KR20010007624A - Composition for preventing and treating atherosclerosis, hyperlipidemia, hepatic diseases and glycosemia, comprising neohesperidin dihydrochalcone - Google Patents

Abstract

PURPOSE: A composition containing neohesperidin dihydrochalcone generally used as a sweetener is provided for preventing and treating arteriosclerosis, hyperlipemia, liver diseases, and glycosemia. CONSTITUTION: Neohesperidin dihydrochalcone(C28H36O15, M.W. 612.60) is obtained by extracting from the rind or outer layer of grapefruit or by chemically changing Naringin, and it is sweeter 1,500 to 2,000 times than sugar per unit weight. Therefore it has been usually used as a sweetener not a medicinal material. The composition containing an effective amount of neohesperidin dihydrochalcone, that is, 0.1 to 500 mg/kg, preferably 1 to 100 mg/kg a day, is orally or topically administrated into a patient and the amount of administration may be variable by patient's age, weight, sex, health, diet, severity of disease, and so on. The composition containing neohesperidin dihydrochalcone may be added into foods and drinks in an amount of 0.01 to 20 wt.%, preferably 0.1 to 5 wt.% of diosmin.

Description

COMPOSITION FOR PREVENTING AND TREATING ATHEROSCLEROSIS, HYPERLIPIDEMIA, HEPATIC DISEASES AND GLYCOSEMIA, COMPRISING NEOHDPERCHAIN DIHY

The present invention relates to a composition for the prevention and treatment of atherosclerosis, hyperlipidemia, liver disease, hyperglycemia comprising neohesperidin dihydrochalcone.

Citrus peels contain a wide variety of bioflavonoids from Table 1, which are known to have very beneficial pharmacological activity.

Citrus peels are known to have various pharmacological activities, for example, lipid metabolism improving effects, anticancer and antiviral effects associated with cardiovascular diseases such as blood pressure and atherosclerosis. For example, hesperidin or hesperetine is known to have capillary fortification, reduced permeability, lowered blood pressure, lowered blood cholesterol, antiplatelet aggregation, anti-inflammatory and antiviral effects (Meyer, OC, Angiology, 45, 579-584 (1994); Struckmann JR, et al., Angiol., 45, 419-428 (1994); Matsubara, Y., et al., Japan Organic Synthesis Chem. Association Journal., 52, 318-327 (1994. Mar.); Galati EM, et al., Farmaco., 51 (3), 219-221 (1996, Mar.); Monforte MT., Et al., Farmaco., 50 (9) , 595-599 (1995, Sep.); JP 95-86929; JP 95-86930; Chung, MI, et al., Chin. Pharm. J. (Taipei)., 46, 429-437 (1994, Nov. Galati, EM, et al., Farmaco., 40 (11), 709-712 (1994, Nov.); and Emim JA, et al., J. Pharm.Pharmacol., 46 (2), 118- 122 (1994). Bioflavonoids are known to have antioxidant, hypotensive, anticancer and antiviral effects (Saija, A., et al., Free Radical Biol. Med., 19, 481-486 (1995); Matsubara, Y., et al. Galati EM, et al., Supra; Felicia V., et al., Nutr. Cancer, 26, 167-181 (1996); European Patent Publication No. 0352147 A2; and Kaul, TN, et. al., J. Med. Virol., 15, 71-79 (1985)), naringenin and naringin are known to have cholesterol-lowering, anticancer and anti-gastric ulceration (protection from alcohol) (Monforte MT. et al., Supra; JP 95-86929; JP 95-86930; Felicia V. et al., Supra; European Patent Publication No. 0352147 A2; and Martin MJ et al., Pharmacol., 49, 144-150 (1994) .

In addition, citrus rinds and seeds contain limonoids, which have been shown to have anticancer activity (Lam, LKT et al., Inhibition of chemically induced carcinogenesis by citrus limonoids.In Food Phytochemicals for Cancer Prevention, Vol. I, ACS Symposium series No. 546, Huang, MT, O. Osawa, CT Ho, R. Rosen (ed). 1993).

Therefore, citrus peels, extracts thereof, or purified bioflavonoids separated therefrom are useful for developing health-producing foods or pharmaceuticals for preventing adult diseases.

One of the bioflavonoids, neohesperidine dihydrochalcone (C ₂₈ H ₃₆ O ₁₅ , MW 612.60), is obtained from grapefruit rinds or linds, or by chemical modification of naringin, by weight to 1,500. It has been developed as a food sweetener because it has a sweet taste of 2,000 to 2,000 times (US Pat. No. 4,085,232). Therefore, neohesperidin dihydrochalcone is not toxic even if administered to animals for a long time, and according to the results of subchronic toxicity, no problem was observed even when ingested at 750 mg / kg / day (BAR Lina et. Al. Food Chem. Toxicol., 7, 500-513 (1990)). However, neohesperidine dihydrochalcone has not been reported for its pharmacological activity other than its effect as a sweetener.

The present inventors have therefore become interested in the pharmacological activity of neohesperidin.

Hamstring and 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 and foam cells along with fat are formed and deposited on the blood vessel wall, and plaque is formed, and atherosclerosis, which inhibits blood circulation, is easy to come. Ross, R., Nature, 362, 801-809 (1993).

Reducing the amount of cholesterol in the blood is a way to suppress the absorption of cholesterol. Acyl CoA: cholesterol-o-acyltransferase (ACL) 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 the level of LDL-cholesterol 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. There is a method of inhibiting the action of methyl glutaryl coenzyme A (HMG-CoA) reductase. HMG-CoA reductase is an enzyme that mediates the synthesis of mevalonic acid, an intermediate step in the biosynthetic pathway of sterols and isoprenoid compounds. Hypercholesterolemia is caused by slowing the rate of cholesterol synthesis by decreasing the activity of this enzyme. (hypercholesterolemia) can be used effectively (William, WP, Cardiovascular Pharmacology, Kanu Chatterjee (ed), Wolfe Pullishing, 8.6-8.7 (1994)). HMG-CoA reductase activity inhibitors currently in use are derived from the genus Penicillium or Aspergillus, such as lovastatin and simvastatin developed by Merck, USA, and pravastatin, developed by Sankyo, Japan. There is this. However, statins have adverse effects on the central nervous system (Saheki, AT et al., Pharm. Res., 11, 304-311 (1994)), and lovastatin and simvastatin increase LDL receptor activity in the blood by increasing liver LDL receptor activity. Although it lowers cholesterol, it has been reported to have side effects such as increased amounts of various enzymes, increased creatine kinase, and rhabdomyolysis (Farmer, JA et al., Baillers-clin. Endocrinol. Metab., 9, 825-847 (1995)). Therefore, the development of new HMG-CoA reductase activity inhibitors that are inexpensive and have no side effects are urgently needed.

On the other hand, the liver is deteriorated due to excessive intake of food or alcohol containing fat components and infection of hepatitis B or C virus, and when left untreated, hepatitis, cirrhosis, liver cancer, and the like progress. In particular, excessive fat intake or excessive alcohol intake causes fatty liver to accumulate lipids in liver tissues, such as glutamate-oxaloacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT), and γ-glutamyl transpeptidase (γGTP). (T. Banciu, et al., Med. Interne., 20, 69-71 (1982); and A. Par, et al., Acta. Med. Acad. Sci. Hung., 33, 309). 319 (1976).

Hayashi et al. Report that green tea extract improves rat liver function by preventing an increase in rat serum GOT and GPT (M. Hayashi, et al., Nippon Yakuri gaku Zasshi, 100, 391-399 (1992). ).

The inventors of the present invention, while searching for and researching pharmacologically active substances rich in herbal medicines, food ingredients, and fruits and vegetables that appear to be relatively safe, the bioflavonoids hesperidin, hesperetin, naringin and naringenin, and citrus peel extracts containing them in abundance, blood cholesterol Has been patented with the finding that it significantly reduces the activity of ACAT, inhibits ACAT activity, strongly inhibits the formation or deposition of macrophage-lipid complexes on the arterial endothelial surface, and has therapeutic and prophylactic effects on liver disease (WO98 / 16220, WO98 / 16221, Korean Patent Application No. 98-10888, Korean Patent Application No. 98-10889).

The present inventors conducted a study and examined the activity of neohesperidin dihydrochalcone belonging to bioflavonoids such as hesperidin and naringin, and as a result, hepatic function protection such as atherosclerosis inhibitory effect, hyperlipidemia inhibitory effect, fatty liver formation and hepatitis inhibitory effect, etc. It was confirmed that the action, and the hypoglycemic effect has completed the present invention.

An object of the present invention to provide a pharmaceutical composition for preventing and treating atherosclerosis.

Another object of the present invention to provide a food or beverage composition for preventing and treating atherosclerosis.

Another object of the present invention to provide a pharmaceutical composition for preventing and treating hyperlipidemia.

Another object of the present invention to provide a food or beverage composition for preventing and treating hyperlipidemia.

Another object of the present invention to provide a pharmaceutical composition for preventing and treating liver disease.

Another object of the present invention to provide a food or beverage composition for preventing and treating liver disease.

Another object of the present invention to provide a pharmaceutical composition for preventing and treating hyperglycemia.

Another object of the present invention to provide a food or beverage composition for preventing and treating hyperglycemia.

Figures 1a, 1b and 1c is an arterial endothelial picture of the control group, lovastatin administration group and neohesperidin dihydrocalcon administration group rabbits, respectively.

Figures 2a, 2b and 2c is a picture of liver dissection of the control group, lovastatin administration group and neohesperidin dihydrocalcon administration group rabbits, respectively.

In accordance with the above object, the present invention provides a pharmaceutical composition for preventing and treating atherosclerosis comprising neohesperidin dihydrochalcone as an active ingredient with a pharmaceutically acceptable carrier. Also provided are food compositions and beverage compositions for preventing and treating atherosclerosis, including neohesperidin dihydrochalcone.

According to another object, there is provided a pharmaceutical composition for preventing and treating hyperlipidemia comprising neohesperidin dihydrochalcone as an active ingredient with a pharmaceutically acceptable carrier. Also provided are food compositions and beverage compositions for preventing and treating hyperlipidemia comprising neohesperidin dihydrochalcone.

According to another object, there is provided a pharmaceutical composition for preventing and treating liver disease comprising neohesperidin dihydrochalcone as an active ingredient with a pharmaceutically acceptable carrier. Also provided are food compositions and beverage compositions for the prevention and treatment of liver disease, including neohesperidin dihydrochalcone.

According to another object, there is provided a pharmaceutical composition for preventing and treating hyperglycemia comprising neohesperidin dihydrochalcone as an active ingredient with a pharmaceutically acceptable carrier. Also provided are food compositions and beverage compositions for preventing and treating hyperglycemia, including neohesperidin dihydrochalcone.

Hereinafter, the present invention will be described in detail.

Neohesperidine dihydrochalcone used as the active ingredient in the present invention can be extracted from grapefruit rind and skin or synthesized from naringin by known synthetic methods. Many methods for their extraction and synthesis have been published in the literature and patents such as the Merck Index.

Neohesperidin dihydrocalcon has a prophylactic effect as well as the treatment of atherosclerosis, hyperlipidemia, liver disease and hyperglycemia. According to the results of the toxicity test by the present inventors, neohesperidin dihydrochalcone was found to have little toxicity when administered orally to rats at a dose of 1000 mg / kg, and does not show any side effects on the function of organs including the liver. .

For the prevention and treatment of atherosclerosis, hyperlipidemia, liver disease and diabetes, neohesperidin dihydrocalcone can be mixed with a pharmaceutically acceptable carrier as an active ingredient to prepare a composition for the prevention and treatment of these diseases. The pharmaceutical composition may further include conventionally used excipients, disintegrants, sweeteners, lubricants, flavoring agents, etc., tablets, capsules, powders, granules, suspensions, emulsifiers, syrups, liquids by conventional methods Or in dosage unit forms or as multi-dose pharmaceutical preparations, such as preparations for parenteral administration.

The composition for the prevention and treatment of atherosclerosis, hyperlipidemia, liver disease and hyperglycemia containing neohesperidin dihydrochalcone of the present invention as an active ingredient can be parenterally or orally administered as desired, and per kg of body weight per day It may be administered in portions of one to several times so as to be administered in an amount of 0.1 to 500 mg, preferably 1 to 100 mg. Dosages 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.

Neohesperidin dihydrochalcone may also be added to foods or beverages for the purpose of preventing or treating atherosclerosis, hyperlipidemia, liver disease and hyperglycemia. In this case, neohesperidine dihydrocalcon may be added in an amount of 0.01 to 20% by weight, preferably 0.1 to 5% by weight based on the raw materials in the manufacture of food or beverage. Foods to which these substances may be added for the development of dietary supplements include, for example, meat, juices, chocolates, snacks, confectionary, pizza, ramen, other noodles, gums, dairy products, including ice cream, various soups. , Tomato paste, tea, alcoholic 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 Animal Administration, ACAT Inhibitory Effect, and HMG-CoA Reductase Inhibitory Effect of Neohesperidin Dihydrochalcone

(1) animal administration

20 Sprague Dawley rats, 3 weeks old, were purchased from the Korean Experimental Animal Center in Eumseong-gun, Chungbuk, Korea, and were bred for one week in experimental animal feed, and at 4 weeks of age (weight: 90-110 g), egg masses It was divided into two groups by randomized block design. Two groups of rats each received a diet containing 1% cholesterol (control), 1% cholesterol and 0.05% neohesperidin dihydrochalcone in the normal diet (AIN-76 experimental animal diet), respectively. The composition of the diets ingested in group 2 is shown in Table 2.

Composition of Experimental Diet (%) Dietary group Control Neohesperidindihydrochalcon group Casein 20 20 D, L-methionine 0.3 0.3 Corn starch 15 15 Sucrose 49 48.95 Cellulose powder 5 5 Mineral mixtures 3.5 3.5 Vitamin mixtures One One Choline tartaric acid 0.2 0.2 Corn oil 5 5 cholesterol One One Neohesperidin Dihydrocalcon - 0.05 gun 100 100

All diets correspond to a high cholesterol diet (1%, wt / wt), neohesperidin dihydrocalcone was purchased from Sigma Chemical Company, St. Louis, Mo, USA, and AIN, the laboratory diet of experimental 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, and their dietary intake was recorded daily and weighed every 7 days. Analysis of the data in the breeding journal after animal breeding was completed showed no significant difference between the two groups in terms of dietary intake and weight gain and showed normal growth.

(2) Determination of total cholesterol, HDL-cholesterol and neutral lipid content in blood of experimental animals

After 8 weeks of breeding, blood was collected at the expense of rats in each experimental group. The collected blood was allowed to stand for 2 hours and then centrifuged at 3,000 rpm for 15 minutes to separate serum from the upper layer, which was stored in a cryogenic freezer and used for blood analysis. Blood analysis was performed using a hematology analyzer (CIBA Corning 550 Express, USA) to measure changes in total cholesterol, HDL-cholesterol, neutral lipids, etc., and then analyzed differences between the groups.

Effects of Neohesperidin Dihydrochalcone on Blood Lipid Levels in Rats Dietary group lipid content Control Neohesperidin dihydrocalcon administration group Total Cholesterol (mg / dl) 135 ± 28 115 ± 10 HDL-cholesterol (mg / dl) 18 ± 4 19 ± 3 Neutral Lipid (mg / dl) 57 ± 13 52 ± 8 (%) 13 16

As shown in Table 3, blood cholesterol was reduced by 15% in rats treated with neohesperidin dihydrochalcone. Therefore, it can be seen that neohesperidin dihydrochalcone, a citrus-derived bioflavonoid, has an effect of lowering animal cholesterol.

(3) ACAT enzyme inhibitory effect assay

(Step 1) Preparation of the microsome

Liver was harvested at the sacrifice of rats of the control group and 0.05% neohesperidin dihydrochalcone group. 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. Supernatants were transferred to 5 ml ultracentrifuge tubes (Beckman) and centrifuged at 100,000 × g for 1 hour at 4 ° C. 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 was suspended with 1 ml of solution 1. The protein concentration in the suspension was measured by the method of Lowry et al., And the protein concentration was adjusted to 4 to 8 mg / ml. This suspension was stored in a deep freezer.

(Step 2) ACAT Activity Measurement

6.67 μl of a 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, followed by 300 mg per 10 ml volume. Distilled water was added to contain the 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 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 water bath at 37 ° C. for 30 minutes.

10 μl of oleyl-CoA solution (0.3 mg / ml) prepared by mixing oleyl-CoA and radiolabeled 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 a mixed solution of isopropanol and heptane (mixture ratio 4: 1 (v / v)), 300 µl of heptane and 200 µl of 0.1 M potassium phosphate (pH 7.4), followed by vigorous mixing with vortex. After that, it was left at room temperature for 2 minutes.

200 µl of the supernatant was placed in a scintillation bottle, and 4 ml of scintillation liquid (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 units by calculating the hourly radiation dose from the measured radiation dose. The results are shown in Table 4 below.

Effects of Neohesperidin Dihydrocalcon Administration on ACAT Activity sample % Inhibition of ACAT activity Control 0 0.05% neohesperidin dihydrochalcone group 20

As shown in Table 4, neohesperidin dihydrochalcone inhibits ACAT activity by about 20%, it can be seen that inhibits the absorption of cholesterol in the body.

(4) Determination of HMG-CoA Reductase Inhibitory Activity of Neohesperidin Dihydrochalcone

In order to measure the activity of HMG-CoA reductase, in the present invention, a method published by Hulker et al. (J. Lipid Res. 14: 625-641 (1973)) was used. The principle of measurement is to measure the coenzyme-A (CoA-SH) produced when HMG-CoA is reduced to mevalonate by the action of HMG-CoA reductase, and convert it to the activity of HMG-CoA reductase by spectrophotometer. . Specific methods and results are shown below.

(Step 1) Preparation of the microsome

Liver was harvested at the expense of the rats of the control group and 0.05% neohesperidin dihydrocalkun administration group. 3 g of liver tissue was washed with 100 ml of cold saline (0.15 M NaCl) and 100 ml of cold buffer A (0.1 M triethanolamine HCl, 0.2 M EDTA and 2 mM dithiothritol), followed by 2 ml / g. Cold buffer A was added, disrupted and homogenized using a homogenizer. This homogenate was centrifuged at 15,000 × g for 15 minutes. The supernatant was transferred to a new tube and ultracentrifuged at 100,000 × g for 60 minutes to obtain a precipitate. The supernatant was discarded and the precipitate was washed with 5 ml of cold buffer A and then ultracentrifuged at 100,000 × g for 60 minutes to obtain a precipitate. The precipitate was washed with 1 ml cold buffer A and placed in a 1.5 ml tube and stored at -70 ° C.

(Step 2) HMG-CoA reductase inhibitory activity measurement

The reactive mass used to measure HMG-CoA reductase activity is as follows: ① buffer B: 0.1M triethanolamine, HCl / 0.02M EDTA (pH 7.4), ② HMG-CoA solution: 150 μmoles / culture mixture, and ③ NADPH solution: 2 μmoles / culture mixture.

The suspension (microsome) obtained in (step 1) was mixed with the reactor, put into a centrifuge tube and reacted at 37 ° C. for 30 minutes, and then reacted at 37 ° C. for 30 minutes, followed by 20 μl of 0.01 M sodium arsenite (sodium). arsenite) solution was treated and left for 1 minute. 100 μl of citric acid buffer (2M citrate, 3% sodium tungstate, pH 3.5) was treated and reacted at 37 ° C. for 10 minutes and centrifuged at 25,000 × g for 15 minutes to remove proteins. 1 ml of the supernatant was taken into a stoppered tube, and 0.2 ml of 2M tris-hydrochloric acid solution (pH 10.6) and 0.1 ml of 2M tris-hydrochloric acid solution (pH 8.0) were added to adjust the pH of the reaction to 8.0. 20 μl of DTNB buffer solution (3mM DTNB, 0.1M triethanolamine and 0.2M EDTA, pH 7.4) was added and mixed well, and then the absorbance was measured at a wavelength of 412 nm with a spectrophotometer. -CoA reductase activity) was calculated. Based on this, the inhibition of HMG-CoA reductase activity of the control group and neohesperidin dihydrocalkun administration group was measured.

The results are shown in Table 5.

Effect of Neohesperidin Dihydrochalcone on HMG-CoA Reductase Activity Expression Lee HMG-CoA Reductase Inhibitory Activity (%) Control 0 Neohesperidin dihydrocalcon administration group 30

Example 2 Effect of Neohesperidin Dihydrocalcon on Blood Lipid Metabolism

Neohesperidin dihydrochalcone was administered orally to men in their mid-fifties with capsules for 60 days in an amount of 10 mg / kg per day, and then blood cholesterol and triglycerides were measured. As a result, blood cholesterol and triglycerides decreased by 20% and 15%, respectively. Thus neohesperidin dihydrocalcon has a therapeutic effect on hyperlipidemia.

Example 3 Animal Administration of Neohesperidin Dihydrochalcone and Its Inhibitory Effect on Intra-arterial Accumulation of Macrophage-lipid Layer and Hepatic Function Protection Effect

(1) animal administration

Experimental animals were tested on 30 healthy male rabbits from New Zealand White, weighing 2.5-2.6 kg (prepared by Yeonam Horticulture and Livestock University), temperature 20 ± 2 ℃, humidity 55 ± 5%, and illumination. Animals were bred in 12L / 12D animal cages and divided into three groups. Three groups of rabbits each had three different high-cholesterol diets, namely normal diets (solid diet RC 4 diet for rabbits; 7.6%, crude protein 22.8%, crude fat 2.8%, crude meal 8,8%, crude fiber 14.4%, Soluble nitrogen free 42.7%, cholesterol 1%) to 1% cholesterol (control); 1% cholesterol and 0.05% neohesperidine dihydrochalcone; And a diet containing 1% cholesterol and the control drug lovastatin (1 mg / kg), respectively, was taken for 6 weeks as shown in Table 6 below.

Experimental group using high cholesterol diet Experimental group Laboratory animals Marisu Duration of administration Dosing method One Rabbit male 6 heads 6 Weeks 1% Cholesterol (Control) Diet 2 Rabbit male 6 heads 6 Weeks 1% cholesterol + lovastatin (1 mg / kg) (control) 3 Rabbit male 6 heads 6 Weeks 1% Cholesterol + 0.05% Neohesperidin Dihydrochalcone

All diets correspond to the high cholesterol diet (1%, wt / wt), neohesperidin dihydrochalcone and lovastatin were purchased from Sigma Chemical company (St. Louis, Mo) of the United States, the rabbit dietary formula Solid feed RC 4 diet was purchased from Oriental Yeast Co., Japan.

(2) blood chemical analysis

The anesthetized by intramuscular injection of ketamine (ketamine, 50 mg / kg) in the thighs of the three dietary group rabbits bred for 6 weeks in (1), the rabbits of each dietary group were sacrificed and blood was collected from the heart. The collected blood was allowed to stand for 2 hours and then centrifuged at 3,000 rpm for 15 minutes to separate serum from the upper layer and stored in a cryogenic freezer before being used for blood analysis. Blood analysis was performed using a hematology analyzer (CIBA Corning 550 Express, USA) to measure changes in GOT, GPT, γGT, total cholesterol, HDL-cholesterol, neutral lipids, and creatin kinase, and then between diets. The difference was analyzed.

(3) analysis of aortic fatty gland

After the experiment, cut the chest of each group of rabbits and cut about 5cm downward along the aortic arch. Then, remove the fat from the perivascular vessel and cut the central part of the aorta along the longitudinal axis. A picture was taken. Fat gland staining was performed by using a method of Esper et al. (Esper E. et al., J Lab Clin Med, 121, 103-110 (1993)) and washing the dissected aorta with anhydrous propylene glycol three times for 2 minutes each, and propylene glycol It was transferred to saturated red oil (ORO) solution and stained for 30 minutes. The stained aorta is washed twice with 85% propylene glycol twice for 3 minutes each to remove excess stain, washed with physiological saline, photographed, and projected to the entire aorta with an image analyzer (LEICA, Q-600, Germany). The difference in the degree of atherosclerotic plaque formation between the groups of animals was analyzed by calculating the ratio (%) of the staining site (fatty gland area) to the area.

(4) Histological observation of each organ

At the end of the experiment, the rabbit was incised at the time of necropsy and the aorta was cut and used for analysis of the fatty gland in (3). After confirmation, half of each organ was rapidly frozen and the other half was placed in 10% neutral formalin and fixed for at least 24 hours. Wash the fixed tissues with running water sufficiently, gradually dehydrate with 70%, 80%, 90% and 100% ethanol, pass through paraffin, and embed them using a embedding device (SHANDON, Histocentre 2, USA). Tissue sections were prepared with a knitting machine (LEICA, RM2045, Germany) to a thickness of about 4 μm, subjected to H & E staining, clarified with xylene, sealed with permount, and observed under an optical microscope (100 × magnification).

(5) measuring the shape of liver tissue

To determine the effect of high cholesterol diet and the administration of each test substance on liver tissue, Fogg F. and Nanji AA.Toxicology and Applied Pharmacology, 136, 87-93 (1996) Hepatocellular specimens of each experimental group were examined under a microscope with reference to (Keegan A. et al., Journal of Hepatology, 23, 591-600 (1995)), and abnormal fat-containing cells in hepatic lobe based on the central hepatic vein. According to the distribution ratio of, it was classified into 1+ (0-25%), 2+ (26-50%), 3+ (51-75%) and 4+ (75-100%), and the scores were analyzed. .

The blood analysis values obtained in the above (2) to (5), the aortic adipose lines and the measurement of the shape of abnormal fat-containing cells of liver tissue, etc., were analyzed and tested for the difference of significance between each experimental group using a computer statistical program (Microsoft excel, version 7.0). (student t-test).

The results are shown in Table 7 below.

Inhibition of hyperlipidemia, arteriosclerosis, fatty liver formation and liver function improvement by neohesperidin dihydrochalcone administration Analysis result Total Cholesterol (mg / dl) Neutral Lipid (mg / ㎗) GOT (IU / ℓ) GPT (IU / ℓ) γGTP (IU / ℓ) Area of fat line measurement in artery (%) Abnormal Fatty Liver Cell Ratio Control 1143 ± 260 56 ± 38 77 ± 8 65 ± 9 4.2 ± 0.9 35 ± 14 3.2 ± 0.3 Lovastatin group 1210 ± 263 66 ± 17 125 ± 19 73 ± 9 12.4 ± 0.8 5 ± 4 3.4 ± 0.5 Neohesperidin dihydrocalcon administration group 1293 ± 89 79 ± 43 53 ± 23 42 ± 20 7.8 ± 5 12 ± 7 2.7 ± 0.3

As shown in Table 7, the neohesperidine dihydrochalcone treated group has almost no atherosclerotic plaque formed in the arterial endothelium compared to the control group, and the neohesperidine dihydrokalcon has almost the same efficacy as the lovastatin. Therefore, the neohesperidin dihydrocalcon of the present invention can be used as a blood lipid lowering agent (hyperlipidemia prevention and treatment) and atherosclerosis prevention and treatment.

In addition, the neohesperidin dihydrocalcon-administered group of the present invention has a GOT, a factor of liver function, about 31% lower than that of the control group, and GPT also drops about 35%. In the case of fatty liver cells of the liver, fatty liver cells are severely formed in the liver tissue in the lovastatin-administered group and the control group, but fatty liver formation is greatly suppressed in the neohesperidin dehydrokalcon-administered group of the present invention.

Figures 1a, 1b and 1c is an arterial endothelial picture of the control group, lovastatin administration group and neohesperidin dihydrocalcon administration group rabbits, respectively. As shown in Figure 1a, the control group is deposited atherosclerotic plaque due to the formation of a large number of macrophage-lipid complexes on the surface of the arterial endothelium, as shown in Figures 1b and 1c, the lovastatin and neohesperidin dehydrokalcon administration group A thin layer of atherosclerotic plaque is deposited on the surface. Therefore, neohesperidin dihydrochalcone of the present invention can be seen that the effect of preventing atherosclerosis is strong even under very high blood cholesterol.

Figures 2a, 2b and 2c is a picture of the liver anatomy of each control group, lovastatin administration group and neohesperidin dihydrocalcon administration group rabbits. As shown in Figures 2a and 2b, the control rabbit's liver contains cells containing excessive fat around the central vein, and in the lovastatin-administered rabbit liver, the central vein and almost all cells around it form fatty liver cells. As shown in 2c, most liver cells were normal in the liver of the neohesperidin dihydrochalcone-treated rabbit and only some liver cells formed fatty liver cells. Therefore, it can be seen that the neohesperidin dihydrochalcone of the present invention strongly inhibits fatty liver formation.

Example 4 Hypoglycemic Effect of Neohesperidin Dihydrochalcone

(1) animal experiment

Experimental animals were used to raise 20 rats of 3 weeks-old Sprague-Dawley male rats in a laboratory diet (Lab. Chow pellet), and when they weighed 280 g, they were specific to pancreatic β-cells and had a significant effect on other organs. Diabetes was induced using streptozotocin (Junod A, et al., Diabetogenic action of streptozotocin: Relationship of dose to metabolic response. J Clin Invest, 48, 2129-2139 (1969)), which was reported to be absent. In other words, streptozotocin (Sigma chemical Co.) was dissolved in citric acid buffer solution at pH 4.5 so that the maximum injection amount was 1 ml or less and injected into the muscle at the amount of 45 mg / kg body weight. After 24 hours after injection, blood was measured by taking tail blood, and it was 350-400 mg / dl, indicating that diabetes was induced.

After fasting for 6 hours on day 1 after the induction of diabetes, the blood was collected from the tail vein to confirm the hyperglycemia, and then divided into two groups by the randomized block design. Two groups of rats each received two different diets, a diet containing the basic diet (AIN-76 semi-tablet diet) (control) and the diet containing 0.05% neohesperidin dihydrochalcone for 5 weeks.

Rats of all diets were kept for 5 weeks on the diet in an animal laboratory at constant temperature (25 ± 2 ° C), humidity (50 ± 5%) and natural lighting. All diets were consumed freely and water was supplied daily to maintain cleanliness to avoid bacterial growth.

The animals were fasted for 12 hours the next morning after animal breeding was completed, the animals were anesthetized with ether, then intraperitoneally administered and blood collected from the inferior vena cava. Blood was centrifuged at 3000 rpm for 15 minutes at 4 ° C to separate serum and stored at -20 ° C. In addition, the liver, heart and kidneys of the rats were extracted and washed several times with physiological saline to remove the surface blood and weighed and stored at -20 ℃.

Blood glucose was measured using a blood glucose meter (Glucocard II GT-1620, Kyto Daiichi Kagaku Co., LTP, model 5616239), and the results are shown in Table 8 below.

Effects of Neohesperidin Dihydrochalcone on Blood Glucose Levels in Rats Diet Glucose (mg / dl) % Glucose reduction Control 730 ± 65 0 0.05% neohesperidin dihydrochalcone administration group 550 ± 40 25

As shown in Table 8, 0.05% neohesperidin dihydrocalcon lowers blood sugar in rats by about 25%.

(2) human body experiment

Two patients with hyperlipidemia (males in their 50s) with blood cholesterol of 230 to 270 mg / dl and neutral lipids of 200 to 220 mg / dl, respectively, were treated with neohesperidin dihydrochalcone in capsules of 6 to 10 mg / kg per day. The dose was administered for 3 months, and after 2 months, the blood sugar dropped 30%.

Example 5: Preparation of Hard Capsule

Hard gelatin capsules were prepared by conventional methods by mixing the following ingredients:

Amount (mg / capsule)

Neohesperidin Dihydrocalcon 200

Vitamin C 50

Lactose 150

-----------------------------------------

400 total

The ingredient powder was mixed well and filled into hard gelatin capsules to a total amount of 400 mg. By taking 1 to 10 capsules per day, arteriosclerosis, hyperlipidemia, liver disease and hyperglycemia can be prevented or treated.

In addition, the compositions may further include other ingredients beneficial to health besides vitamin C.

Example 6: Preparation of food using neohesperidin dihydrochalcone

0.01-10% (w / w) neohesperidine dihydrochalcone was added at the time of ice-cream preparation.

Example 7: Preparation of orange juice using neohesperidin dihydrochalcone

0.01-0.5% (w / w) neohesperidine dihydrochalcone was added at the time of orange juice preparation.

Neohesperidin dihydrocalcon inhibits the formation of macrophage-lipid complexes on the arterial endothelial surface in mammals, inhibits the synthesis of HMG-CoA reductase, a cholesterol synthase, inhibits cholesterol synthesis, and inhibits ACAT activity to inhibit cholesterol Inhibition of absorption, lowering serum GOT and GPT, inhibiting fatty liver formation, and lowering blood sugar, the composition of the present invention comprising neohesperidin dihydrochalcone, arteriosclerosis, hyperlipidemia, liver disease, diabetes It can be usefully used as a medicine and food for the prophylaxis and treatment of.

Claims (5)

A pharmaceutical composition for preventing and treating atherosclerosis, comprising an effective amount of neohesperidine dihydrochalcone as an active ingredient together with a pharmaceutically acceptable carrier. The method of claim 1, The effective amount of the neohesperidin dihydrocalcon is 0.1 to 500 mg / kg body weight / day composition. A pharmaceutical composition for preventing and treating hyperlipidemia, comprising an effective amount of neohesperidine dihydrochalcone as an active ingredient together with a pharmaceutically acceptable carrier. A pharmaceutical composition for preventing and treating liver disease, comprising an effective amount of neohesperidine dihydrochalcone as an active ingredient together with a pharmaceutically acceptable carrier. A pharmaceutical composition for preventing and treating hyperglycemia, comprising an effective amount of neohesperidine dihydrochalcone as an active ingredient, together with a pharmaceutically acceptable carrier.