KR20000017664A - Acyl coa: cholesterol-o-acyltransferase inhibitory composition comprising naringin or naringenin - Google Patents

Abstract

PURPOSE: A composition is provided to be usefully used as preventing and treating agents of various cardiovascular diseases by reducing low density lipoprotein(LDL) cholesterol in blood by impeding the ACAT(acyl CoA:cholesterol-O-acyltransferase) activation in the body. CONSTITUTION: The pharmacological composition is for ACAT(acyl CoA:cholesterol-O-acyltransferase) impediment containing naringin or naringenin with a carrier pharmacologically allowable as activation element. The naringin or the naringenin can be extracted from citrus fruits or can be compounded by a compounding method. The naringin or naringenin impedes the ACAT activation, and reduces the cholesterol in blood of a mouse. And an ACAT activation impeding agent composition can be produced by mixing the naringin or naringenin as an effective element with the carrier allowable pharmacologically or as a food addition.

Description

ACYL COA: CHOLESTEROL-O-ACYLTRANSFERASE INHIBITORY COMPOSITION COMPRISING NARINGIN OR NARINGENIN

The present invention relates to an acyl coei: cholesterol-O-acyltransferase (ACAT) inhibitor composition comprising naringin (C27H32O14, molecular weight 580.53) and naringenin (C15H12O5, molecular weight 272.25).

Coronary cardiovascular disease currently accounts for more than 30% of all deaths and is a serious problem in developed countries such as the United States and Europe. Meanwhile, in developing countries, heart disease is on the rise due to westernization of diet and lack of exercise. If the amount of cholesterol in the blood is high, macrophage, foam cells, etc. are formed and deposited in the blood vessel walls, and plaques are formed, and atherosclerosis, which inhibits the blood circulation, is known to be prone to occur. Ross, R., Nature, 362 , 801-809 (1993)). Reducing the amount of cholesterol in the blood is a way to suppress the absorption of cholesterol. ACAT is an enzyme that converts cholesterol into cholesteryl esters in human tissues. The formation of macrophage or smooth muscle cell-derived foam cells is an important factor in experimental and clinical atherosclerosis. Foam cells contain large amounts of cholesterol esters formed by ACAT and carried by LDL in the blood. Because foam cells in the arterial vessel walls are often found with increased ACAT activity, ACAT inhibitors are likely to be protective agents of atherosclerosis. Inhibition of ACAT activity in the liver may also lower LDL-cholesterol levels in the blood (Witiak, DT and DR Feller (eds), Anti lipidemic Drugs: Medicinal, Chemical, and Biochemical Aspects , Elsevier, pp159-195 (1991)).

Other ways to reduce the amount of cholesterol in the blood include 3-hydroxy-3-, an enzyme that inhibits the action of cholesterol ester transfer protein (CETP) that mediates cholesterol transfer between lipoproteins or is involved in cholesterol biosynthesis in the liver. A method of inhibiting the action of methyl glutaryl coenzyme A (HMG-CoA) reductase. The present inventors conducted research on pharmacologically active substances of substances rich in herbal medicines, food ingredients, fruits and vegetables that are considered to be relatively safe.

Naringenin is a substance obtained by removing glycosides from naringin, a natural product present in citrus fruits such as oranges and tangerines, and when administered to rats, naringenin has been reported to lower blood cholesterol concentrations at a concentration of 0.5 to 1%. 283154 (October 29, 1996). However, the literature reported that natural products containing glycosides such as naringin have no effect on cholesterol lowering. In addition, the amount of cholesterol in the blood is controlled through a lot of regulation in the animal body, which does not mention the specific mechanism by which blood cholesterol drop occurs.

The present inventors have found that bioflavonoids in citrus fruits have many types of activity beneficial to the human body, and therefore they are responsible for cholesterol metabolism, especially cholesterol biosynthesis-related enzymes (eg, HMG-CoA reductase) or cholesterol absorption related to atherosclerosis. As a result of continuing the study to determine whether related enzymes (eg, ACAT or CETP) and related activities, the present invention was completed by finding that naringin and naringenin inhibit ACAT activity.

An object of the present invention to provide a pharmaceutical composition for inhibiting acyl coay: cholesterol-O-acyltransferase (ACAT) activity containing naringin or naringenin.

Another object of the present invention to provide a food or beverage composition for inhibiting ACAT activity comprising naringin or naringenin.

In the present invention, there is provided a pharmaceutical composition for inhibiting acyl coei: cholesterol-O-acyltransferase (ACAT) activity comprising naringin or naringenin as an active ingredient together with a pharmaceutically acceptable carrier. Also provided are food compositions and beverage compositions for inhibiting ACAT comprising naringin or naringenin.

Naringin or naringenin can be extracted from citrus fruits or synthesized by known synthetic methods. Citrus fruits in the present invention refers to tangerine, orange, lemon, grapefruit, citron or tanza. Flavonoids contained in citrus fruits may vary in active ingredient ratio depending on the maturity of the fruit or the type of fruit, and their extraction methods are disclosed in the literature and patents such as the Merck Index.

Naringin or naringenin inhibit ACAT activity, and some of the decrease in blood cholesterol in rats may be due to a decrease in ACAT activity. Toxicity studies studied to date have shown that naringin or naringenin is virtually nontoxic when administered orally to rats at a dose of 1000 mg / kg. In addition, naringin or naringenin show no adverse effects on the function of organs, including the liver.

In order to inhibit the activity of ACAT, naringin or naringenin may be mixed with a carrier that is pharmaceutically or as an acceptable food additive as an active ingredient to prepare an ACAT activity inhibitor composition. The composition may further include conventionally used excipients, disintegrants, sweeteners, lubricants, flavoring agents and the like, and may be prepared by tablets, capsules, powders, granules, suspensions, emulsifiers, syrups, solutions or by conventional methods or It may be formulated in a unit dosage form or in multiple dosage form pharmaceutical preparations, such as preparations for parenteral administration.

The ACAT inhibitor composition containing naringin or naringenin of the present invention as an active ingredient can be parenterally or orally administered as desired, and 0.001 to 10 g, preferably 0.1 to 1 g per kg of body weight per day. The amount can be administered in one to several portions. Dosage levels for a particular patient may vary depending on the patient's weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion, severity of the disease, and the like.

Naringin or naringenin may also be added to foods or beverages for the purpose of inhibiting ACAT activity. Examples of foods to which naringin or naringenin may be added for the development of dietary supplements include, for example, various foods, meats, beverages, chocolates, snacks, snacks, pizzas, ramen noodles, other noodles, gums, ice creams, alcohols. Beverages, vitamin complexes and dietary supplements.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following Examples are only for illustrating the present invention, and the scope of the present invention is not limited to these.

Example 1 Dietary Composition of Animals and Animal Administration of Naringin and Naringenin

30 Sprague-Dawley rats of 3 weeks of age were purchased from the Korean Experimental Animal Center in Negative Chungcheongbuk-do and were bred for 1 week in experimental animal feed, and at 4 weeks of age (90-110 g), Divided into three groups by randomized block design. Three groups of rats each had three different high cholesterol diets, namely 1% cholesterol (control) in normal diet (AIN-76 experimental animal diet); 1% cholesterol and 0.1% naringin; A diet containing 1% cholesterol and 0.1% naringenin was taken. The composition of the diets ingested in the three groups is shown in Table 1 below.

All diets correspond to a high cholesterol diet (1%, wt / wt), naringin and naringenin were purchased from Sigma Chemical company, St. Louis, Mo. AIN-, the laboratory diet of rats. Cellulose, mineral and vitamin mixture components in the 76 dietary composition were purchased from TEKLAD premier, Madison, Wisconsin, USA.

All dietary rats were given free diet with water for 6 weeks. The dietary intake was recorded daily and weighed every 7 days. After the animal breeding was completed, the data on the breeding journals were analyzed.

Example 2: Blood Total Cholesterol, HDL-Cholesterol, and Neutral Lipid Content in Experimental Animals

Measure

The effects of experimental diet on blood cholesterol and triglyceride content in rats were investigated as follows.

Blood samples were taken from three dietary rats, and plasma HDL fractions were isolated from the HDL-cholesterol reagent (Sigma Chemical Co. Cat. No. 352-3) including dextran-sulfate. Total cholesterol and HDL-cholesterol concentrations were measured by enzymatic coloration using Sigma's Total Cholesterol Kit (Cat. No. 352-100) (Allain et al., Clin. Chem., 20 , 470-475 (1974)). It was. Neutral lipid concentrations were measured using Sigma Diagnostic Kit (Cat. No. 339-50) (Bucolo, G. and David, H., Clin. Chem., 19 , 476-482 (1973)). The results are shown in Table 2 below, plasma total cholesterol concentration was reduced by 32% compared to the control group in naringin administration group, 18% compared to the control group in naringenin administration group.

Example 3: Effect of Naringin and Naringenin Administration on ACAT Enzyme Activity

(Step 1) Preparation of the microsome

Liver was harvested at the expense of three groups of rats. 1 g of liver tissue was homogenized with 5 ml of solution 1 (0.1 M potassium phosphate pH 7.4, 0.1 mM EDTA and 10 mM β-mercaptoethanol). This homogenate was centrifuged at 3,000 × g for 15 minutes at 4 ° C. The supernatant was transferred to a new tube and centrifuged again at 15,000 × g at 4 ° C. for 15 minutes. The supernatant was transferred to a 5 ml ultracentrifuge tube (Beckman) and centrifuged at 100,000 × g at 4 ° C. for 1 hour. The supernatant was discarded and the pellet was suspended with 3 ml of solution 1, then the solution was centrifuged at 4Ox at 100,000xg for 1 hour. The supernatant was discarded and the pellet suspended in 1 ml of solution 1. The protein concentration in the suspension was measured by Lowry et al. And the protein concentration was adjusted to 4-8 mg / ml. This suspension was stored in a deep freezer.

(Step 2) ACAT Activity Measurement

6.67 μl of cholesterol solution dissolved in acetone at a concentration of 1 mg / ml was mixed with 6 μl of 10% Triton WR-1339 in acetone, and acetone was evaporated using nitrogen gas. Distilled water was added to contain cholesterol.

10 μl of the aqueous solution of cholesterol obtained above, 10 μl of 1 M potassium phosphate (pH 7.4), 5 μl of 0.6 mM bovine serum albumin (BSA), 10 μl of the microsome obtained in step 1 and 55 μl of distilled water were mixed. (90 μl total). The mixture was pre-reacted in a waterbath at 37 ° C. for 30 minutes.

10 μl of oleyl-CoA solution (0.3 mg / ml) prepared by mixing radiolabeled oleyl-CoA and oleyl-CoA was added to the pre-reacted mixture, and the resulting mixture was water bathed at 37 ° C. for 30 minutes. Reaction at To this was added 500 μl of isopropanol: heptane = 4: 1 (v / v) solution, 300 μl of heptane and 200 μl of 0.1 M potassium phosphate (pH 7.4), followed by vigorous mixing with vortex, followed by room temperature. It was left for 2 minutes at.

200 μl of the supernatant was placed in a scintillation bottle, and 4 ml of scintillation solution (manufactured by Lumac) was mixed and the radiation dose was measured by a scintillation counter. ACAT enzyme activity was expressed in picomolar / min / mg protein by calculating the radiation dose per hour from the measured radiation dose. The results are shown in Table 3.

As can be seen in Table 3, administration of 0.1% naringin reduced ACAT activity in rat liver 21%. In addition, administration of 0.1% naringenin reduced ACAT activity by 33%.

Example 4 Oral Toxicity of Naringin

Specific pathogens free of 7-8 weeks of age, 6 females weighing 25-29 g and 6 males weighing 34-38 g, temperature 22 ± 1 ° C, humidity 55 ± 5%, illumination 12L / 12D was bred in the animal room. Mice were allowed to acclimate for about a week before being used for the experiment. Feed for experimental animals (for Cheil Jedang Co., Ltd., mice and rats) and drinking water were supplied after sterilization and free ingestion.

Naringin was prepared at a concentration of 100 mg / ml using 0.5% Tween 80 as a solvent, followed by oral administration of 0.2 ml per 20 g of mouse body weight. Samples were administered orally once and observed for side effects or lethality for 10 days after administration. That is, on the day of dosing, changes in general symptoms and the presence or absence of dead animals were observed at least once in the morning, at least 1 pm, 4 hours, 8 hours, 12 hours, and the next day until the 10th day of administration. In addition, the animals were killed and dissected at 10 days of administration, and the internal organs were visually examined. Changes in body weight were measured at daily intervals from the day of administration to observe the weight loss of animals caused by naringin.

The purpose of this experiment was to provide information on available drug doses in general pharmacology and drug efficacy tests and to derive basic data on toxicity by identifying the degree of acute toxicity in oral routes of naringin. Got it.

In acute oral toxicity, no lethal animals were observed for 10 days at the dose of 1,000 mg / kg naringin. An autopsy of the surviving animals was performed 10 days later, and there were no gross lesions, and normal body weight gain was observed without any weight loss for 10 days from the day after the administration.

In conclusion, naringin was not toxic upon oral administration at the above concentrations.

The following formulation examples are merely illustrative of the formulation of the present invention and do not limit the scope of the invention.

My example

Hard gelatin capsules were prepared using the following ingredients:

Amount (mg / capsule)

Active Ingredients (Naringin) 20

Dry starch 160

Magnesium stearate 20

200 mg total

ACAT activity inhibitor composition comprising naringin or naringenin can be usefully used as a prophylactic and therapeutic agent for various cardiovascular diseases since it inhibits ACAT activity in the animal to reduce blood low density lipoprotein (LDL) cholesterol.

Claims (2)

A food composition for inhibiting acyl CoA: cholesterol-O-acyltransferase (ACAT) activity, comprising an effective amount of naringin or naringenin. The method of claim 1, Food composition in the form of a drink.