KR101504722B1 - A pharmaceutical composition comprising Kamolonol as a active ingredient for prevention or treatment of cardiovascular diseases - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for the prevention and treatment of cardiovascular diseases, which comprises a camolinol compound as an active ingredient. More specifically, the present invention relates to a pharmaceutical composition comprising a camolinol compound as an active ingredient as an extract isolated from a ferula assafoetida To a pharmaceutical composition for the prevention and treatment of cardiovascular diseases and a method for producing a camolinol compound.
The camolinol compound or its pharmaceutically acceptable salt of the present invention has excellent inhibitory effect on rookinase and has almost no cytotoxicity. Therefore, it is a preventive or therapeutic agent for cardiovascular diseases such as hypertension, heart failure, myocardial infarction, Can be usefully used.

Description

[0001] The present invention relates to a pharmaceutical composition for prevention and treatment of cardiovascular diseases, which comprises a camolinol compound as an active ingredient,

The present invention relates to a pharmaceutical composition for the prevention and treatment of cardiovascular diseases comprising, as an active ingredient, a camolinol compound, and more particularly, to a pharmaceutical composition for preventing and treating cardiovascular diseases which comprises a camolol compound as an active ingredient To a pharmaceutical composition for the prevention and treatment of cardiovascular diseases and a method for producing a camolinol compound.

The prevalence of ischemic cardiovascular disease is increasing due to obesity, an increase in the elderly population, coronary artery disease, hypertension, and environmental factors. In fact, the prevalence of ischemic cardiovascular disease is estimated to increase to 40% Heart failure, myocardial infarction treatment for the treatment of diseases such as hypertension, except for the underlying disease is almost no condition.

Rho kinase (ROCK) is recognized as a factor that regulates the shape and motility of cells through regulation of the actin cytoskeleton. Recently, there has been a growing interest in the cardiovascular field for several reasons It is a pharmacological target. Rokinase is a major regulator of calcium (Ca ²⁺ ) -independent mechanism of vascular smooth muscle systolic phase and is a heterotrimeric G protein receptor associated with angiotensin II, 5-HT, UII, Thromboxane A2, , Heart hypertrophy, hypertension, vascular smooth muscle cell proliferation and migration have been reported as a pharmacological target for cardiovascular therapies including heart failure, angina and hypertension. The expression of rookinase is directly promoted by stimuli (angiotensin II, IL-β, and PKC / NF-kB) that cause an inflammatory response, and in fact, inflammatory lesions and arteriosclerotic lesions in experimental animals and humans, And an increase in activity was observed (Fig. 1). In particular, correlation with various pathologic markers of cardiovascular disease has been demonstrated, including vascular endothelial dysfunction, macrophage counts and vascular complications in the blood vessels, contraction of smooth muscle longitudinal contraction, and stress fibrosis induction. The need for the development of the inhibitors of roxinase has been shown in various animal studies to date that rocynease inhibitors have almost exclusively functions as medicines which contain almost all the effects of various drugs except cholesterol lowering effect of statin .

 Currently, the clinically available locinase inhibitor is only fasudil, which was marketed by Asahi Kasei Pharma, Japan in 1995 as a calcium sensitizer and later found to be a ROCK inhibitor through pharmacokinetic studies. In Japan, intravenous fasadillin is used to treat cerebral vasoconstriction, and fasudil oral medication is undergoing some clinical trials in Japan and the United States. However, there is still a need for studies on unstable angina and myocardial infarction.

Recent research trends have shown that Rho kinase subtype-2, which is highly selective for PKA, PKC, RSK, and MSK of the same family, and has excellent inhibitory effects against rokynia, ROCK2) with ligand binding pockets, such as pyridine, 1H-indazole, isopuinolin and tetrahydroisoquinoline.

Kamolonol is an active substance of the sesquiterpene coumarin family extracted from ferula assafoetida . In addition, the effects of camorinol or direct administration of rotinase subtype-2 on cardiovascular diseases including myocardial fibrosis and heart failure mitigation have not been reported yet. In the present invention, it was discovered for the first time that the camolinol compound is inferior to the excellent locinase subtype-2. The inventors of the present invention found that by inhibiting the locinase type 2-activity in the camorin heart cells, The present invention can be used as an effective ingredient of a therapeutic agent for diseases.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

Korean Patent Publication No. 2010-0084812, Jul. 28, 2010

 Dastan D et al., Disesquiterpene and sesquiterpene coumarins from Ferula pseudalliacea, and determination of their absolute configurations. Phytochemistry. 2012 Jun; 78: 170-8  Effects of Angelica gigas Nakai, Angelica gigas Nakai, and Astragali on smooth muscle relaxation and iNOS expression. Journal of Korean Oriental Medicine. 2000, 21 (2): 60-67

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of cardiovascular diseases using an extract / separation compound from a natural product. It is another object of the present invention to provide a process for producing the above compound.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating cardiovascular diseases comprising, as an active ingredient, a camolonol compound represented by Chemical Formula 1 or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

A) preparing an extract by extracting an ascorbic acid with a C1-C4 lower alcohol or a mixed solvent thereof;

b) filtering the extract of step a) and concentrating under reduced pressure;

c) sequentially fractionating the extract obtained in step b) with n-hexane, dichloromethane and butanol;

d) separating and purifying the dichloromethane extract obtained in step c) by chromatography; And

e) subjecting the second fraction obtained in step d) to silica gel column chromatography again to obtain a camolinol compound of formula (1); ≪ RTI ID = 0.0 > a < / RTI >

The camolinol compound or its pharmaceutically acceptable salt of the present invention has excellent inhibitory effect on rookinase and has almost no cytotoxicity. Therefore, it is a preventive or therapeutic agent for cardiovascular diseases such as hypertension, heart failure, myocardial infarction, Can be usefully used.

BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a diagram showing the action mechanism of camorinol as an active ingredient of the present invention. Fig.
FIG. 2 is a 1 H-NMR spectrum of a camolone as an active ingredient of the present invention.
FIG. 3 is a 13 C-NMR spectrum of the camorinol as an active ingredient of the present invention.
4 is a graph showing the effect of inhibiting the activity of locinase subtype-2 at various concentrations of camorinol, an active ingredient of the present invention.
FIG. 5 is a graph showing the inhibition pattern of camorinol, an active ingredient of the present invention, in response to inhibition of type 2 of locinase.
FIG. 6 is a graph showing the inhibitory effect of angiotensin-induced MYPT and MLC on phosphorylation according to the concentration of camorinol as an active ingredient of the present invention.
FIG. 7 is a graph showing the cardiac hypertrophy inhibitory effect according to the concentration of camorinol, an active ingredient of the present invention.
Fig. 8 is a graph showing the effect of camorinol, an active ingredient of the present invention, on inhibition of actin stress fiber formation.

Hereinafter, the present invention will be described in detail.

The present invention relates to a method for producing camolinol represented by the following formula (1), which comprises reacting camolinol, 7 - [[(1R, 2R, 4R, 4aS, 5R, 8aS) -4-hydroxy-1,2,4a, 5-tetramethyl- -3,4,5,7,8,8a-hexahydro-2H-naphthalen-1-yl] methoxy] chromen-2- one or a pharmaceutically acceptable salt thereof as an active ingredient for the prevention of cardiovascular diseases Or a pharmaceutical composition for therapeutic use.

[Chemical Formula 1]

The camolinol compound according to the present invention can be extracted or isolated from the ferula assafoetida , but is not limited thereto.

However, no activity related to ROCK mediated diseases or cardiovascular diseases has yet been reported. In the present invention, these active ingredients are extracted and used as a cardiovascular preventive or therapeutic agent. The camolinol compound of formula (1) of the present invention can be extracted or separated from the bottom or commercially available or can be prepared by a method known in the art.

The camolinol compound inhibits ROCK (Rho kinase) to inhibit ROCK-mediated cardiovascular disease, ischemic myocardium, ischemic brain disease, obesity-induced cardiovascular disease, ischaemia, hypertensive heart disease, valve disease, cardiomyopathy, , Peripheral vascular disease, angina pectoris, heart failure, coronary heart disease, arteriosclerosis, myocardial infarction or arrhythmia, but is not limited thereto.

The present invention also relates to a method for producing an extract, comprising the steps of: a) extracting an ascorbic acid with a C1-C4 lower alcohol or a mixed solvent thereof;

b) filtering the extract of step a) and concentrating under reduced pressure;

c) sequentially fractionating the extract obtained in step b) with n-hexane, dichloromethane and butanol;

d) separating and purifying the dichloromethane extract obtained in step c) by chromatography; And

e) subjecting the second fraction obtained in step d) to silica gel column chromatography again to obtain a camolinol compound of formula (1); ≪ RTI ID = 0.0 > 1, < / RTI >

Preferably, in the step of extracting, the solvent is methanol, nucleic acid, dichloromethane, butanol or a mixed solvent thereof. In the step of separating and purifying the dichloromethane fraction by chromatography, dichloromethane, methanol, Nucleic acid, ethyl acetate or a mixed solvent thereof is preferably used.

In step a), the step of extracting the ascites with a C1-C4 lower alcohol or a mixed solvent thereof is carried out.

The extraction solvent is preferably 2 to 20 times as much as the weight of the ovine cells. Extraction can be carried out by any conventional extraction method, preferably by boiling for 2 to 24 hours at 40 to 120 ° C, or by using a C1 to C6 alcohol or a mixed solvent of water and C1 to C6 alcohol as an extraction solvent , It can be left at room temperature for 1 to 5 days for extraction. The extract obtained by the above method can be used as an agate extract by further filtration, purification, concentration and the like using a filter paper or the like.

Next, in step b), the extract of step a) is filtered and concentrated under reduced pressure. Preferably, the extract is cooled with methanol for several days, filtered, and the filtrate is concentrated under reduced pressure.

In the step c), water is added to the sea tangle extract obtained in step b), preferably the methanol extract of the sea tangle, and the resulting suspension is successively fractionated with hexane, dichloromethane and butanol. At this time, a usual fraction extraction method can be used.

Next, in step d), the step of separating and purifying the dichloromethane-soluble extract obtained in step c) by chromatography is carried out.

In the next step e), the second fraction obtained in step d) is further subjected to silica gel column chromatography to obtain a camolinol compound of formula (1).

In the step chromatography, dichloromethane, methanol, nucleic acid, ethyl acetate, or a mixed solvent thereof may be used as the mobile phase, and the reaction can be carried out by a conventional method. At this time, when a mixed solvent is used, a mixed solvent of dichloromethane and methanol, or a mixed solvent of hexane and ethyl acetate may be preferably used. Chromatography can be carried out once to several times until a single compound is purified, and concentration and recrystallization can be carried out if necessary.

More specifically, the dichloromethane fraction was dissolved in dichloromethane, followed by separation and purification using silica gel column chromatography. Four fractions were obtained by using a mixed solvent of dichloromethane / methanol (100: 0 → 50: 0 → 10: 1) as a mobile phase, and the second fraction was separated and purified again by silica gel column chromatography , And the solvent used may be eluted and separated by using a gradient elution method in which the concentration is sequentially increased from nonpolar to polar, using nucleic acid / ethyl acetate to obtain a final camolanol. The camolinol obtained was verified by direct comparison of physical spectral properties ( ¹ H-NMR and ¹³ C-NMR).

The pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration or the like.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . The daily dosage of the pharmaceutical composition of the present invention is, for example, 0.001-100 mg / kg.

The pharmaceutical composition of the present invention may be formulated into a conventional preparation using pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs May be prepared in unit dosage form or may be manufactured by intrusion into a multi-dose container. Typical formulations are, for example, oral (tablets, capsules, powders), intraoral, sublingual, rectal, vaginal, intranasal, topical or parenteral (intravenous, intracameral, intramuscular, subcutaneous And intravenous) administration formulations. For example, a compound according to the present invention may be in the form of tablets containing starch or lactose, in the form of capsules containing the active ingredient alone or as an excipient, or in the form of an elixir or suspension containing a chemical that flavors or colors For example, orally, buccally or sublingually. Liquid preparations may contain suspending agents (e. G., A mixture of a semisynthetic glyceride such as methylcellulose, witepsol or apricot kernel oil and a PEG-6 ester, or a mixture of PEG-8 and caprylic / Such as a glyceride mixture, such as a mixture of glycerides (e.g., a mixture of glycerides). It is also most preferred to use it as a sterile aqueous solution form when injected parenterally, for example, intravenously, intraperitoneally, intramuscularly, subcutaneously, and intrathecally, wherein the solution is administered in order to have isotonicity with the blood It may contain other substances (for example, salts or monosaccharides such as mannitol, glucose).

The camorinol of the present invention can be used in the form of a pharmaceutically acceptable salt, and as the salt, an acid addition salt formed by a pharmaceutically acceptable free acid can be used. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Derived from organic acids such as acetic acid, benzoic acid, citric acid, lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, fumaric acid, and the like. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinate, maleic anhydride, maleic anhydride, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzene sulfonate, toluene sulfonate, chlorobenzene sulfide Propyl sulphonate, naphthalene-1-yne, xylenesulfonate, phenylsulfate, phenylbutyrate, citrate, lactate,? -Hydroxybutyrate, glycolate, maleate, Sulfonate, naphthalene-2-sulfonate or mandelate.

The acid addition salt according to the present invention can be obtained by a conventional method, for example, by dissolving the compound of the formula (1) in an organic solvent such as methanol, ethanol, acetone, methylene chloride, acetonitrile and the like, , Or may be prepared by drying, or after the solvent and excess acid are distilled off under reduced pressure, followed by drying or crystallization in an organic solvent.

In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or an alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. In addition, the corresponding silver salt is obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate). In addition, the present invention includes both the camolinol compound represented by Chemical Formula 1 and pharmaceutically acceptable salts thereof, as well as possible solvates, hydrates, stereoisomers and the like which can be prepared therefrom.

In addition, the compounds of the present invention may contain one or more asymmetric carbon atoms and may exist in racemic and optically active forms. All such compounds and enantiomers are included within the scope of the present invention.

Hereinafter, preferred embodiments and experimental examples are provided to facilitate understanding of the present invention. However, the following examples and experimental examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited by the examples and experimental examples.

Preparation of Camolinol Compound

The camolonol according to the present invention can be prepared by solvent extraction, separation and purification from commercially available avirulent extracts. The avi extract was purchased from Arjuna Natural Extracts, Ltd., Kerala, India, and was certified by Professor Kim Young-gyun and stored in the plant sample room of the Chemical Research Institute. The extract was air-dried for 7 days using 20 liters of methanol and then filtered. The filtrate was concentrated under reduced pressure. 460 g of the extract was suspended in water and then subjected to solvent extraction successively using nucleic acid (56 g), dichloromethane (290 g) and butanol (50 g). Thereafter, the dichloromethane fraction (5 g) was dissolved in 100 mL of dichloromethane, followed by separation and purification using silica gel column chromatography. Four fractions were obtained using a mixed solvent of dichloromethane / methanol (100: 0 → 50: 0 → 10: 1) as a mobile phase, and the second fraction (570 mg) was re-run on silica gel column chromatography Separation and purification were carried out. At this time, the solvent used was a gradient elution method in which the concentration was sequentially increased from nonpolar to polar, and eluted and separated using nucleic acid / ethyl acetate to obtain a final 120 mg of camolinol. The physical properties (1H-NMR and < 13 > C-NMR) of the obtained camolinol were the same as those shown in FIG. 2 and FIG.

Lokinase type 2 inhibition confirmation experiment

The inhibitory activity of camorinol represented by the formula (1) according to the present invention on the locinase subtype-2 was measured by a differential molding photolysis method (IMAP-TR- FRET Screening Express Kit).

The buffer solution was prepared by adding 5 mM DTT to the phosphorylation reaction solution (5 times diluted with reaction buffer containing 0.01% Tween 20, supplied by MDS), fluorescent detection solution (binding solutions A and B provided by MDS) (MDS) and 100 μg / ml of enzyme-conjugated protein (MDS) were added to each well. The resulting mixture was mixed with 7: 3 each, IMAP binding reagent 1/600 and turbid donor 1/400. (# 14-451; Upstate) and 10 mM ATP (Sigma-aldrich Co., St. Louis, Mo., USA) were prepared. (Final reaction concentration: 0.1 μg / ml), 4 μM (final reaction concentration: 1 μM) and 12 μM (final reaction concentration: Reaction concentration: 3 [mu] M). The buffer solution used in all dilutions and preparations was a phosphorylation reaction solution and the fluorescence detection solution was used to induce a differential fluorescence reaction at the end.

The prepared sample was dispensed using a 16-channel pipet (Finnpipette, Thermo Scientific, Essex, UK) into white microplates (Multiwell 384 well plates, # 3572, Corning Life Sciences, Lowell, MA, USA) The reactants were dispensed so that the total phosphorylation reaction volume per each well was 20 占 퐇. As a negative control, 5 μl of 5% DMSO, 5 μl of substrate and 5 μl of phosphorylation reaction solution were used. As a positive control, 5 μl of 5% DMSO, 5 μl of substrate, 5 [mu] l of the NAasease-2 solution was used. As the experimental group, 5 μl of the compound prepared in Example, 5 μl of the substrate solution and 5 μl of the Lokinasease type-2 solution were used. Before the phosphorylation reaction, pretreatment was performed between the compound and the enzyme for about 10 minutes, and 5 쨉 l of ATP was added to induce the phosphorylation reaction. Since each test substance, enzyme, substrate and ATP account for 25% of the total volume during the reaction, they were prepared at a high concentration of 4 times each before the addition. After incubation for 3 minutes with weak shaking, phosphorylation reaction was induced at room temperature for 45 minutes. To induce the differential fluorescence reaction, 60 μl of fluorescent detection solution prepared in advance was added to induce a differential fluorescence reaction. In the fluorescence detection solution, a nanoparticle composed of trivalent metal ions, a linker and a sensitizer (Tb-donor) labeled with a fluorescent substance Tb (Turbium) were mixed, so that the chain coupling reaction between the nanoparticle, the linker and the Tb donor A differential fluorescence reaction is induced. The time-resolved fluorescence resonance energy transfer (TR-FRET) values were measured using a multilabel counter (Envision, PerkinElmer, Turku, Finland) after leaving for 3 hours at room temperature : 495 nm, 520 nm, excitation wavelength: 340 nm), to a differential property and calculating the fluorescent reaction rate and inhibition rate, and as a result the concentration of a test substance Rocky better inhibit the subtype -2 50% in vitro according to the following equation It was expressed as the IC ₅₀ value.

[Equation 1]

&Quot; (2) "

As shown in the attached FIG. 4, the efficacy of various concentrations of camorinol (0.01 μM to 30 μM) inhibited the activity of Lokinase-type-2 in a concentration-dependent manner and IC ₅₀ value was 2.27 μM .

Analysis of the inhibition pattern of camorinol inhibition by Lokinase subtype 2

To analyze the inhibition pattern of camorinol inhibition by Lokinase subtype 2 (Final reaction concentration: 0.6 μg / ml) and 4 μM substrate (final reaction concentration: 1 μM) and 0.4 μg / ml of Lokinasease subtype 2 The final reaction concentration was 0. 3, 6 μM). The phosphorylation reaction was induced according to the above method (Experimental Example 1), and the differential fluorescence reaction rate at 0, 30, 60 and 120 minutes was measured to measure the reaction rate . The initial reaction rate (the amount of phosphorylated substrate per minute, nM / min) and the amount of phosphorylated substrate (ATP) per minute were calculated using the regression line between the differential fluorescence response rate and the phosphorylated substrate amount, The inhibition pattern of camorinol against the inhibition of subtype 2 of rotamase was analyzed by applying double reciprocal plots on the basis of the amount (μM)

As shown in FIG. 5, the inhibitory mode of camolinol against the inhibition of the locinase type 2 was analyzed as an ATP-competitive inhibitor of ATP, and it was confirmed that camolinol binds to the ATP- And inhibited the activity of subtype 2.

MYPT / MLC phosphorylation inhibition confirmation experiment

In order to examine the inhibitory effect of camolinol on the direct locinase subtype-2 represented by the above formula according to the present invention, myosin phosphatase (MYPT), known as a direct substrate of Lokinnae subtype-2, Western blotting was performed using a rat cardiac cell line (H9c2) activated with angiotensin II for the evaluation of the pharmacological effect of inhibition of myosin light chain (MLC) phosphorylation.

Cardiac cells (H9c2) were pretreated with camolinol for 1 hour and activated intracellular signaling with angiotensin II for 15 minutes. The medium was removed and the total protein extract of the cells was obtained by treating the PRO-PREP protein extraction solution provided by the domestic company Intron. SDS-PAGE was performed using 8% or 12% glycine, and the proteins were transferred to a nitrocellulose membrane. Then, an anti-phospho-MYPT antibody and an anti-phosphate-MLC antibody (cell signaling) MYPT and MLC were phosphorylated and inhibited by phosphorylation. Western blotting was performed using anti-MYPT antibody and anti-MLC antibody in the same manner to show that the same amount of protein was analyzed.

As shown in FIG. 6, it was shown that the phosphorylation of MYPT and MLC induced by angiotensin II was inhibited by camolinol (> 3 μM) in a concentration-dependent manner by the direct inhibition of rookinase have. The validation experiment was performed with Y27632 (0.3 μM), which is known as a reference material of Locinasease-2.

Cellular hypertrophy inhibition test

One of the major factors of cardiac hypertrophy, which is a direct pathologic sign of heart failure, is the overactive activity of the locinase subtype-2. Therefore, an experiment for verifying the inhibitory effect of ROKNEA on the cell hypertrophy of camorinol was carried out as follows.

Cardiac cells (H9c2) were pretreated with the test substance represented by the above formula for 1 hour, and angiotensin II (100 nM) inducing cardiac hypertrophy was continuously administered for 4 days. After washing with PBS (phosphate buffered saline), the cells were fixed with 1% glutaraldehyde (Sigma, St. Louis, Md., USA) for 30 minutes. The fixed cells were stained with 0.1% Crystal Viole (Sigma) for 10 min. Cell images were collected using a microscope equipped with a digital camera (Nikon, Tokyo, Japan) and Image-Pro PLUS software (MediaCybernetics, Silver Spring , MD, USA) was used to analyze the number and size of cells. At this time, it was confirmed that the angiotensin II inducing factor of cardiac hypertrophy induces about 44% of cardiac hypertrophy by continuously administering angiotensin II for 4 days. Based on the cardiac cell status induced by angiotensin II The percent inhibition of cardiac hypertrophy was expressed as a percentage (%), and the inhibitory effect of each concentration on cardiac hypertrophy was shown in FIG. 7 as a graph.

As shown in FIG. 7, it was confirmed that the concentration-dependent inhibition of cardiac hypertrophy induced by camorinol angiotensin II was observed, and a statistically significant inhibitory effect was confirmed especially from 3 μM or more. This confirms the validation experiment with Y27632 (0.3 μM), a reference compound known as the conventional Lokinase type 2 inhibitor.

Test for inhibition of actin stress fiber formation

Actin stress fibrosis is a direct morphological phenomenon of cardiac cells and vasoconstriction following myosin L chain phosphorylation and is an experiment to verify the inhibition effect of rochynase type 2 by inhibition of actin stress fibrosis formation of camorinol Was carried out as follows.

The test material represented by the above formula was pretreated with cardiac cells (H9c2) cultured in a chamber slide (16 well) for 1 hour and activated intracellular signaling with angiotensin II for 2 hours to induce F- Actin stress fiber formation. After washing with PBS (phosphate buffered saline), cells were fixed with 4% paraformaldehyde (Sigma, St. Louis, MD, USA) for 30 minutes. The immobilized cells were resuspended in 0.5% triton X-100 solution for 5 minutes and then treated with a blocking solution containing 1% BSA (boine serum albumin) for 30 minutes at room temperature. F-actin stress fibers and nuclei were stained using Alexa fluor 586 phalloidin and Hoechst staining reagents and cell images (200-fold) were collected on a microscope equipped with a digital camera (Nikon, Tokyo, Japan).

As shown in FIG. 8, it was confirmed that the formation of F-actin stress fibers strongly induced by angiotensin II (300 nM) was suppressed in a concentration-dependent manner by the pretreatment of camorinol for 2 hours, And statistically significant inhibitory effects were confirmed. In particular, it was confirmed that inhibition of intracellular central actin was more intensified than that of coarse peripheral actin, as in the case of a typical locinase inhibitor-2 inhibitor.

An example of a preparation for a composition comprising the camorinol of the present invention as an active ingredient is illustrated below.

[Formulation Example 1]

Preparation of pharmaceutical preparations

1-1: Manufacture of powder

2 g of the camolinol compound according to the present invention

Lactose 1 g

After mixing the above components, the mixture was packed in an airtight container to prepare a powder.

1-2: Preparation of tablets

100 mg of the camolinol compound according to the present invention

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

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

1-3: Preparation of capsules

100 mg of the camolinol compound according to the present invention

Corn starch 100 mg

100 mg of milk

2 mg of magnesium stearate

After mixing the above components, the capsules were filled in gelatin capsules according to the conventional preparation method of capsules.

1-4: Preparation of injection solutions

10 [mu] g / ml of the camolinol compound according to the present invention

Until dilute hydrochloric acid BP pH 3.5

Sodium chloride BP injected up to 1 ml

A suitable volume of the solution was prepared by dissolving the camolinol compound according to the present invention in sodium chloride BP, adjusting the pH of the resulting solution to pH 3.5 with dilute hydrochloric acid BP, adjusting the volume using injectable sodium chloride BP, . The solution was filled in a 5 ml type I ampoule made of transparent glass, sealed in an upper lattice of air by dissolving the glass, sterilized by autoclaving at 120 DEG C for 15 minutes or longer, and an injection solution was prepared.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

A pharmaceutical composition for preventing or treating heart failure comprising as an active ingredient a camolonol compound represented by the following formula (1) or a pharmaceutically acceptable salt thereof:
[Chemical Formula 1]

The pharmaceutical composition according to claim 1, wherein the compound is extracted or separated from the fermented ascapoetida .
The pharmaceutical composition according to claim 1, wherein the compound inhibits ROCK (Rho kinase).
delete a) extracting the ascites with a C1-C4 lower alcohol or a mixed solvent thereof to prepare an extract;
b) filtering the extract of step a) and concentrating under reduced pressure;
c) sequentially fractionating the extract obtained in step b) with n-hexane, dichloromethane and butanol;
d) separating and purifying the dichloromethane extract obtained in step c) by chromatography; And
e) subjecting the second fraction obtained in step d) to silica gel column chromatography again to obtain a camolinol compound of formula (1); ≪ / RTI >
6. The process according to claim 5, wherein the solvent in step (a) is methanol, nucleic acid, dichloromethane, butanol or a mixed solvent thereof.
6. The process according to claim 5, wherein dichloromethane, methanol, nucleic acid, ethyl acetate or a mixed solvent thereof is used as a mobile phase in step (d) for separating and purifying the dichloromethane fraction by chromatography.
8. The process according to claim 7, wherein the mixed solvent is a mixed solvent of dichloromethane and methanol.

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