KR100564800B1 - Composition for preventing and treating cardiovascular diseases containing compound from extract of lettuce - Google Patents

Abstract

The present invention relates to a composition for the prevention and treatment of cardiovascular diseases containing a compound isolated from the lettuce extract as an active ingredient, more specifically by containing a compound isolated from the lettuce extract represented by the following formula (1) as an active ingredient, It relates to a cardiovascular treatment composition that inhibits the activity of the ACAT enzyme to increase blood HDL levels and lower LDL levels to prevent deposition and increase of blood cholesterol. By using the composition according to the present invention, it is possible to prevent various cardiovascular diseases due to an increase in blood cholesterol, such as hypercholesterolemia, hyperlipidemia, arteriosclerosis, angina pectoris, myocardial infarction or cerebral infarction.

<Formula 1>

Lettuce extract, cardiovascular disease, cholesterol

Description

Composition for preventing and treating cardiovascular diseases containing compound from extract of lettuce

₁ shows the spectrum of H ₁ -NMR of a compound isolated from lettuce.

Figure 2 shows the spectrum of ¹³ C-NMR of the compound isolated from lettuce.

Figure 3 shows the effect on the total cholesterol content of a compound isolated from a high fat diet and lettuce.

Figure 4 shows the effect on triglyceride content of a compound isolated from a high fat diet and lettuce

Figure 5 shows the effect on the HDL content of the compound isolated from diet and lettuce

The present invention relates to a composition containing a compound isolated from lettuce as an active ingredient, and more particularly, to a composition capable of preventing and treating various cardiovascular diseases by inhibiting the absorption of cholesterol in the blood by containing the composition. will be.

Cholesterol is an essential component of cells and plays an important role in cell growth, differentiation and development. In adults, cholesterol demand is about 1,300 mg per day, of which about 1,000 mg of cholesterol is produced by almost every cell in the body, about 300 mg of cholesterol is consumed by food, and excess cholesterol from food is used to meet the needs of cells. And remain (Gurr, MI, Proceedings of Nutrition Society , 47: 277 (1981); Goldstein, JL et al., Circulation , 76: 504 (1987)). These cholesterols wander through low-density lipoproteins, which are transformed into modified forms, adsorbed into the vascular lining, and then converted into foam cells, fat streaks, and atheros, which reduce the ability of blood vessels to contract and expand. Cause arterial disease, stroke, peripheral vascular narrowing, high blood pressure (Witztum JL and Steinberg D., J. Clin. Invest. 88: 1785-1792 (1991)). In addition, when the amount of cholesterol in the blood is high, it is known that atherosclerosis, which causes the formation and deposition of macrophages and foam cells along with fat on the blood vessel wall, forms plaque, which inhibits blood circulation (Ross, R., Nature 362: 801-809 (1993). Therefore, the continuous accumulation of such cholesterol eventually closes the arteries and leads to serious cardiovascular diseases such as various heart diseases and angina pectoris. The Lipid Research Clinics-Coronary Primary Prevention Trial (LRH-CPPT) study on the relationship between cholesterol ingested from food (hereinafter referred to as "diet cholesterol") and cardiovascular disease (The Lipid Research Clinic Program, J. Am. Med. Assoc., 251: 365 (1984)) and the results of the Helsinki Heart Study (Frick, MH et al., N. Eng. J. Med. , 317: 1237 (1987)). If intake is reduced by 1%, cardiovascular disease is reduced by 2%. Dietary cholesterol and endogenous cholesterol are mainly synthesized by the liver and small intestine and contribute to the level of cholesterol circulating in the blood. Therefore, the absorption efficiency of the plasma cholesterol level acts as an important factor and also acts as a risk factor involved in the premature develpment of cardiovascular diseases including atherosclerosis.

The therapeutic agents for cardiovascular diseases thus far can be classified into three types: agents that lower plasma cholesterol, agents that alleviate and alleviate arteriosclerosis, and agents that block or reverse the development of cardiovascular diseases. Among them, in particular, plasma cholesterol lowering agents are most widely used, and mainly act to lower the level of LDL-cholesterol or total cholesterol in the blood.

For this reason, studies have been actively conducted to develop effective drugs that can lower the total cholesterol level in plasma for the prevention of cardiovascular diseases. As a result, several substances have been developed that inhibit cholesterol biosynthesis in the human body. However, in order to improve side effects caused by these current drugs, research has recently been focused on finding compounds that can only selectively regulate blood cholesterol, and the representative one is ACAT (acyl-coenzyme A: cholesterol acyltransferase). ) Inhibitors.

ACAT is an enzyme that converts cholesterol into cholesteryl esters in human tissues. When atherosclerosis foam cells are formed by ACAT, these foam cells are transported by LDL in the blood. It contains a lot of esters. In other words, foam cells in the arterial vessel wall are often found with increased ACAT activity, and inhibiting ACAT activity in the liver can lower the level of LDL-cholesterol in the blood (Witiak DT and DR Feller, Medical, Chemical). and Biochemical Aspects, Elsevier, 159-195 (1991))

The mechanism of action of ACAT occurs largely in three parts of the intestine, liver, and blood vessel wall cells. First, in the intestine, ACAT converts ingested cholesterol into the form of esters to facilitate its absorption into the intestine. Second, cholesterol absorbed from the outside or biosynthesized in the body is accumulated in a carrier called VLDL (very lowdensity lipid) in the liver and then supplied to each organ through the blood vessels, whereby cholesterol is converted into cholesterol ester form by ACAT. Cholesterol accumulation is possible. Third, in the cells that make up the arteriovascular wall, ACAT converts cholesterol into its ester form to promote the accumulation of cholesterol in the cell, which is a direct cause of atherosclerosis. Therefore, the drug that inhibits the activity of ACAT may first reduce the amount of cholesterol that enters the body by inhibiting the absorption of intestinal cholesterol, and secondly, by inhibiting the release of cholesterol into the blood vessels from the liver to reduce blood cholesterol levels. Third, by preventing the accumulation of cholesterol in the blood vessel wall cells, it is expected that not only directly prevents arteriosclerosis but also prevents and treats various cardiovascular diseases. Moreover, ACAT inhibitors have the potential to be used as a direct treatment of atherosclerosis by promoting the conversion of already accumulated cholesterol esters into cholesterol.

Therefore, the development of new plant materials and substances that can exhibit the effect of inhibiting ACAT activity without safely exhibiting side effects has been continually required, and the present inventors have tried to prevent and treat various cardiovascular diseases including atherosclerosis. While studying the anti-absorption agent of cholesterol and neutral lipids, the extract of lettuce has ACAT inhibitory activity, and furthermore, the compound of formula (I) isolated from lettuce extract showed ACAT inhibitory activity to complete the present invention.

An object of the present invention is to provide a composition for the prevention and treatment of cardiovascular diseases that can regulate the rise of cholesterol in the blood by inhibiting the absorption of cholesterol.

Another object of the present invention is to provide a dietary supplement containing the composition.

Still another object of the present invention is to provide a method of preparing an active ingredient of the composition for preventing and treating the cardiovascular system.

The present invention provides a composition for the prevention and treatment of cardiovascular diseases containing a compound isolated from lettuce as an active ingredient and a dietary supplement containing the composition.

The present invention also provides a method for extracting the above compound from lettuce.

Hereinafter, the present invention will be described in detail.

The present invention is characterized by providing a composition for the prevention and treatment of cardiovascular diseases containing a compound of formula (1) as an active ingredient.

In one embodiment of the present invention, to obtain the compound of Formula 1, it was prepared by extracting from lettuce. Lactuca sativa was used in this example. The production method was extracted with 100% methanol, extracted again with 80% methanol, concentrated under reduced pressure, concentrated extraction with water and acetyl ethanol, and separated and purified by chromatography. Specifically, silica gel or ODS column chromatography was used.

Cardiovascular diseases and symptoms that can be treated with the compound of formula 1 according to the present invention include diseases caused by increased intravascular cholesterol and deposition. Specifically, it may be used to prevent or treat hypercholesterolemia, hyperlipidemia, arteriosclerosis, angina pectoris, myocardial infarction, cerebral infarction, etc. by inhibiting the absorption of cholesterol in the blood.

The cardiovascular prevention and treatment composition according to the present invention may contain a compound prepared as described above as an active ingredient, and a pharmaceutically acceptable carrier is added according to a pharmaceutical preparation method known in the art. It may be prepared as.

Examples of the carrier in the above, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, poly Vinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In addition, the lipase inhibitor may further include fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers or preservatives.

In addition, the tablets or compounds synthesized according to the present invention can be absorbed by formulating them in unit or multiple dosage forms such as tablets, capsules, powders, granules, suspensions, emulsions or parenteral preparations by conventional methods. It can be used as an agent for preventing and treating hyperlipidemia and preventing and treating various cardiovascular diseases caused by hypercholesterolemia.

Pharmaceutical compositions containing the compound isolated in the present invention as an active ingredient can be administered orally or parenterally as desired. Dosage may be determined by the expert taking into account various factors such as the patient's weight, age, sex, health condition, diet, time of administration, method of administration and excretion, complications, drug mixture and severity of the disease.

In addition, the compound of formula 1 may be added to food for the purpose of lowering blood cholesterol and preventing cardiovascular diseases. Foods to which the compound of formula 1 may be added include, for example, various foods, meats, beverages, snacks, confections, noodles, shells, ice creams, alcoholic beverages, tea bags, instant teas, granules, flavors, tablets and capsules. Etc., but is not limited thereto.

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

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

<Example 1> Isolation and Purification of Lettuce Extract

(1-1) Preparation of Lettuce Extract

20 kg of lettuce (Lactuca sativa) was added to 72 liters of 100% methanol and extracted at room temperature for 24 hours. Then, 22.5 liters of 80% methanol was added and extracted again. After filtration and concentration under reduced pressure, 120 g of lettuce extract was obtained.

(1-2) Isolation and Purification of Lettuce Extract

The concentrate was partitioned and extracted with 2 L of water and 2 L of acetyl ethanol, followed by TLC (Thin Layer Chromatography; Merck Co. Ltd.) using a solvent in which a ratio of n-hexane: acetyl ethanol was mixed at a ratio of 1: 1 with respect to the acetyl ethanol fraction. , 25TLC aluminiun sheet silica gel 60 F ₂₅₄ ) was developed, dried, and labeled with fluorescent or absorbed UV lamps (Spectroline, ENF-240C / F, specronics corporation, USA). After dipping in 10% aqueous sulfuric acid solution, the resultant was dried and heated with an alcohol lamp to confirm the color development and R _f value.

56 g of acetyl ethanol fractions were developed with sequential changes of CHCl ₃ : MeOH from 7: 1 to 5: 1 and 3: 1, followed by silica gel column chromatography (Merck Co. Ltd., silica gel 60 (63-200). Μm) and 750 g) were collected, and 70 ml each. This aliquot was identified by TLC to obtain eight subfractions, and n-hexane: acetyl ethanol was changed from 5: 1 to 3: 1 as solvent for the second fraction, and CHCl ₃ : MeOH was changed to 10: 1. Silica gel column chromatography was carried out to obtain six small fractions. Take 100 mg from the fourth fraction and change the acetone: methanol: water from 3: 7: 1 to 9: 3: 1 as the solvent, and then use OSD column chromatography (octadecyl silicagel, Merck Co. Ltd., LiChroprep RP-18 (40). ˜63 μm) and 50 g) were used to isolate and purify the first 70 mg of the fraction.

Melting point: 132 ° C; White Powder (CHCl ₃ )

¹ H-NMR (400 MHz, CDCl ₃ ): (Table 1, FIG. 1)

¹³ C-NMR (100 MHz, CDCl ₃ ): (Table 1, FIG. 2)

IRv (KBr, CHCl ₃ ): 3334, 2954, 2923, 2868, 1669 cm ^-1

EI / MS: m / z = 29 [M ⁺ ]

<Example 2> Determination of the ACAT inhibitory activity of the compound of Formula 1 in vitro. )

8 μl of microsomal protein solution (17.3 mg / ml in 10 mM HEPES buffer, pH 7.4), 0.8 μl of 100 mM DTT, 4 μl of 400 μM BSA, sample solution for measurement (water: dimethylsulfoxide) = 9: 1 (v / v)) 4 μl, 1.5 M phosphate buffer (pH 7.4) 8 μl, 100 μM 14C-oleoyl-CoA (11.1 MBq / mmol) and 5 μl of water were added thereto, followed by reaction at 30 ° C. for 5 minutes. 200 μl of methanol was added to stop the reaction between the enzyme and the compound, followed by preparative TLC using a developing solvent of ether: diethyl ether: acetyl acid = 90: 14: 2 (Rf = 0.54), followed by iodine spraying. The cholesterol ester band (cholesterol ester band) was aliquoted and placed in a scintillation vial, and then 4 ml of EX-H solution was added to measure radioactivity. The ACAT activity was compared with the control group by calculating the hourly radiation dose from the measured radiation dose, and the picomol / min / mg protein unit. As a result, the ACAT activity inhibition rate was 0% of the control group. It was confirmed that 91.6% of the compounds isolated from lettuce.

<Example 3> Measurement of plasma lipid content in mice

(3-1) Dietary composition and sampling of mice

After breeding mice (C57BL / 6, 5 weeks old, male, Daehan Biolink) for 1 week as a general solid feed, and after an adaptation period, the normal diet group, the high-fat diet group, and the high-fat diet group containing 3% of the material extracted from the lettuce Eight animals per group were classified. Experimental diet was prepared as shown in Table 2 with reference to the AIN-76 dietary composition, and was bred for 4 weeks by freely ingested with water.

After the breeding mice were fasted for 12 hours, anesthetized with diethyl ether and blood was collected from the heart using a heparin-treated syringe. Plasma was separated from the collected blood by centrifugation at 8,000 rpm for 15 minutes and stored at -20 ° C until the measurement. The total cholesterol, triglyceride and HDL contents were measured.

(3-2) Determination of Total Cholesterol Content

Total cholesterol content in plasma was measured by enzymatic method (Sale et al., Anal. Biochem. , 142: 347-350, 1984) using a solution AM 202-K (Asan Pharmaceutical Co., Ltd., Korea) for measuring total cholesterol . It was. 20 μl of plasma, reagent blank, and standard solution and 3 ml of enzyme solution were mixed well and allowed to stand at 37 ° C. for 5 minutes, and then the absorbance was measured at a wavelength of 500 nm within 60 minutes. The total cholesterol content was obtained by multiplying the ratio of the sample absorbance to the standard solution absorbance by the standard solution concentration (300 mg / dL). As shown in FIG. 3, the high fat diet resulted in an increase in total cholesterol content of 29.5% over the normal diet. The total cholesterol content increased by the high fat diet decreased to the level similar to the normal diet group in the high fat diet group with lettuce extract.

(3-3) Triglyceride content measurement

The triglyceride content in plasma was determined by enzymatic method (Biggs et al., Clin. Chem. , 21: 437-441, 1975) using Asanset triglyceride measuring solution AM 157S-K (Asan Pharmaceutical Co., Ltd., Korea) . Was measured. 20 μl of plasma, reagent blank, standard solution, and 3 ml of enzyme solution were mixed well and left at 37 ° C. for 10 minutes, and then the absorbance was measured at a wavelength of 550 nm within 60 minutes. The triglyceride content was calculated by multiplying the ratio of the sample absorbance to the standard solution absorbance by the standard solution concentration (300 mg / dL). As shown in FIG. 4, the high fat diet resulted in an increase of triglyceride content of 31.9% over the normal diet. The total cholesterol content increased by the high fat diet decreased to the level similar to that of the normal diet group in the high fat diet group with lettuce extract.

(3-4) HDL content measurement and LDL content estimation

HDL content in plasma was precipitated β-lipoprotein (LDL, VLDL) by using Ezediel Cholesterase AM 203-K (Asan Pharmaceutical Co., Ltd., Korea), and then the HDL cholesterol in the supernatant Obtained by the enemy method. The plasma and 0.2 ml of the separation solution were mixed well, and allowed to stand at room temperature for 10 minutes, followed by centrifugation at 3000 rpm for 10 minutes. 3 ml of enzyme solution was well mixed with each 0.1 ml of the supernatant, reagent blank, and standard solution, which was allowed to stand at 37 ° C. for 5 minutes. The absorbance was measured at a wavelength of 500 nm with the reagent blank as a control. The HDL content was obtained by multiplying the ratio of the sample absorbance to the absorbance of the standard solution by the standard solution concentration (50 mg / dl) and the dilution factor 2. LDL content was determined by the following equation (Friedewald et al., Clin. Chem., 18: 499-502).

As shown in Figure 5, the high-fat diet group significantly reduced HDL content in the blood 27.8% compared to the normal diet group, the HDL content reduced by the high-fat diet rather than the normal diet group in the high-fat diet group added lettuce extract More than 12.4%. Since HDL plays a role in transporting and removing unnecessary cholesterol from the blood, an increase in the content of HDL means that lettuce extract can lower blood cholesterol. As shown in Figure 5, the high-fat diet group significantly increased the LDL content in the blood nearly two times compared to the normal diet group, the LDL content increased by the high-fat diet was added to the normal diet group in the high-fat diet group added with lettuce extract Decreased to a level similar to Since LDL is a modified form of LDL cholesterol that easily adheres to the blood vessel walls and causes atherosclerosis, a decrease in LDL means a decrease in the deposition of cholesterol in the blood.

Compounds isolated from lettuce inhibit ACAT, an enzyme that esterifies cholesterol, increasing blood HDL levels and lowering LDL levels to prevent the deposition and increase of blood cholesterol. Therefore, it is effective in the prevention and treatment of cardiovascular diseases such as hypercholesterolemia, arteriosclerosis, angina pectoris, myocardial infarction or cerebral infarction as well as hypercholesterolemia. In addition, compared to drugs that are chemically synthesized and sold, they are not addictive and can be added to various food compositions and used as various supplements for reducing blood cholesterol concentration.

Claims (6)

delete delete delete Blood cholesterol lowering health functional food containing the compound of formula 1 as an active ingredient. <Formula 1>

The lettuce is extracted with 100 vol.% Methanol, then extracted with 80 vol.% Methanol, concentrated under reduced pressure and concentrated, followed by separation and purification. <Formula 1>

The method of claim 5, wherein the separation and purification are sequentially performed from CHCl ₃ : MeOH (volume: volume) to 7: 1 (volume: volume) and 5: 1 (volume: volume) and 3: 1 (volume: volume). Varying and spreading, silica gel column chromatography was carried out, and each of 70 ml was collected; For the second fraction of this aliquot, n-hexane: acetyl ethanol is changed from 5: 1 (volume: volume) to 3: 1 (volume: volume) as solvent, and CHCl ₃ : MeOH is 10: 1 (volume: Volume), to conduct silica gel column chromatography to obtain 6 small fractions; Take 100 mg from the fourth aliquot of the small aliquot and change the acetone: methanol: water from 3: 7: 1 (volume: volume) to 9: 3: 1 (volume: volume) as a solvent and perform OSD column chromatography. Method for preparing a compound of formula 1 comprising the step of separating and purifying the first fraction 70mg. <Formula 1>

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