention has been completed.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for the prevention and treatment of cardiovascular diseases, characterized in that it contains a triterpenoid saponin-based compound or a pharmaceutically acceptable salt thereof as an active ingredient.

In order to solve the above problems,

The present invention provides a pharmaceutical composition for the prevention and treatment of cardiovascular diseases, comprising a triterpenoid saponin-based compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

Wherein R1 is alpha-L-rhamnopyranosyl (1 → 2) -alpha-L-arabinopyranosyl (α-L-rhamnopyranosyl- (1 → 2) -α-L-arabinopyranosyl) or hydrogen, and R2 is Methanol (CH ₃ OH) or hydrogen, R3 is beta-D-xylpyranosyl (1 → 6) -beta-D-glucopyranosyl (β-D-xylopyranosyl- (1 → 6) -β-D- glucopyranosyl), beta-D-glucopyranosyl and beta-D-glucopyranosyl (1 → 6) -beta-D-glucopyranosyl (β-D-glucopyranosyl- (1 → 6) -β-D-glucopyranosyl).

Hereinafter, the present invention will be described in detail.

The pharmaceutical composition for preventing and treating cardiovascular diseases of the present invention is characterized by containing a triterpenoid saponin-based compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof as an active ingredient.

<Formula 1>

Wherein R1 is alpha-L-rhamnopyranosyl (1 → 2) -alpha-L-arabinopyranosyl (α-L-rhamnopyranosyl- (1 → 2) -α-L-arabinopyranosyl) or hydrogen, and R2 is Methanol (CH ₃ OH) or hydrogen, R3 is beta-D-xylpyranosyl (1 → 6) -beta-D-glucopyranosyl (β-D-xylopyranosyl- (1 → 6) -β-D- glucopyranosyl), beta-D-glucopyranosyl and beta-D-glucopyranosyl (1 → 6) -beta-D-glucopyranosyl (β-D-glucopyranosyl- (1 → 6) -β-D-glucopyranosyl).

The triterpenoid saponin-based compound is not limited thereto but is preferably 3- O -alpha-L-ramnopyranosyl (1 → 2) -alpha-L-arabinofyranosyl-hederagenin 28- O -beta -D-xylpyranosyl (1 → 6) -beta-D-glucopyranosyl ester, 3beta-hydroxyolean-12-ene-28-phosphoric acid and 3- O -alpha-L-ramnopyranosyl (1 → 2) -alpha-L-arabinopyranosyl-hederagenin 28- O -beta-D-glucopyranosyl (1 → 6) -beta-D-glucopyranosyl ester have. Specifically, the 3- O -alpha-L-ramnopyranosyl (1 → 2) -alpha-L-arabinopyranosyl-hederagegenin 28- O -beta-D-xylpyranosyl (1 → 6)- Beta-D-glucopyranosyl ester (3- O -α-L-rhamnopyranosyl (1 → 2) -α-L-arabinopyranosyl-hederagenin 28- O- β-D-xylopyranosyl (1 → 6) -β-D- glucopyranosyl ester) may be represented by the following Chemical Formula 2, and 3beta-hydroxyolean-12-ene-28-ophosphoric acid (3β-hydroxyolean-12-en-28-oic acid) may be represented by the following Chemical Formula 3. 3- O -alpha-L-ramnopyranosyl (1 → 2) -alpha-L-arabinopyranosyl-hederagegenin 28- O -beta-D-glucopyranosyl (1 → 6) -beta -D-glucopyranosyl ester (3- O -α-L-rhamnopyranosyl (1 → 2) -α-L-arabinopyranosyl-hederagenin 28- O- β-D-glucopyranosyl (1 → 6) -β-D-glucopyranosyl ester) may be represented by the following formula (4).

The triterpenoid saponin-based compound may be isolated and purified from a plant or part of a plant. Preferably from Aralia elata , Pulsatilla cernua (Zhang QW et al., Yao Xue Xue Bao , Oct: 35 (10), 756-759, 2000) using methods known in the art Can be isolated and purified. Most preferably, it can be separated and purified from Aralia elata . Specifically, all or part of the plant (root, leaf, stem) is extracted with purified water or an organic solvent to obtain an extract from the triterpenoid saponin-based compound can be isolated and purified from the known method. Examples of the organic solvent include, but are not limited to, various solvents such as methanol, ethanol, propanol, butanol, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane, cyclohexane and petroleum ether. Preferably, methanol, ethanol and butanol can be used. Separation and purification of the triterpenoid saponin-based compound from the plant extract can be performed by using a single column or column chromatography packed with various synthetic resins such as silica gel or activated alumina, or high performance liquid chromatography. However, the isolation and purification of the triterpenoid saponin-based compound according to the invention is not limited to the above-described method.

In an embodiment of the present invention, methanol extract of Aralia elata was fractionated with butanol and water to prepare butanol extract, and the butanol extract was separated and purified by silica gel column chromatography to obtain a triterpenoid saponin-based compound (<exemplary embodiment). See Example 1).

The triterpenoid saponin-based compound according to the present invention has an excellent vasorelaxant effect and an excellent activity for lowering blood pressure.

In an experimental example of the present invention, the blood pressure change was measured by administering the triterpenoid saponin-based compound to congenital hypertension mice and mice induced with high blood pressure by angiotensin II, and thus increased blood pressure to a degree similar to that of rozatan, which is a conventional hypertension treatment agent. Was suppressed (see <Experimental Example 1>). Furthermore, as a result of specifying the degree of vascular relaxation by administering the triterpenoid saponin-based compound to the mouse, it was found that blood vessels were relaxed better than lozatan (losartan), a conventional hypertension treatment agent (see <Experimental Example 2>).

In addition, the triterpenoid saponin-based compound according to the present invention has the activity of increasing eNOS (endothelial nitric oxide synthase, nitric oxide synthase) expression. The nitric oxide synthase synthesizes nitric oxide (NO) in vascular endothelial cells, thereby relaxing blood vessels, regulating blood flow, and protecting blood vessel walls from thrombus formation (Cannon RO). , Clin Chem . 44: 1809-1819, 1998). Therefore, when the activity of the nitric oxide synthase is inhibited and abnormalities in the function of vascular endothelial cells may cause cardiovascular diseases, and in particular, endothelial abnormalities during the pathogenesis of hypertension, a representative example of cardiovascular diseases. dysfunction) (Albrecht et. al., J. Pathol . 199: 817, 2003).

In one experimental example of the present invention, the triterpenoid saponin-based compound was administered to a high blood pressure-induced mouse to analyze the expression level of the nitric oxide synthase, and a control group administered physiological saline and rozatan, a conventional hypertension therapeutic agent It was found that the expression of eNOS was significantly increased compared with the case of administration of NO, and it was found that the secretion of NO was increased (see <Experimental Example 3>).

In addition, the triterpenoid saponin-based compound according to the present invention has an activity capable of reducing P47 ^phox expression and inhibiting the generation of reactive oxygen by NAD (P) H oxidase. The P47 ^phox is a component of NAD (P) H oxidase, and when its expression is reduced, the generation of free radicals by NAD (P) H oxidase may be inhibited, thereby preventing or treating cardiovascular disease (Boggrell SA). et al., Drug News Perspect . 17 (9): 615-632, 2005; Inoguchi T., Curr Drug Targets 6 (4): 495-501, 2005; Muzaffar S. et al., Trends Cardiovasc Med. 15 (8) ): 278-282).

In one experimental example of the present invention, administration of the triterpenoid saponin-based compound to the hypertensive-induced hypertensive mice and the angiotensin II-induced mice showed that the expression of P47 ^phox and the generation of free radicals were inhibited. <Experiment 4>).

As described above, the triterpenoid saponin-based compound according to the present invention can induce vascular endothelial cell activation by decreasing blood pressure and lowering blood pressure and increasing eNOS (endothelial nitric oxide synthase) expression and inducing vascular endothelial cell activation. Of P47 ^phox , a component of P) H oxidase Since there is an activity that can suppress the generation of free radicals by NAD (P) H oxidase by reducing the expression can be useful for preventing or treating cardiovascular diseases.

The cardiovascular disease is a disease caused by abnormalities of the heart and the vascular system, and includes a disease caused by abnormalities of heart and blood vessels, central blood vessels and lower organs such as aorta, and peripheral blood vessels of tissues. Specifically, the cardiovascular disease is not limited thereto but preferably refers to arteriosclerosis, hypertension, angina pectoris, hyperlipidemia, myocardial infarction and heart failure.

In the case of the pharmaceutical composition, the triterpenoid saponin-based compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof may be included alone or one or more pharmaceutically acceptable carriers, excipients or diluents may be further included. can do.

As used herein, “pharmaceutically acceptable salts” means salts suitable for use in contact with tissues of humans and lower animals without causing excessive toxicity, irritation, allergic reactions, etc. within the scope of medical judgment. Kinds of such pharmaceutically acceptable salts are well known in the art. For example, the pharmaceutically acceptable salts are described in detail in the literature (SM Berge et al., J. Pharmaceutical Sciences , 1977, 66: 1). Pharmaceutically acceptable salts can be broadly divided into acid addition salts and basic addition salts. Acid addition salts of the triterpenoid saponin-based compounds according to the invention can be prepared in situ during the final separation and purification of the compounds of the invention or by reacting the free base functional groups with suitable organic acids in separate reaction systems. Representative acid addition salts include, but are not limited to, acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsulfonate, digluconate, glycerol Phosphate, hemisulfate, heptanoate, hexanoate, fumarate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate (isothionate), lactate, maleate, methanesulfonate, Nicotinate, 2-naphthalenesulfonate, oxalate, palmitoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, Phosphate, glutamate, bicarbonate, p-toluenesulfonate and udecanoate It is included. Examples of acids that can be used to form acid addition salts include inorganic acids such as hydrochloric acid, bromic acid, sulfuric acid and phosphoric acid and organic acids such as oxalic acid, maleic acid, succinic acid and citric acid. Basic addition salts may be used to convert carboxylic acid moieties in situ during the final separation and purification of the compounds of the invention into suitable bases (e.g., hydroxides, carbonates or bicarbonates of pharmaceutically acceptable metal cations). Nate), ammonia, organic primary amines, secondary amines or tertiary amines. Pharmaceutically acceptable basic addition salts include, but are not limited to, alkali metal or alkaline earth metal cations such as lithium, sodium, potassium, calcium, magnesium and aluminum salts, nontoxic quaternary ammonia and amine cations (ammonium, tetramethyl Ammonium, tetraethylammonium, methylammonium, dimethylammonium, trimethylammonium, triethylammonium, diethylammonium and ethylammonium). Other representative organic amines useful for the formation of base addition salts include ethyleneamine, ethanolamine, diethanolamine, piperidine, piperazine and the like.

The method of administering the pharmaceutical composition to a mammal is not particularly limited and may be administered orally or parenterally, for example. Parenteral administration methods include, but are not limited to, subcutaneous, intravenous, intramuscular or intraperitoneal administration. Preferably the pharmaceutical composition of the present invention may be administered orally.

The pharmaceutical composition may be formulated into a preparation for oral or parenteral administration according to the route of administration as described above.

In the case of preparations for oral administration, the compositions of the present invention may be formulated using methods known in the art as powders, granules, tablets, pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions and the like. Can be. For example, oral formulations can obtain tablets or dragees by combining the active ingredients with solid excipients and then grinding them, adding suitable auxiliaries and processing them into granule mixtures. Examples of suitable excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol and starch, cellulose, including starch, corn starch, wheat starch, rice starch and potato starch, etc. Fillers such as cellulose, gelatin, polyvinylpyrrolidone, and the like, including methyl cellulose, sodium carboxymethylcellulose, hydroxypropylmethyl-cellulose, and the like. In addition, crosslinked polyvinylpyrrolidone, agar, alginic acid or sodium alginate and the like may optionally be added as a disintegrant. Furthermore, the pharmaceutical composition may further include an anticoagulant, a lubricant, a humectant, a perfume, an emulsifier, a preservative, and the like.

Formulations for parenteral administration may be formulated in the form of injections by methods known in the art. These formulations are described in Remington's Pharmaceutical Science, 15th Edition, 1975. Mack Publishing Company, Easton, Pennsylvania 18042, Chapter 87: Blaug, Seymour, a prescription generally known in all pharmaceutical chemistries.

Suitable dosages of the pharmaceutical compositions according to the invention are about 0.1 mg / kg body weight / day to 2000 mg / kg body weight / day, more preferably about 0.2 mg / kg body weight / day to 250 mg / kg body weight / day. However, the dosage of the composition according to the present invention may be appropriately selected depending on various factors such as the route of administration, the age, sex, weight and severity of the patient.

As described above, the triterpenoid saponin-based compounds according to the present invention can induce activation of vascular endothelial cells by relaxing blood vessels, lowering blood pressure and increasing eNOS (endothelial nitric oxide synthase) expression. Of P47 ^phox , a component of NAD (P) H oxidase There is an activity that can suppress the generation of reactive oxygen by NAD (P) H oxidase by reducing the expression. Therefore, the triterpenoid saponin-based compound or a pharmaceutically acceptable salt thereof may be usefully used for preventing or treating cardiovascular diseases.

Hereinafter, the present invention will be described in detail by way of examples.

However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

&Lt; Example 1 >

Isolation of Triterpenoid Saponins Compounds with Blood Pressure Lowering Activity from Aralia elata

Aralia elata Seemen (AE) was collected from Halla Mountain in Jeju Island and used for the experiment. Samples were taken, dried in the shade, and finely chopped and leached by adding 10 L of 80% methanol to 1 kg of arbor. The leached sample was extracted with ultrasonic decomposition for 24 hours, and then filtered using a vacuum suction filter. The solvent extract obtained by repeating the above procedure three times was concentrated with a rotary concentrator to obtain a methanol extract (100 g). The methanol extract obtained above was suspended in water and extracted by adding butanol using a separatory funnel to obtain a butanol fraction (46 g). The butanol fractions were charged with silica gel and then loaded into a column (5 × 17 cm) to perform column chromatography with chloroform: methanol: water sequentially changing from 7: 3: 1 to 6: 4: 1 to 10 Fractions were obtained.

The obtained fractions were orally administered (500 mg / kg) to congenital hypertensive mice (SHR) once, and then measured by indirect method without change of blood pressure (constrictor blood pressure; SBP) and heart rate for 24 hours.

As a result of the experiment, a fraction having blood pressure lowering activity was selected and structural identification was performed using NMR according to a method known to those skilled in the art. As a result, triterpenoid saponins having blood pressure lowering activity of Chemical Formulas 2 to 4 below The compound was separated. Specifically, 3- O -alpha-L-ramnopyranosyl (1 → 2) -alpha-L-arabinofyranosyl-hederagenine 28- O -beta-D-xylpyranosyl (1) → 6) -beta-D-glucopyranosyl ester (3- O -α-L-rhamnopyranosyl (1 → 2) -α-L-arabinopyranosyl-hederagenin 28- O- β-D-xylopyranosyl (1 → 6)- β-D-glucopyranosyl ester), 3beta-hydroxyolean-12-ene-28-ophosphoric acid represented by formula (3) and 3 represented by formula (4) O -alpha-L-ramnopyranosyl (1 → 2) -alpha-L-arabinopyranosyl-hederagenin 28- O -beta-D-glucopyranosyl (1 → 6) -beta-D-glucose Pyranosyl ester (3- O -α-L-rhamnopyranosyl (1 → 2) -α-L-arabinopyranosyl-hederagenin 28- O- β-D-glucopyranosyl (1 → 6) -β-D-glucopyranosyl ester) The compound of Formula 2 is referred to as aralia saponin 1, and Formula 3 is represented by oleanolic acid ester and 4 was named dipsacoside B.

NMR Results of Aralia Saponin 1 Carbon no. δ _C Carbon no. δ _C δ _H One 39.02 3-Ο-Arabinose 2 26.16 C-1 104.25 5.03 (d, J = 5.6 Hz) 3 81.04 C-2 75.84 4 43.38 C-3 74.45 5 47.65 C-4 69.23 6 18.19 C-5 65.48 7 32.78 3-Ο-Rhamnose 8 39.91 C-1 101.63 6.10 (br. S) 9 48.18 C-2 72.22 10 36.87 C-3 72.48 11 23.35 C-4 73.98 12 122.95 C-5 69.74 13 144.10 C-6 18.55 1.54 (d, J = 6.0 Hz) 14 42.12 28-Ο-Glucose 15 28.26 C-1 95.58 6.15 (d, J = 8.0 Hz) 16 23.85 C-2 73.80 17 47.03 C-3 78.60 18 41.68 C-4 71.01 19 46.20 C-5 78.05 20 30.78 C-6 69.05 21 33.96 28-Ο-Xylose 22 32.56 C-1 105.47 4.81 (d, J = 7.2 μs) 23 63.97 C-2 74.74 24 13.95 C-3 77.76 25 16.21 C-4 70.71 26 17.58 C-5 67.02 27 26.12 28 176.62 29 33.19 30 23.71

NMR Results of Oleanolic Acid Ester Carbon no. δ _C Carbon no. δ _C δ _H One 38.91 Glc-1 95.72 6.32 (d, J = 8.0 Hz) 2 26.25 2 74.10 3 74.3 3 79.33 4 39.53 4 71.05 5 55.77 5 78.86 6 18.50 6 62.17 7 33.2 8 39.94 9 48.00 10 36.96 11 23.73 12 123.2 5.41 (br. S) 13 144.0 14 42.16 15 28.28 16 23.76 17 47.04 18 41.78 19 46.21 20 30.86 21 34.12 22 32.86 23 28.26 24 17.12 25 15.66 26 17.55 27 26.24 28 176.3 29 33.24 30 23.75

NMR results of dipsacoside B Carbon no. δ _C Carbon no. δ _C δ _H One 39.39 3-Ο-Arabinose 2 26.61 C-1 104.61 5.07 (d, J = 6.0 Hz) 3 81.32 C-2 76.15 4 43.86 C-3 74.96 5 48.54 C-4 69.65 6 18.57 C-5 65.97 7 33.17 3-Ο-Rhamnose 8 40.26 C-1 101.97 6.19 (br. S) 9 48.04 C-2 72.65 10 37.24 C-3 72.87 11 24.23 C-4 74.42 12 123.14 C-5 70.06 13 144.33 C-6 18.96 1.59 (d, J = 6.0 Hz) 14 42.50 28-Ο-Glucose 15 28.68 C-1 95.95 6.21 (d, J = 8.4 Hz) 16 23.76 C-2 74.20 17 47.38 C-3 79.00 18 42.03 C-4 71.17 19 46.54 C-5 78.27 20 31.11 C-6 78.27 21 34.33 28-Ο-Xylose 22 32.91 C-1 105.50 4.98 (d, J = 7.6 Hz) 23 64.28 C-2 75.46 24 14.37 C-3 78.76 25 16.61 C-4 71.78 26 17.96 C-5 78.68 27 26.43 C-6 62.90 28 176.72 29 33.48 30 24.06

<Formula 2>

<Formula 3>

<Formula 4>

Experimental Example 1

Blood pressure lowering effect of triterpenoid saponins compound of the present invention

Congenital hypertension using aralia saponin 1 and oleanolic acid ester isolated in <Example 1> using an osmotic minipump at a dose of 30 mg / kg / day The mice were injected into the abdominal cavity for 7 days and the change in blood pressure was observed and the results are shown in FIG. 1A. At this time, the case of administration of physiological saline as a negative control, the case of administering the same amount of lozatan (losartan), a conventional hypertension treatment as a positive control

As described in FIG. 1A, when the aralia saponin 1 and the oleanolic acid ester isolated in Example 1 were administered, the blood pressure was significantly decreased after 1 day of administration. It showed a similar effect as that of rozatan (30 mg / kg / day), which is a conventional hypertension treatment.

On the other hand, in the mice with hypertension induced by angiotensin II, the compound isolated in Example 1 was administered in the same manner as described above, and then blood pressure change was observed, and the results are shown in FIG. 1B.

As shown in FIG. 1B, when the compound isolated in Example 1 was administered, blood pressure increase by angiotensin II was significantly inhibited, and similar effect to that of rozatan, which is a conventional hypertension treatment agent, was observed.

Therefore, it was found that the compound isolated in Example 1 may be usefully used for treating and preventing hypertension.

Experimental Example 2

Vasorelaxant effect of triterpenoid saponins compounds of the present invention

In order to determine whether the compound of the present invention has a blood pressure-lowering effect in the <Experimental Example 1> because of the vascular relaxation activity, the following further experiments were carried out.

Thoracic artery was extracted by sacrificing congenital hypertensive mice used in Experimental Example 1, and Krebs solution (NaCl 120mM, KCl 4.75mM, Glucose 6.4mM, NaHCO ₃ 25mM, KH ₂ PO ₄ 1.2mM, MgSO ₄ 1.2mM, After transferring to Ca Pet ₂ 1.7mM) filled Petri dishes, connective tissue and perivascular tissues such as fat were removed and a 3 mm thoracic aortic ring was made. Suspension was carried out with a stainless tissue holder specially prepared in an organ bath filled with oxygen-saturated Krebs solution. At this time, the temperature in the organ bath was maintained at 37 ℃ and the pH of the solution was maintained at 7.4. Resting tension was applied to the extracted thoracic artery (1 g), followed by stabilization for 1 hour while changing the bath solution every 15 minutes. Vascular contraction and relaxation reactions connect the other side of the fixed aortic ring to an isometric force displacement transducer (FT03, Grass, AD instruments, USA), to the physiograph recorder (PowerLab / 400, AD instruments, USA). Recorded and analyzed with Chart5 for Windows program (AD instruments, USA). At this time, endothelial cell-dependent vascular relaxation test was performed by pre-constricting blood vessels with norepinephrine 10 ^-7 M in blood vessels in which endothelial cells were intact, and then acetylcholine was applied cumulatively to observe the relaxation response of each group. The results using the congenital hypertension mouse of the experimental results are shown in FIG.

As shown in FIGS. 2A and 2B, when the compound isolated in Example 1 was administered, it showed higher endothelial cell-dependent vasorelaxation effect than conventional losartan.

<Experimental Example 3>

The effect of promoting the expression of endothelial nitric oxide synthase (eNOS) and increasing the NO secretion of triterpenoid saponins compounds

Expression of eNOS in the aorta to confirm whether the compound of the present invention of Example 1 is due to the increased expression of eNOS (endothelial nitric oxide synthase) in <Experimental Example 2> The degree was observed.

The thoracic artery extracted in Experimental Example 2 was homogenized buffer (50 mM Tris-HCl, pH 7.5, 150 mM Sodium chloride, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 2mM EDTA, 5µg / ml aprotinin, 5 μg / ml leupeptin, 1 mM DTT, 1 mM PMSF) was added to homogenize. The homogenate was centrifuged at 12,000 g, 4 ° C. for 20 minutes to obtain a supernatant, and the expression of eNOS in the supernatant was analyzed by Western blot. Specifically, the protein (40 ㎍ / sample) contained in the supernatant was quantified by Bradford assay and electrophoresed with 8% SDS-polyacrylamide using a mini-gel system (Bio-Rad Laboratories, USA). After the electrophoresis, the protein was recovered using a 45 μm PVDF membrane (Millipore, USA), and the membrane was blocked in TTBS (TBS + 0.1% Tween 20) solution containing 5% skim milk, and the anti-mouse eNOS antibody was used as the primary antibody. (1: 1000 dilution; BD Transduction Laboratories, USA) and anti-mouse α-actin antibody (1: 5000 dilution; Santa Cruz Biotechnology) were used, and the secondary antibody was HRP coupled mouse IgG (Horseradish peroxidase-conjugated goat anti). -mouse IgG, 1: 5,000 dilution; Santa Cruz Biotechnology) was used to confirm the expression level of eNOS by ECL system (Amersham Biosciences, USA) and analyzed using MCID Analysis 7.0 software (MCID ^™ , USA). The experimental results are shown in FIG. 3.

As shown in FIG. 3, when the compound isolated in <Example 1> was administered, eNOS expression was significantly increased in the aorta compared to the control group administered with saline, and loartan (losartan), which is a conventional hypertension treatment There was no significant change in eNOS expression when administered.

On the other hand, the following experiments were further performed to determine whether to increase the secretion of nitric oxide (NO) by increasing the expression of the endothelial nitric oxide synthase (eNOS).

Specifically, the blood of the mouse used in Experimental Example 1 was collected in a test tube containing EDTA, and centrifuged to separate plasma, and twice the amount of methanol collected was added thereto, followed by centrifugation at 10,000 rpm for 10 minutes. The supernatant was separated and the NOx was quantified by an NO detector-HPLC system (ENO20; Eicom Co., Kyoto, Japan). The results are shown in FIG. 4.

As shown in FIG. 4, all of the compounds isolated in Example 1 increased the secretion of nitric oxide (NO), indicating that NOx was contained in the blood.

Therefore, the compound isolated in Example 1 may increase eNOS protein expression, thereby increasing the secretion of nitric oxide (NO), thereby relaxing blood vessels, lowering blood pressure, regulating blood flow or forming blood clots in blood vessels. It was found that it can be inhibited.

Experimental Example 4

P47 of the triterpenoid saponins compound of the present invention ^phox  Expression inhibition and free radical reduction

The thoracic artery extracted in Experimental Example 2 was analyzed for p47 ^phox expression using the same Western blot method as in Experimental Example 3. Anti-mouse eNOS antibody (1: 1000 dilution; BD Transduction Laboratories, USA) and anti-mouse α-actin antibody (1: 5000 dilution; Santa Cruz Biotechnology) were used as primary antibodies. The results using the congenital hypertension mice of the experimental results are shown in FIGS. 5A and 5B, and the results using the mice induced by high blood pressure by angiotensin II are described in FIGS. 5C and 5D.

As shown in FIG. 5, when the compound isolated in Example 1 was administered to congenital hypertension mice, it was found that the expression of P47 ^phox was more effectively inhibited than that of lozatan, a conventional antihypertensive drug, and angiotensin. When administered to mice induced by hypertension II, it was found that the expression of P47 ^phox was suppressed similarly to loartan, which is a conventional antihypertensive drug.

Meanwhile, the abdominal aorta of the mouse used in <Experimental Example 1> was extracted and fixed without forming a frozen block using an OCT (Optimal Cutting Temperature) compound, and the frozen slicer (CM 3050 S, LEICA) was 30 μm thick. , Germany), and then reacted with DHE (dihydroethidium (10 μM) for 30 minutes in a shaded humidified laboratory at 37 ° C., followed by fluorescence microscopy (excitation, 515-560 nm; emission, 590 nm, LEICA, Germany). The degree of fluorescence expression was analyzed using MCID Analysis 7.0 software (MCID ^™ , USA), and the results using congenital hypertension mice are shown in FIGS. 6A and 6B, and hypertensive mice were induced by angiotensin II. The results using are described in Figures 6C and 6D.

As shown in FIG. 6, when the compound isolated in Example 1 was administered to congenital hypertension mice, it was found that the generation of active oxygen was more effectively inhibited than that of lozatan, which is a conventional antihypertensive drug, and angiotensin. In the case of administration to mice with hypertension induced by II, it was found that the generation of free radicals was more effectively suppressed than that of lozatan, which is a conventional hypertension treatment agent.

Therefore, the compound isolated in Example 1 can reduce the expression of P47 ^phox , which is a component of the NAD (P) H oxidase, thereby inhibiting the generation of free radicals caused by NAD (P) H oxidase. It can be effectively used in the prevention and treatment of related diseases (Boggrell SA et al., Drug News Perspect. 17 (9) 615-632, 2005, Inoguchi T., Curr Drug Targets 6 (4): 495-501, 2005, Muzaffar S. et al., Trends Cardiovasc Med. 15 (8): 278-282).

<Manufacture example 1>

Preparation of a medicament containing the triterpenoid saponin-based compound as an active ingredient in the present invention

<1-1> Preparation of Tablet

Aralia saponin 1, oleanolic acid ester or dipsacoside B 25 mg of the present invention is a lactose for excipients 26 mg and 3.5 mg avicel (microcrystalline cellulose), sodium starch glyconide as a disintegration aid, and lactic acid L as a binder -HPC (low-hydrosyprophylcellulose) with 8 mg in a U-type mixer and mixed for 20 minutes. After mixing was completed, 1 mg of magnesium stearate was further added as a lubricant and mixed for 3 minutes. Tablets were prepared by tableting and film coating after a quantitative test and a moisture test.

<1-2> Preparation of Syrup

2 g of acid addition salt, 0.8 g of saccharin, and 25.4 g of sugar of Aralia saponin 1, oleanolic acid ester or dipsacoside B of the present invention were dissolved in 80 g of warm water. After cooling the solution, 8.0 g of glycerin, 0.04 g of flavor, 4.0 g of ethanol, 0.4 g of sorbic acid, and an appropriate amount of distilled water were mixed. Water was added to the mixture to 100 ml.

&Lt; 1-3 > Preparation of capsules

A capsule is prepared by mixing Aralia saponin 1, oleanolic acid ester or dipsacoside B 50 mg, lactose 50 mg, starch 46.5 mg, talc 1 mg, and an appropriate amount of magnesium stearate and filling it into a hard gelatin capsule of the present invention. It was.

<1-4> Preparation of Injection

Arali saponin 1, oleanolic acid ester of the present invention or 1 g of hydrochloride of dipsacoside B, 0.6 g of sodium chloride and 0.1 g of ascorbic acid were dissolved in distilled water to obtain a final volume of 100 ml. The solution was filled in ampoules and heat sterilized.

The triterpenoid saponin-based compounds according to the present invention can induce vascular endothelial cell activation by relaxing blood vessels, lowering blood pressure, increasing endothelial nitric oxide synthase (eNOS) expression, and inducing vascular endothelial cells. Of P47 ^phox , a component of H oxidase There is an activity that can suppress the generation of reactive oxygen by NAD (P) H oxidase by reducing the expression. Therefore, the triterpenoid saponin-based compound or a pharmaceutically acceptable salt thereof may be usefully used for preventing or treating cardiovascular diseases.

1A is a graph showing the blood pressure lowering activity of the compound by administering a triterpenoid saponins compound of the present invention to congenital hypertensive mice (SHR: control group administered with saline solution, OA: oleanoic acid ester) , AS: Aralia saponin 1, LOS: Losartan, a therapeutic agent for hypertension, ^*** : p <0.001 compared with SHR).

FIG. 1B is a graph illustrating the blood pressure lowering activity of the triterpenoid saponins compound of the present invention by administering to the mouse hypertension induced by angiotensin II (CON: control group administered physiological saline) Ang II: When angiotensin II was administered, Ang II + OA 30: Angiotensin II and oleanolic acid ester (30 mg / kg / day), Ang II + AS1 30: Angiotensin II and Aralia saponin 1 (30 mg / kg / day), Ang II + LOS 30: angiotensin II and losartan (30 mg / kg / day), ⁺⁺⁺ : p <0.001, ^* : p <0.05 compared to control group , ^** : p <0.01 compared with AngII, ^*** : p <0.001 compared with AngII).

FIG. 2A is a graph illustrating the vascular relaxation activity of the triterpenoid saponins compound of the present invention to congenital hypertensive mice (CON: control group administered physiological saline solution, OA: oleanoic acid ester) , AS: Aralia saponin 1, LOS: Losartan, a therapeutic agent for hypertension, ^* : p <0.05 compared to SHR, ^** : p <0.01 compared to SHR).

Figure 2B is a graph of the triterpenoid saponins of the present invention administered to angiotensin II-induced hypertension induced mice to confirm the vasorelaxant activity of the compound (CON: control group administered physiological saline, Ang II: When angiotensin II was administered, Ang II + OA 30: Angiotensin II and oleanolic acid ester (30 mg / kg / day), Ang II + AS1 30: Angiotensin II and Aralia saponin 1 (30 mg / kg / day), Ang II + LOS 30: angiotensin II and losartan (30 mg / kg / day), ⁺⁺⁺ : p <0.001, ^* : p <0.05 compared to control group , ^** : p <0.01 compared with AngII, ^*** : p <0.001 compared with AngII).

3 is a graph showing the effect of triterpenoid saponins compounds of the present invention on the expression of endothelial nitric oxide synthase (eNOS) in blood vessels (SHR: control group administered with saline solution, OA: oleanoic acid Ester, AS: Aralia saponin 1, LOS: Losartan, a drug for treating hypertension, ^* : p <0.05 compared to SHR, ^** : p <0.01 compared to SHR)

Figure 4 is a graph showing the effect of triterpenoid saponins compounds of the present invention on the secretion of NO (OAE: oleanolic acid ester, AS1: Aralia saponin 1, LOS: high blood pressure therapeutic agent Rosatan ( losartan), DSB: deep sacoside B)

5 is a graph showing the effect of triterpenoid saponins compound of the present invention on p47 ^phox expression (SHR: control group administered with saline solution, OA: oleanolic acid ester, AS: aralia saponin 1, LOS: losartan, a treatment for hypertension, Ang II: angiotensin II, SD: control group administered with saline solution).

A, B: congenital hypertensive mice ( ^** : p <0.01 compared to SHR, ^*** : p <0.001 compared to SHR)

C, D: High blood pressure induced mice by angiotensin II ( ⁺⁺ : p <0.01 compared to control, ^*** : p <0.001 compared to Ang II)

6 is a graph showing the effect of triterpenoid saponins compounds of the present invention on the amount of active oxygen generation (SHR: control group administered with saline solution, OA: oleanolic acid ester, AS: aralia saponin 1, LOS: losartan, a treatment for hypertension, Ang II: angiotensin II, SD: control group administered with saline solution).

A, B: congenital hypertensive mice ( ^** : p <0.01 compared to SHR, ^*** : p <0.001 compared to SHR)

C, D: High blood pressure induced mice by angiotensin II ( ⁺⁺ : p <0.01 compared to control, ^*** : p <0.001 compared to Ang II)

Claims (4)

A pharmaceutical composition for preventing and treating cardiovascular diseases, comprising a triterpenoid saponin-based compound represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient; <Formula 1>

Wherein R ₁ is an alpha -L- ramno pyrazol nosil (1 → 2) - alpha -L- arabino pyrazol nosil (α-L-rhamnopyranosyl- (1 → 2) -α-L-arabinopyranosyl) and, R ₂ is Methanol (CH ₃ OH), R ₃ is beta-D-xylpyranosyl (1 → 6) -beta-D-glucopyranosyl (β-D-xylopyranosyl- (1 → 6) -β-D-glucopyranosyl ) Or beta-D-glucopyranosyl (1 → 6) -beta-D-glucopyranosyl (β-D-glucopyranosyl- (1 → 6) -β-D-glucopyranosyl). The triterpenoid saponin-based compound according to claim 1, wherein the triterpenoid saponin-based compound is 3- O -alpha-L-ramnopyranosyl (1 → 2) -alpha-L-arabinofyranosyl-hederagenin 28- O -beta- D-xylpyranosyl (1 → 6) -beta-D-glucopyranosyl ester or 3- O -alpha-L-ramnopyranosyl (1 → 2) -alpha-L-arabinofyranosyl-hederagenine 28- O -beta-D-glucopyranosyl (1 → 6) -beta-D-glucopyranosyl ester. According to claim 1, wherein the triterpenoid saponin-based compound is a pharmaceutical composition, characterized in that the extract from the elm. The pharmaceutical composition of claim 1, wherein the cardiovascular disease is selected from the group consisting of arteriosclerosis, hypertension, angina pectoris, hyperlipidemia, myocardial infarction, stroke and heart failure.

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