KR102568917B1 - Composition supportive for treating Cardiovascular Diseases comprising 2S-2`methoxy-kurarinone - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for assisting the treatment of cardiovascular diseases, comprising 2S-2'methoxy-kurarinone as an active ingredient; Health food composition for improving cardiovascular disease; And it relates to a method for preventing or treating cardiovascular disease using the same.
The pharmaceutical composition for supplementing the treatment of cardiovascular disease, containing 2S-2'methoxyclarinone as an active ingredient of the present invention, suppresses the increase in PCSK9 expression and increases the production of LDLR, which is a side effect of the administration of statin, a cardiovascular drug. It has an increased activity of AMPK reduced by statins, so it can be usefully used for adjuvant treatment of cardiovascular diseases.

Description

Composition supportive for treating Cardiovascular Diseases comprising 2S-2`methoxy-kurarinone as an active ingredient, a pharmaceutical composition for assisting in the treatment of cardiovascular diseases

The present invention relates to a pharmaceutical composition for assisting the treatment of cardiovascular diseases, comprising 2S-2'methoxy-kurarinone as an active ingredient; Health food composition for improving cardiovascular disease; And it relates to a method for preventing or treating cardiovascular disease using the same.

In most countries, cardiovascular disease is one of the leading causes of morbidity and mortality (Pepys, Hirschfield et al., 2006; Van Gaal, Mertens et al., 2006). About one-third of men develop major cardiovascular disease before age 60, and fewer women (about 10 to 1 ratio). As we get older (after age 65), the disease increases even more. Vascular disease such as coronary disease, stroke, restenosis and peripheral vascular disease are leading causes of death and disability worldwide (Bonora and Targher, 2012; Brown, 2013). Although diet and lifestyle can accelerate the development of cardiovascular disease, genetic predisposition to dyslipidemia is a major factor in cardiovascular disease and death.

Atherosclerosis is known to be caused by various endogenous and exogenous factors, such as weakening of the arterial blood vessel itself, damage caused by infection, and accumulation of cholesterol due to lipoprotein (Vaziri, Navab et al., 2010; Brunt, Wong et al., 2015). Complex factors that cause damage and degeneration of the arterial wall promote the formation of arteriosclerosis, but among the risk factors, hypertension, hyperlipidemia, and smoking independently cause arteriosclerosis. In particular, in the case of hyperlipidemia, in which cholesterol levels in the blood are high due to excessive intake of animal fat, phagocytes located between the inner lining of the artery and the muscle layer contain more oil in the cytoplasm, and fat is accumulated on the arterial wall (Martinez-Hervas, Real et al. al., 2010).

As a method of lowering the blood lipid concentration, a diet that suppresses a high-fat diet, exercise therapy, and drug therapy are recommended. However, it is difficult to strictly manage and implement diet or exercise therapy, and there are many cases where the effect is limited. Lipid concentration lowering agents developed so far include cholesterol-lowering drugs such as bile acid-binding resins, HMG-CoA reductase inhibitors, and neomycin, and neutral fat-lowering drugs such as fibric acid derivatives, nicotinic acid, and fish oil. there is.

However, these drugs have side effects such as liver toxicity, gastrointestinal disorders and cancer. AMP-activated protein kinase (AMPK) is a heterotrimeric complex that includes a catalytic α subunit and a regulatory β/γ subunit that senses low energy states by monitoring the ratio of ATP to AMP.

Hyperlipidemia (高脂血症; hyperlipidemia) refers to a state in which the amount of fat in the blood is increased due to poor metabolism of fats such as neutral fat and cholesterol. Serum lipids are composed of cholesterol, triglycerides, phospholipids, and free fatty acids.

These move in the body in the form of lipoproteins, and lipoproteins are classified into four types: chylomicron, very low-density lipoprotein (VLDL), low-density lipoprotein (LDL), and high-density lipoprotein (HDL). The causes of hyperlipidemia include genetic predisposition and secondary factors such as hypothyroidism, jaundice, nephrotic syndrome or diabetes. In the fasting state, the liver synthesizes VLDL and exports triglycerides to peripheral cells, some of which are converted to LDL. LDL carries the largest amount of cholesterol in the blood and supplies cholesterol to peripheral cells, but it is the most dangerous lipoprotein that causes coronary atherosclerosis. Therefore, hyperlipidemia can be treated with thorough diet and drug therapy.

Among the treatments for cardiovascular diseases, statins are also known as HMG-CoA reductase inhibitors, and refer to a group of antihyperlipidemia drugs whose drug names end in -statin. Statins are widely used drugs for dyslipidemia and hyperlipidemia, and are known to significantly reduce cardiovascular abnormalities and mortality caused by these diseases.

However, side effects of the statin-based drugs include elevated liver levels and rhabdomyolysis, and it is important to study supplementary drugs that can supplement them. In particular, if the target level of LDL-C is not achieved even with statins, the dose of statins may be increased to the maximum, but combined therapy with other drugs is recommended. Although the effect can be expected, side effects can occur as much as that, so research on the combined administration of auxiliary drugs that can compensate for this is in the spotlight.

Accordingly, the present inventors obtained 2S-2`methoxy-kurarinone, a flavonoid component of high ginseng, and studied its activity, and found that side effects were alleviated and activity was further increased when combined with a statin-based substance, resulting in the present invention. Completed.

One object of the present invention is to provide a pharmaceutical composition for assisting the treatment of cardiovascular disease, containing 2S-2'methoxy-kurarinone as an active ingredient.

Another object of the present invention is to provide a method for preventing or treating cardiovascular disease, comprising the step of administering to a non-human subject in combination with a statin to the composition.

Another object of the present invention is to provide a health food composition for improving cardiovascular disease, containing 2S-2'methoxyclarinone as an active ingredient.

A detailed description of this is as follows. Meanwhile, each description and embodiment disclosed in the present invention may also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed herein fall within the scope of the present invention. In addition, it cannot be seen that the scope of the present invention is limited by the specific descriptions described below.

One aspect of the present invention for achieving the above object provides a pharmaceutical composition for assisting the treatment of cardiovascular disease, containing 2S-2'methoxy-kurarinone as an active ingredient.

In the present invention, when 2S-2'methoxyclarinone is administered with statin, PCSK9 expression is rather reduced while LDLR expression is treated with statin alone regarding the side effect of increasing PCSK9 expression as well as LDLR when treated with statin drugs The effect of improving to a significant level compared to one case was confirmed.

Therefore, by confirming the possibility of co-treatment as an adjuvant of statin-based drugs, it was confirmed that it can be used for preventing or treating cardiovascular diseases according to the reduction of fatty acids and cholesterol in the blood.

The term of the present invention, “2S-2`methoxy-kurarinone” is one of the active ingredients of gosam, a kind of flavonoid component, and is a compound having the structure of Formula 1 below, with molecular formula C ₂₇ H ₃₂ O ₆ , The molecular weight is 452.54.

[Formula 1]

The term of the present invention, "Gosam" is a perennial herb that grows in mountains and fields throughout Korea. It is also called the thief's cane, neosam, and bang's pavilion tree. The growing environment is sunny, the soil is rich in humus, and it grows in a place with good drainage. The height is about 1m, and the leaf has 15 to 40 small leaves attached to the stem and is 15 to 25 cm long. has hair. The stem turns black and branches at the top. The root is yellowish brown, thick, and tastes very bitter. The flowers are light yellow, and many flowers with a length of about 1.5 to 1.8 cm hang at the end of the main stem and on the branches. The fruit is 7-8 cm long and about 0.8 cm wide in September-October, and inside is chestnut brown and round, and contains seeds about 0.5 cm in diameter. In oriental medicine, dried roots are called gosam, which is bitter in taste and has the efficacy of ginseng, so it is prescribed for indigestion, neuralgia, hepatitis, jaundice, and hemorrhoids. In the private sector, the stems and leaves are decocted and used as insecticides. The taste is bitter and the nature is cold. It is also used when the stomach is weak, when it is difficult to urinate due to accumulation of heat and when it hurts, dysentery, genital itching, and treats. As a plant of the same genus, there is a sandpiper, which is very similar in appearance. Distributed in Korea, Japan, China, and Siberia.

The term of the present invention, "statin (statin) or statin-based drug", acts as a competitive inhibitor of HMG-CoA (3-hydroxy-3-methylglutaryl-coenzyme A) reductase involved in the early stage of cholesterol biosynthesis, thereby reducing serum cholesterol As a concentration-lowering drug, it inhibits cholesterol synthesis in hepatocytes and increases low-density lipoprotein receptor (LDLR) expression to lower blood LDL-C (low density lipoprotein-cholesterol) levels. In addition, it has the effect of reducing triglyceride levels and increasing HDL-C (high density lipoprotein-cholesterol) levels, so it is commonly used for lipid control in patients with dyslipidemia. In the present invention, the statin may be atorvastatin, rosuvastatin, fluvastatin, pravastatin, pitavastatin or simvastatin. It is not particularly limited thereto as long as it has an inhibitory activity of HMG-CoA reductase.

However, side effects of statin drugs that appear in proportion to their effects include elevated liver levels and rhabdomyolysis. Mechanism studies have shown that LDLR increases but PCSK9 also increases proportionally. research on is important. Specifically, the side effect may be a concomitant increase in PCSK9. In particular, if the target level of LDL-C is not achieved even with statins, the dose of statins may be increased to the maximum, but combined therapy with other drugs is recommended. Effects can be expected, but side effects can occur, so research on the combined administration of auxiliary drugs that can compensate for this is in the spotlight, and better prevention of cardiovascular disease through increased LDLR and reduced activity of PCSK9 during combined administration Or a therapeutic effect can be expected.

As used herein, the term "treatment" refers to any activity that inhibits or delays cardiovascular disease by co-administering a composition containing the 2S-2'methoxyclarinone or a statin thereto. For the purpose of the present invention, the treatment may be understood as an act of improving symptoms of cardiovascular disease or alleviating pathological symptoms by using the pharmaceutical composition of the present invention, but is not particularly limited thereto.

The term of the present invention, "pharmaceutical composition for auxiliary treatment" means a composition that can improve or alleviate the side effects of cardiovascular disease treatment. As an example, it refers to a substance that can be co-administered with a statin drug to alleviate its side effects to enhance the preventive or therapeutic effect of cardiovascular disease, but is not limited thereto.

In one embodiment of the present invention, as the composition containing 2S-2`methoxyclarinone as the pharmaceutical composition for therapeutic support is co-administered with statins, LDLR (low- Regarding the increase of PCSK9 (Proprotein convertase subtilisin/kexin type) expression along with the increase of density lipoprotein receptor), the effect as a cardiovascular treatment adjuvant was confirmed by simultaneously achieving the effect of reducing PCSK9 expression and increasing LDLR expression.

In another embodiment of the present invention, when the composition is treated in combination with statins, reduced AMPK ((Adenosine monophosphate-activated protein kinase) phosphorylation due to statins is treated in combination with 2S-2`methoxyclarinone Therefore, it was confirmed that it could be alleviated, and this confirmed the possibility of using 2S-2`methoxyclarinone as a therapeutic adjuvant in the treatment of cardiovascular diseases using statins (FIG. 3).

As used herein, the term "cardiovascular disease" refers to diseases occurring in the heart and major arteries, and major diseases include hyperlipidemia, hypertension, angina pectoris, coronary heart disease, myocardial infarction, arteriosclerosis, atherosclerosis, stroke, arrhythmia, etc. In addition, the accumulation of cholesterol in blood vessels (total cholesterol, LDL cholesterol, triglyceride increase, HDL cholesterol decrease) can be cited as a representative cause.

The term "PCSK9 (Proprotein convertase subtilisin/kexin type 9)" of the present invention is an enzyme encoded by the PCSK9 gene on human chromosome 1, and is ubiquitous in many tissues and cell types. PCSK9 binds to LDL receptor LDLR. When PCSK9 binds to LDLR after LDLR-LDL complexes are absorbed into cells in the liver and other cell membranes, PCSK9 degrades LDLR so that LDLR can no longer bind to LDL particles on the cell membrane surface. to prevent playback. Thus, blocking or inhibiting the production of PCSK9 can regenerate more LDLR and lower the concentration of LDL particles in the blood.

In one embodiment of the present invention, in order to confirm the inhibitory effect of 2S-2'methoxyclarinone on the increase in PCSK9 expression, which is a side effect of statins, the expression level of PCSK9 was compared during co-treatment with statins (FIG. 2 , Figures 4 and 5). Through this, it was confirmed that 2S-2'methoxyclarinone suppresses the expression of PCSK9 more than when statin alone treatment, and it can be used as an auxiliary treatment that can reduce or alleviate the side effects of statin.

The term "Low density lipoprotein receptor (LDLR)" of the present invention is a protein that binds to LDL, the main cholesterol carrier in the blood, and mediates the endocytosis of cholesterol-rich LDL to maintain the plasma level of LDL. do LDLR is a cell-surface receptor that recognizes the apoprotein B100 incorporated in the outer phospholipid layer of LDL particles. Endocytosis of LDL through LDLR occurs in all nucleated cells, but mainly in the liver, which removes about 70% of LDL in the blood. .

In one embodiment of the present invention, the expression pattern of LDLR was confirmed when 2S-2'methoxyclarinone was co-treated with statin (Figs. 1, 4 and 5).

Through this, 2S-2`methoxyclarinone has the effect of increasing the expression level of LDLR compared to statin treatment alone, so it can lower the level of cholesterol in blood, and therefore, in the treatment of cardiovascular disease, the use of statins It was confirmed that it can be used as an auxiliary treatment that can improve the effect.

The term "AMP-activated protein kinase (AMPK)" of the present invention refers to an enzyme comprising a regulatory β/γ subunit and a catalytic α subunit that senses a low energy state by monitoring the ratio of ATP to AMP, plays a role in maintaining energy homeostasis. AMPK is activated by phosphorylation of threonine 172 by LKB1 and CaMKK (Ca2+ /calmodulin-dependent kinase kinase), which are upper enzymes of AMPK during muscle contraction and exercise (phospho-AMPK, pAMPK). Depending on the activation of AMPK, effects such as fatty acid oxidation in liver and skeletal muscle, promotion of glucose uptake and inhibition of cholesterol synthesis appear. Specifically, glucose uptake in skeletal muscle occurs by enhancing GLUT4 translocation to the plasma membrane. In addition, AMPK is a target molecule of leptin and adiponectin, two hormones derived from adipose tissue, and these hormones are key regulators of energy metabolism and glucose homeostasis (Ewart and Kennedy, 2012; Penumathsa et al 2009; Samovski et al. 2012).

AMPK is a type of serine/threonine kinase that is activated when intracellular energy (ATP) is deficient and increases intracellular energy production. Activated AMPK stimulates catabolic processes that generate ATP, such as beta-oxidation of fatty acids, and inhibits processes that consume ATP, such as lipogenesis.

In one embodiment of the present invention, the phosphorylation patterns of AMPK and ACC were confirmed when 2S-2'methoxyclarinone was co-treated with statins (FIG. 3).

Through this, it was confirmed that 2S-2`methoxyclarinone improved the increase in phosphorylation of AMPK and ACC compared to the case of statin alone treatment, and depending on the type of statin, it was confirmed that the decrease in AMPK phosphorylation was alleviated and the catabolism of fatty acids was improved. By stimulating the process and inhibiting adipogenesis, it was confirmed that in the treatment of cardiovascular diseases, it can be used as an adjuvant treatment that can improve the effect of statins.

Another aspect of the present invention for achieving the above object in the present invention provides a pharmaceutical composition for preventing or treating cardiovascular disease comprising 2S-2'methoxy-kurarinone and a statin as active ingredients.

The definitions of 2S-2'methoxy-kurarinone, statins, and cardiovascular diseases are as described above.

The term "prevention" of the present invention refers to any activity that suppresses or delays the onset of cardiovascular disease by administration of a composition. For the purpose of the present invention, the prevention may be understood as an act of inhibiting or delaying the onset of cardiovascular disease using the pharmaceutical composition of the present invention, but is not particularly limited thereto.

2S-2`methoxyclarinone of the pharmaceutical composition of the present invention may be used in combination with statin, wherein the ratio may be 1:1 to 1:100 or 1:1 to 100:1, but is not limited thereto.

In one embodiment of the present invention, the ratio of 2S-2'methoxyclarinone:statin may be 1:1 to 1:10, or 1:1 to 1:2, but is not limited thereto.

In one embodiment of the present invention, a composition containing 2S-2'methoxyclarinone and a statin is a therapeutic agent for cardiovascular disease, while reducing PCSK9 expression with respect to an increase in PCSK9 expression, which is a side effect of statin, while reducing LDLR expression by statin alone Due to the significantly improved effect compared to treatment, it is possible to improve, improve, or increase the therapeutic effect of the cardiovascular disease by improving or alleviating the side effects of statins.

In addition, in another embodiment of the present invention, by confirming that the reduced AMPK phosphorylation due to statins can be alleviated by the combined treatment with 2S-2'methoxyclarinone, the therapeutic effect of cardiovascular disease is improved and improved Or it was confirmed that it could be increased.

The pharmaceutical composition may further include a pharmaceutically acceptable carrier, excipient, or diluent commonly used in the preparation of pharmaceutical compositions, and the carrier may include a non-naturally occurring carrier. Specific examples of the carrier, excipient, and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, Microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil may be used, but is not limited thereto.

In addition, the pharmaceutical compositions may be formulated into tablets, pills, powders, granules, capsules, suspensions, solutions for internal use, emulsions, syrups, sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried agents, and suppositories, respectively, according to conventional methods. It may have any one formulation selected from the group consisting of, and may be various oral or parenteral formulations. When formulated, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants. Solid preparations for oral administration may include tablets, pills, powders, granules, capsules, etc., and the solid preparations may contain at least one excipient such as starch, calcium carbonate, sucrose or lactose, Gelatin and the like may be used. In addition to simple excipients, lubricants such as magnesium stearate and talc may be used. Liquid formulations for oral administration may include suspensions, internal solutions, emulsions, syrups, etc., and various excipients such as wetting agents, sweeteners, aromatics, and preservatives in addition to water and liquid paraffin, which are commonly used simple diluents can be used Sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations or suppositories may be used as preparations for parenteral administration. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, etc. may be used, but is not limited thereto.

Another aspect of the present invention for achieving the above object in the present invention is a method for preventing or treating cardiovascular disease, comprising administering 2S-2'methoxyclarinone in combination with statin to a non-human subject to provide.

The definitions of 2S-2'methoxyclarinone, statin, treatment, and prevention are as described above.

In one embodiment of the present invention, by co-administering statin to a composition containing 2S-2'methoxyclarinone, it was confirmed that the production of LDLR was increased and the effect of inhibiting the expression of PCSK9 was confirmed, and the effect of increasing AMPK phosphorylation activity was confirmed. there was.

This confirms that the increase in PCSK9 expression of statins can be suppressed by the combined administration of statins and 2S-2`methoxyclarinone, which are therapeutic agents for cardiovascular diseases, and have significant effects on the decreased AMPK increase and the increase in LDLR production. By doing so, it suggests that it can be usefully used for the prevention or treatment of cardiovascular diseases.

As used herein, the term "administration" means introducing the pharmaceutical composition to a subject in an appropriate manner. Specifically, 2S-2`methoxyclarinone and a statin-based drug may be administered in combination using the pharmaceutical composition of the present invention. The statin drug may be atorvastatin, rosuvastatin, fluvastatin, pravastatin, pitavastatin or simvastatin, but is not limited thereto. don't In addition, 2S-2`methoxyclarinone and statin-based drugs may be administered simultaneously, sequentially, or in reverse order to an individual.

As used herein, the term "individual" refers to all animals, such as rats, mice, and livestock, including humans who have or may develop cardiovascular disease. The animal may be not only humans but also mammals such as cows, horses, sheep, pigs, goats, camels, antelopes, dogs, and cats that require prevention or treatment of symptoms similar thereto, but are not limited thereto.

The pharmaceutical composition of the present invention can be administered in a pharmaceutically effective amount.

The term "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is dependent on the type and severity of the subject, age, sex, drug activity, It may be determined according to factors including sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used concurrently, and other factors well known in the medical field.

The pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. In addition, single or multiple administrations may be possible. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art.

In addition, the pharmaceutical composition may be administered orally or parenterally (for example, intravenously, subcutaneously, intraperitoneally or topically applied) according to the desired method, and the dosage is the patient's condition and weight, the degree of disease , Depending on the drug form, administration route and time, it can be appropriately selected by those skilled in the art. As a specific example, the pharmaceutical composition may generally be administered once or several times a day, but a preferred dosage may be appropriately determined by those skilled in the art according to the condition and body weight of the subject, the severity of the disease, the type of drug, the route of administration and the period of administration. can be chosen appropriately.

Another aspect for the object of the present invention provides a food composition for assisting in improving cardiovascular disease, containing 2S-2`methoxyclarinone as an active ingredient.

The definitions of 2S-2'methoxyclarinone, statins, and cardiovascular diseases are as described above.

The food composition of the present invention can be consumed on a daily basis, and unlike general drugs, it is made of natural substances and has no side effects that can occur when taking drugs for a long time, so it is very useful for preventing or improving cardiovascular diseases. can be used

As used herein, the term "for improvement" refers to all activities that alleviate or reduce parameters related to the side effects of statins, for example, symptoms, by ingestion of the food composition.

As used herein, the term "food" refers to meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, chewing gum, dairy products including ice cream, various soups, beverages, tea, drinks, and alcoholic beverages. , vitamin complex, health functional food and health food, etc., and includes all foods in the usual sense.

The health functional food (health functional food) is the same term as food for special health use (FoSHU), and in addition to supplying nutrients, it is processed to efficiently display bioregulatory functions, and has high medical effects. means food.

Here, 'function (sex)' means obtaining useful effects for health purposes such as regulating nutrients for the structure and function of the human body or physiological functions. The health food (health food) means a food that has an active health maintenance or promotion effect compared to general food, and health supplement food (health supplement food) means food for the purpose of health supplement. In some cases, the terms health functional food, health food, and health supplement food may be used interchangeably.

Specifically, the health functional food is a food prepared by adding 2S-2`methoxyclarinone to food materials such as beverages, teas, spices, gum, confectionery, etc., or encapsulated, powdered, suspended, etc., to be consumed. In the case of medicine, it means that it brings a specific effect on health, but unlike general medicines, it has the advantage of not having side effects that can occur when taking medicines for a long time by using food as a raw material.

The food of the present invention can be prepared by a method commonly used in the art, and can be prepared by adding raw materials and ingredients commonly added in the art.

In addition, the food composition can be prepared without limitation in various types of formulations as long as they are recognized as food.

In addition, the food composition may further include a physiologically acceptable carrier, the type of carrier is not particularly limited, any carrier commonly used in the art may be used.

In addition, the food composition may include additional ingredients that are commonly used in food compositions to improve smell, taste, and vision. For example, vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, panthotenic acid, and the like may be included. In addition, minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), and chrome (Cr); and amino acids such as lysine, tryptophan, cysteine, and valine.

In addition, the food composition may include preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), bactericides (bleaching powder, high bleaching powder, sodium hypochlorite, etc.), antioxidants (butylhydroxyanisole (BHA), butyl hydroxy Loxytoluene (BHT), etc.), coloring agents (tar color, etc.), coloring agents (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite, etc.), seasonings (MSG sodium glutamate, etc.), sweeteners (dulcin, cyclemate, saccharin) , sodium, etc.), flavoring (vanillin, lactones, etc.), expanding agent (alum, D-potassium hydrogen stannate, etc.), strengthening agent, emulsifier, thickener (thickener), coating agent, gum base agent, foam inhibitor, solvent, improver, etc. food May contain food additives. The additives may be selected according to the type of food and used in an appropriate amount.

The pharmaceutical composition for supplementing the treatment of cardiovascular disease, containing 2S-2'methoxyclarinone as an active ingredient of the present invention, suppresses the increase in PCSK9 expression and increases the production of LDLR, which is a side effect of the administration of statin, a cardiovascular drug. It has an increased activity of AMPK reduced by statins, so it can be usefully used for adjuvant treatment of cardiovascular diseases.

Figure 1 shows the results of LDLR expression upon the combined treatment of 2S-2'methoxy-clarinone and atorvastatin or rosuvastatin.
Figure 2 shows the results of PCSK9 expression upon the combined treatment of 2S-2'methoxy-curarinon and atorvastatin or rosuvastatin.
Figure 3 shows the results of AMPK and ACC phosphorylation expression during the combined treatment of 2S-2'methoxy-clarinone and atorvastatin or rosuvastatin.
FIG. 4 shows the results of PCSK9 and LDLR expression in HepG2 cells pretreated with PCSK9 when 2S-2`methoxy-curarinon and atorvastatin or rosuvastatin were co-treated.
Figure 5 shows the results of PCSK9 and LDLR expression upon co-treatment with 2S-2`methoxy-curarinon and statins-based drugs in PCSK9 pre-treated HepG2 cells.

Hereinafter, the configuration and effects of the present invention will be described in more detail through examples and experimental examples. These examples and experimental examples are only for exemplifying the present invention, but the scope of the present invention is not limited by the examples and experimental examples.

Example 1. Investigation of cell viability

MTT assay was performed to confirm the cell viability of the combined treatment of 2S-2`methoxyclarinone and statin. The MTT screening method is widely used as a cytotoxicity and cell proliferation screening method because many samples can be easily read using an ELISA reader using a 96-well plate. It is a method to reduce water-soluble MTT tetrazolium of yellow color to water-insoluble MTT formazan of purple color.

In order to confirm the effect of the combined treatment of 2S-2`methoxyclarinone and statins on cell viability, HepG2 cells (human liver hepatocellular carcinoma) were treated with DMEM (Welgene, Korea) suspended in a medium at a concentration of 1 x 10 ⁵ /ml, and inoculated into a 96-well plate in 100 μl each. Samples were treated with each concentration after 4 hours. After culturing for 24 hours, 5 μl of MTT (5 mg/ml) was added to each well and cultured for another 4 hours. Then, after removing the medium, 100 μl of DMSO was added to each well to react, and the absorbance was measured at 570 nm. Cell viability was calculated according to Equation 1 below, with the value of the negative control group treated with DMSO as 100%, and each result is shown in Table 1.

[Equation 1]

Cell viability (%) = [(OD570 nm value of extract treatment) / (OD570 nm value of negative control group)] × 100

1-1. Cell viability according to the combined treatment of 2S-2`methoxyclarinone and statin

In order to examine the cell viability by the combined treatment of 2S-2`methoxyclarinone and statin, 2S-2`methoxyclarinone and atorvastatin were treated at 10 μM, and rosuvastatin at 20 μM After each combination treatment, it was reacted for 24 hours. The results of the MTT assay are shown in Table 1.

Cell viability when treated with 2S-2`methoxyclarinone, atorvastatin, and rosuvastatin in combination, respectively sample name Rosuvastatin
(Rosuvastatin) atorvastatin
(Atorvastatin) 2S-2`methoxy-kurarinone Cell viability (%, mean ± standard deviation) - - - 100±1.07 - - + 111.97±2.58 - + - 89.64±2.06 + - - 84.18±3.31 - + + 84.20±2.49 + - + 87.57±0.18

As can be seen from the results of Table 1 above, 2S-2'methoxyclarinone, atorvastatin, and rosuvastatin each showed a cell viability of 80% or more, and 2S-2'methoxyclarinone and statin were treated in combination It was confirmed that all of them showed a cell viability of 80% or more and did not have a significant effect on cytotoxicity. Through this, it was confirmed that 2S-2`methoxyclarinone is safe as a composition for assisting treatment of cardiovascular diseases.

Example 2: LDLR (Low-Density Lipoprotein Receptor: low-density lipoprotein receptor) production investigation

In order to confirm the therapeutic efficacy of cardiovascular disease according to the combined treatment of 2S-2`methoxyclarinone and statin, the amount of LDLR production was investigated.

The LDL receptor is a protein that binds to LDL (low-density lipoprotein), the main carrier of cholesterol in the blood, and is inserted into the cell. LDL receptor gene abnormality was found to be the causative gene of familial hypercholesterolemia, the most common genetic disease in humans. Deficiency of the LDL receptor can lead to atherosclerosis due to an increase in LDL. In order to examine the effect of 2S-2`methoxyclarinone and statins on increasing LDLR production, RT-PCR was performed in HepG2 cells. HepG2 cells were suspended at a concentration of 2.5 x 10 ⁵ cells/ml, inoculated in 1 ml each in a 12-well plate, and maintained for one day.

After treatment with 2S-2`methoxyclarinone and atorvastatin at 10 μM and rosuvastatin at 20 μM, incubation was performed for 24 hours, RNA was recovered, quantified, and stored at -70°C. After synthesizing cDNA, the LDLR result was confirmed by Real Time-PCR. RealTime-PCR and analysis were performed to investigate the amount of LDLR production, and the analysis was performed according to the laboratory protocol. The results are shown in Figure 1.

As a result of confirming the amount of LDLR production according to the combined treatment of 2S-2'methoxyclarinone and statin by the above method, as can be seen in Figure 1, the combination with statin is higher than that of 2S-2'methoxyclarinone alone treatment. It was confirmed that the amount of LDLR production increased during treatment. In addition, this value was increased compared to the case of statin alone treatment, and it was confirmed that the synergistic effect according to the combined treatment was confirmed (Fig. 1A, B).

Example 3. Investigation of PCSK9 (Proprotein convertase subtilisin/kexin type 9) production

HepG2 cell line was treated with 10 μM of 2S-2`methoxyclarinone and atorvastatin, and 20 μM of rosuvastatin, followed by reaction for 24 hours. Subsequently, as a result of measuring PCSK9 at the RNA and protein stages, it was confirmed that when 2S-2`methoxyclarinone was treated alone, the amount of PCSK9 production was reduced compared to the control, but atorvastatin and rosuvastatin were treated alone When it was done, it was confirmed that PCSK9 increased compared to the control. After that, when 2S-2'methoxyclarinone and each statin were treated in combination, it was confirmed that PCSK9, which had been increased by statin, was reduced (FIG. 2).

Due to the above results, it can be confirmed that the negative effect of PCSK9 increase caused by statins can be alleviated by the combined treatment of 2S-2`methoxyclarinone, confirming the possibility of using statins as an adjuvant in the treatment of cardiovascular disease. could

Example 4. AMPK phosphorylation activity investigation

To investigate the effect of combined treatment of 2S-2`methoxyclarinone, atorvastatin, and rosuvastatin on the phosphorylation activation of AMPK (Adenosine monophosphate-activated protein kinase), liver-derived cell lines ( HepG2) was used.

Cells were serum-starved for 6 hours prior to drug treatment and exposed to drugs at the indicated concentrations and times. To prepare total protein, cells were lysed in RIPA lysis buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 5 mM sodium fluoride, 2 mM sodium orthovanadate, 1% NP-40, 0.1% sodium dodecyl sulfate [SDS], 1 mM phenylmethylsulfonyl fluoride [PMSF], and protein inhibitor cocktail [Roche Diagnostics, Heidelberg, Germany]). The protein concentration of the lysate was measured using Bio-Rad dye-binding micro assay. For immunoblotting, 30 μg of cell lysate was separated by SDS-polyacrylamide gel electrophoresis (PAGE). Proteins were transferred onto Polyvinylidene difluoride (PVDF) membranes (Merck Millipore, MA, USA). The membrane was blocked with TBS (10 mM Tris-HCl pH 7.4, 150 mM NaCl) containing 0.1% Tween 20 and 5% skim milk powder and incubated overnight at 4°C with primary antibody diluted in blocking buffer. The membrane was washed and incubated for 1 hour at room temperature with the appropriate secondary antibody. HRP-conjugated secondary antibodies were detected using ECL detection reagent (FIG. 3).

4-1. AMPK phosphorylation activity effect according to the combined treatment of 2S-2`methoxyclarinone and statin

As can be seen from the results of Figure 3, it was confirmed that phosphorylation of AMPK and ACC increased when 2S-2'methoxyclarinone was treated alone. In addition, when treated with statins, phosphorylation of ACC increased, but phosphorylation of AMPK showed different patterns depending on the type of statins. That is, when atorvastatin was treated, AMPK phosphorylation was rather decreased.

However, in the case of combined treatment with 2S-2`methoxyclarinone, it was found that the phosphorylation of AMPK and ACC increased in both cases of treatment with atorvastatin and rosuvastatin. In addition, it was confirmed that the increase in phosphorylation of AMPK and ACC was improved compared to the case of treatment with statin alone.

From the above results, it was confirmed that AMPK phosphorylation, which was reduced by statins, could be mitigated by the combined treatment with 2S-2'methoxyclarinone, and thus, 2S-2'methoxymetholone in the treatment of cardiovascular diseases using statins. The possibility of using toxyclarinone as an adjuvant was confirmed.

Example 5. Effects of PCSK9 and LDLR expression upon combined treatment with 2S-2`methoxyclarinone and statins in HepG2 cells pretreated with PCSK9

Cells dispensed by the method of Example 3 were pretreated with 2 μg/ml of PCSK9, 2S-2`methoxyclarinone and atorvastatin were treated with 10 μM, and rosuvastatin was treated with 20 μM of HepG2 cell line. Reacted for 4 hours. Thereafter, proteins were extracted, and 30 μg of each extracted protein was subjected to electrophoresis on an SDS-polyacrylamide gel, attached to a membrane, and protein expression levels were confirmed using respective antibodies: PCSK9 and LDLR.

In addition, immunoblot experiments were performed to confirm PCSK9 and LDLR when atorvastatin, rosuvastatin, fluvastatin, pravastatin, pitavastatin, or simvastatin and 2S-2'methoxycurarinon were treated in combination. Cells seeded by the method of Example 3 were treated with 2S-2'methoxyclarinone and each of the statins (pravastatin, pitavastatin, fluvastatin, or simvastatin) alone or simultaneously, and proteins were extracted and tested.

As a result of measuring PCSK9 after pretreatment with PCSK9, it was confirmed that PCSK9 increased in the group pretreated with PCSK9, but decreased in the group treated with 2S-2`methoxyclarinone. Thereafter, each statin increased PCSK9, but when 2S-2`methoxyclarinone and each statin were treated in combination, it was confirmed that PCSK9 was significantly reduced compared to the statin alone treatment group (FIG. 4 and FIG. 5) .

As a result of confirming the changes in PCSK9 when 6 widely used statins and 2S-2`methoxyclarinone were used in combination, 2S-2`methoxyclarinone, atorvastatin, rosuvastatin, fluvastatin, pravastatin, Alternatively, it was found that the amount of PCSK9 produced in the combined treatment with simvastatin was reduced compared to the single treatment group (FIG. 5).

In addition, as a result of measuring the expression level of LDLR after pretreatment with PCSK9, it was confirmed that LDLR was decreased by PCSK9 pretreatment in HepG2 cells (FIG. 4). In addition, LDLR expression decreased in cells treated with each statin alone after pretreatment with PCSK9, but in cells treated with 2S-2`methoxycurarinone and each statin in combination, LDLR expression and production were higher than those in the single treatment group. It was confirmed that it significantly increased (FIG. 5).

In other words, 2S-2`methoxyclarinone can be treated in combination with statin drugs to alleviate the side effect of increasing PCSK9 expression as well as LDLR when treated with statin drugs. In this case, PCSK9 expression is rather reduced while LDLR Expression can be significantly improved compared to the case of treatment alone, and it was confirmed that it can be used for preventing or treating cardiovascular diseases by reducing blood fatty acids and cholesterol.

Claims (11)

A pharmaceutical composition for supporting the treatment of cardiovascular disease, containing 2S-2'methoxy-kurarinone as an active ingredient,
The composition is a pharmaceutical composition for adjuvant of statins (statin), a treatment for cardiovascular disease.
delete The pharmaceutical composition according to claim 1, wherein the composition improves or alleviates side effects of statin administration.
The method of claim 1, wherein the statin is selected from the group consisting of Atorvastatin, Rosuvastatin, Fluvastatin, Pravastatin, Pitavastatin, and Simvastatin. Any one or more selected, the pharmaceutical composition.
The pharmaceutical composition according to claim 1, wherein the composition inhibits PCSK9 (Proprotein convertase subtilisin/kexin type) increased by statins.
The pharmaceutical composition according to claim 1, wherein the composition increases the production of low-density lipoprotein receptor (LDLR).
The pharmaceutical composition according to claim 1, wherein the composition increases AMPK (Adenosine monophosphate-activated protein kinase) phosphorylation activity reduced by statins.
The pharmaceutical composition according to claim 1, wherein the cardiovascular disease is any one or more selected from the group consisting of hyperlipidemia, hypertension, angina pectoris, coronary heart disease, myocardial infarction, arteriosclerosis, atherosclerosis, stroke, and arrhythmia.
A pharmaceutical composition for preventing or treating cardiovascular disease comprising 2S-2`methoxyclarinone and statin as active ingredients.
A method for preventing or treating cardiovascular disease, comprising administering to a non-human subject a combination of statin in the composition of any one of claims 1 and 3 to 8.
A health functional food composition containing 2S-2`methoxyclarinone as an active ingredient, for assisting improvement in cardiovascular disease,
The composition is a health functional food composition for adjuvant of statins (statin), a treatment for cardiovascular disease.