KR20010015495A - Rutin and quercetin-containing composition for preventing and treating hyperlipidemia, artherosclerosis and hepatic disease - Google Patents

Abstract

PURPOSE: A pharmaceutical composition containing one of rutin and Quercetin or the mixture is provided to prevent and treat hyperlipidemia, arteriosclerosis and liver diseases. CONSTITUTION: The rutin is extracted from buckwheat or leaves, stems and flowers of the buckwheat by leaving the materials at a base of pH10-12 by using calcium hydroxide, neutralizing and settling the materials, or by using 20-97% alcohol or water at a high temperature and pressure. The buckwheat and the leaves, stems and flowers also can be dried and pulverized to get powdered buckwheat. The resulting buckwheat flour or buckwheat extract containing 0.01-10wt% of the rutin, quercetin or the mixture of them is added to tomato ketchup, sauce and juice, ramen, noodle, bread, cookies and cracker, soup and gravies, ground beef, or dairy products to get functional foods.

Description

RUTIN AND QUERCETIN-CONTAINING COMPOSITION FOR PREVENTING AND TREATING HYPERLIPIDEMIA, ARTHEROSCLEROSIS AND HEPATIC DISEASE} including hyperlipidemia, arteriosclerosis, and liver disease, including rutin and quercetin

The present invention relates to a pharmaceutical composition and a food composition for the prevention and treatment of hyperlipidemia, atherosclerosis, liver disease, containing rutin and quercetin, which is a kind of bioflavonoid which is widely present in various vegetables, fruits and agricultural products. .

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)). Thus, by developing an active substance that reduces blood cholesterol or inhibits macrophages from depositing on the arterial endothelium, arteriosclerosis can be prevented and treated.

Reducing the amount of cholesterol in the blood is a way to suppress the absorption of cholesterol. Acyl CoA: Cholesterol-o-acyltransferase (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. 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. It can be effectively used in therapy (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, excessive intake of foods or alcohol containing a lot of fat components, or infected with hepatitis B or C virus, liver function is reduced, if left untreated, hepatitis, cirrhosis, liver cancer and so on. In particular, excessive fat or alcohol intake from food causes fatty liver, which accumulates lipids in liver tissue, and increases GOT (glutamate-oxaloacetate transaminase), GPT (glutamate-pyruvate transaminase), and γGTP (γ-glutamyl transpeptidase). (T. Banciu, et al., "The Hepatic Component in Alcoholic Encephalopathy", Med. Interne., 20, 69-71 (1982); and A. Par, et al., "Serum Gamma-Glutamyl Transpeptidase: Its Clinical Significance ", Acta. Med. Acad. Sci. Hung., 33, 309-319 (1976).

Hayashi et al. Concluded that the administration of green tea extract to rats prevented the increase of serum GOT and GPT, which could be seen as an effect of green tea on liver function (M. Hayashi, et al. , Nippon Yakurigaku Zasshi, 100, 391-399 (1992)).

Flavonoid compounds, on the other hand, are widely present in nature as polyphenolic antioxidants, and especially in beverages such as vegetables, fruits and wine. In the West, people consume an average of about 25 mg of flavonoids per day, and rutin and quercetin are one of the typical vegetable vegetable flavonoids. High intakes have been reported to reduce the risk of death from heart disease in older adults (M. G. L. Hertog et. Al, Dietary antioxidant flavonoids and risk of coronary heart disease, Lancet. 342: 1007-1011, 1993).

Flavonoids have been reported to have several useful pharmacological activities. For example, citrus-derived bioflavonoids have been reported to have anti-inflammatory, capillary potentiating, anticancer, antiviral and platelet aggregation (O. Benavente-Garcia et. Al. Uses and properties of citrus flavonoids. Agri.Food Chem., 45: 4506-4515, 1997).

Rutin is a glycosided quercetin, which is broken down by microorganisms in the intestine and is broken down into non-glycosided quercetin and absorbed into our body (C. Manach et. Al. Bioavailability, metabolism, and physiological impact of 4-oxo-flavonoids Nutrition Research 16: 517-544, 1996). In the Netherlands, people are known to consume about 25 to 160 mg of flavonoids per day, and they consume large amounts of quercetin, as well as luteolin, kaempferol, apigenin and myricetin ( myricetin).

There are more than 100 bioflavonoids reported so far, but a few of the citrus fruits found are listed in Table 1 below.

Bioflavonoids are known to have a variety of pharmacological activity, for example, lipid metabolism improving effects associated with cardiovascular disease, anticancer and antiviral effects. For example, hesperidin or hesperetine is known to have capillary fortification, decreased vascular permeability, lowered blood pressure, lowered blood cholesterol, prevented platelet 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); EP 0352147 A2 (1990.1.24); 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. , EP 0352147 A2 (1990.1.24); and Martin MJ, et al., Pharmacol., 49, 144-150 (1994).

Rutin is known to be effective in treating high protein edema (see US Pat. No. 5,096,887), and quercetin has been reported to have anticancer and antiviral effects (JP-A-234320). Reference).

While the present inventors studied the pharmacological activity of bioflavonoids, which are abundant in herbal medicines, food ingredients, and vegetables, which are considered to be relatively safe, rutin and quercetin significantly reduce blood cholesterol and inhibit HMG CoA reductase and ACAT. The present invention has been completed by finding that the phagocytic-lipid complex strongly inhibits the deposition or attachment to the arterial endothelial surface and has an effect on protecting the liver function of the animal when administered to the animal.

Accordingly, it is an object of the present invention to provide a pharmaceutical composition for the prevention and treatment of atherosclerosis, hyperlipidemia and liver disease comprising rutin, quercetin or mixtures thereof.

Another object of the present invention is to provide a food and beverage composition for the prevention and treatment of atherosclerosis, hyperlipidemia and liver disease, including rutin, quercetin or mixtures thereof.

Figure 1a, 1b and 1c is an arterial endothelial picture of the control group, lovastatin administration group, routine administration group, respectively,

Figures 2a, 2b and 2c is a photograph showing the fatty liver formation state and liver tissue state of the control group, lovastatin administration group, routine administration group, respectively.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention and treatment of atherosclerosis, hyperlipidemia and liver disease comprising an effective amount of rutin, quercetin or a mixture thereof and a pharmaceutically acceptable carrier.

In order to achieve the above object, the present invention provides a food and beverage composition for the prevention and treatment of atherosclerosis, hyperlipidemia and liver disease with an effective amount of rutin, quercetin or a mixture thereof.

Rutin (C ₂₇ H ₃₀ O ₁₆ , MW610.51) and Quercetin (C ₁₅ H ₁₀ O ₇ , MW302.23) are particularly present in various vegetables such as lettuce, onions, fruits such as citrus fruits, and grains such as buckwheat. However, it can also be used as a powder obtained by extracting from these or drying and pulverizing their flowers, buds, stems and the like. Furthermore, it can synthesize | combine also by a well-known synthesis | combining method (refer to Merck Index 11th edition (1989)).

For example, buckwheat fruit, leaves, stems, flowers, sprouts and the like contain rutin in an amount of 0.1 to 10% by weight. In particular, leaves and stems contain about 0.6% rutin and about 3% in flowers. Therefore, these may be extracted and concentrated in a conventional manner, or may be dried and powdered, and then used in pharmaceutical raw materials and health food raw materials.

Rutin and quercetin have prophylactic effects as well as therapeutic effects of atherosclerosis, hyperlipidemia and liver disease. In addition, the results of the toxicity studies studied so far revealed that these substances were almost non-toxic when administered orally to mice at doses of 1,000 mg / kg and 160 mg / kg, respectively. In addition, these substances show no adverse effects on the function of the organs, including the liver.

For the prevention and treatment of atherosclerosis, liver disease and diabetes, rutin, quercetin or a mixture thereof may 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, emulsions, syrups, etc. by conventional methods It may be formulated in unit dosage forms or in multiple dosage forms of pharmaceutical preparations, such as solutions for parenteral or parenteral administration.

The pharmaceutical composition of the present invention contains rutin, quercetin or a mixture thereof in an amount of 0.01 to 100% by weight, preferably 0.1 to 70% by weight, and may be parenterally or orally administered as desired, and It can be administered once to several times, preferably once to three times in the range of 0.1 to 500 mg / kg body weight, preferably in the range of 0.5 to 100 mg / kg body weight. 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.

The ingredients may also be added to foods or beverages for the purpose of preventing or treating atherosclerosis, hyperlipidemia and liver disease. Foods to which these substances can be added for the development of dietary supplements include, for example, meat, juices, chocolates, snacks, sweets, pizzas, ramen, other noodles, gums, dairy products, including ice cream, various soups. , Tomato paste, tea, alcoholic beverages, vitamin complexes, and the like. The food is mixed with rutin, quercetin, mixtures or extracts thereof in an amount of 0.01 to 10% by weight.

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, but the scope of the present invention is not limited to these.

Example 1 Acute Toxicity Test of Rutin

The purpose of this study was to determine the degree of acute toxicity in oral administration of rutin, to provide information on available drug dose in general pharmacology and efficacy tests, and to derive basic data on toxicity.

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 It was bred in the / 12D animal room. Mice were allowed to acclimate for about a week before being used in the experiment. Feed for experimental animals (JeilJedang Co., Ltd., mice and rats) and beverages were sterilized and supplied freely.

Rutin purchased from Aldrich-Sigma Co., St. Louis, Missouri, USA was dissolved in a 0.5% Tween 80 solution at a concentration of 100 mg / ml, then orally in an amount of 0.2 ml per 20 g of mouse weight. Administered. Samples were administered orally once and observed for side effects or lethality for 10 days after administration. That is, on the day of dosing, the change of general symptoms and the presence or absence of dead animals were observed at least once in the morning, at least once every afternoon from 1 hour, 4 hours, 8 hours, 12 hours after the administration and from the next day until the 10th. In addition, the animals were killed and dissected at 10 days of administration, and the internal organs were visually examined. The weight loss of each animal was measured by measuring the change in body weight every day from the day of administration.

For acute oral toxicity, no lethal animals were observed for 10 days at a routine dose of 1,000 mg / kg. 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, no routine toxicity was observed upon oral administration at these concentrations.

Example 2 Effect on Blood Total Cholesterol, HDL-Cholesterol and Neutral Lipid Content

Step 1: Animal Dose Experiment of Rutin and Quercetin

30 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 old (weight: 90-110 g), egg masses It was divided into three groups by randomized block design. Three groups of rats each had three different high-cholesterol diets: a diet containing 1% cholesterol (control) and 1% cholesterol and 0.05% rutin or quercetin in the normal diet (AIN-76 experimental animal diet). Each diet was ingested. The composition of the diets ingested in the three groups is shown in Table 2.

Dietary group Control group (n = 10) Routine (n = 10) Quercetin (n = 10) Casein 20 20 20 D.L.-Methionine 0.3 0.3 0.3 Corn starch 15 15 15 Sucrose 49 48.95 48.95 Cellulose powder 5 5 5 Mineral mixtures 3.5 3.5 3.5 Vitamin mixtures One One One 2-week choline 0.2 0.2 0.2 Corn oil 5 5 5 Cholesterol One One One Routine - 0.05 - Quercetin - - 0.05 gun 100 100 100

All diets correspond to a high cholesterol diet (1%, wt / wt), routines and quercetin were purchased from Sigma Chemical Company, St. Louis, Mo, USA, and the laboratory diet of the rat AIN-76 Cellulose, mineral mixtures and vitamin mixture components in the 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 three groups in terms of dietary intake and weight gain and showed normal growth.

Stage 2: Determination of Total Cholesterol, HDL-Cholesterol and Neutral Lipid Content in Blood

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

After breeding for 8 weeks according to the method of step 1, the rats of each experimental group were sacrificed and blood was collected. 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 an cryogenic freezer, which was then used for blood analysis. Blood analysis was performed using a blood chemistry analyzer (CIBA Corning 550 Express, USA) to measure changes in total cholesterol (TC), HDL-cholesterol (HDL), triglyceride (TG), and the results are shown in the following table. Same as 3.

Dietary group lipid content Control Routine administration group Quercetin-administered group Total Cholesterol (mg / dl) 135 ± 28 100 ± 10 95 ± 9 HDL cholesterol (mg / dl) 18 ± 4 20 ± 3 21 ± 3 Triglycerides (mg / dl) 57 ± 13 50 ± 10 45 ± 7 HDL cholesterol / total cholesterol (%) 13 20 22

In Table 3, the total cholesterol in blood was reduced by 25% and 30% in the rats treated with Rutin and Quercetin, respectively. Meanwhile, triglycerides decreased by 14% and 21%, respectively.

Example 3: ACAT enzyme activity inhibitory effect assay

Step 1: Preparation of Microsomes

1 g of liver tissue extracted from the sacrificed mice was homogenized using 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 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: Measuring ACAT Activity

6.67 μl of a solution of cholesterol 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 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) and vigorously mixed 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. The ACAT enzyme activity was calculated from the measured radiation dose, and the degree of inhibition of ACAT activity was calculated using this. The results are shown in Table 4.

sample Inhibition of ACAT activity (%) Control 0 Routine 0.1% administration group 25 Quercetin 0.1% group 20

From Table 4, it can be seen that rutin and quercetin inhibit ACAT activity by 20% to 25% and inhibit cholesterol absorption in the body.

Example 4: Determination of HMG-CoA reductase inhibitory activity.

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 by spectrophotometer. It is equivalent. Specific methods and results are shown below.

Step 1: Preparation of Microsomes

After taking 3 g of liver tissue from the sacrificed mice, 100 ml of cold saline (0.15 M NaCl) and 100 ml of cooling buffer A (0.1 M triethanolamine, HCl / 0.2 M EDTA / 2 mM dithiothreitol) The liver tissue was washed with). 2 g of cooling buffer A per 1 g of liver tissue was broken down and completely homogenized with a homogenizer, and then the supernatant was collected by centrifugation (15,000 x g, 15 minutes) of the ground liver tissue. The collected supernatant was ultracentrifuged (100,000 x g, 60 minutes) and the supernatant was removed to obtain microsomal precipitate. The obtained microsomal precipitate was washed with 5 ml of cooling buffer A and again ultracentrifuged (100,000 × g, 60 minutes) to obtain a precipitate. The precipitate was washed with 1 ml of cold buffer A and placed in a 1.5 ml tube and stored at -70 ° C.

Step 2: measuring HMG-CoA reductase activity

Reactors used to measure HMG-CoA reductase activity are as follows; ① buffer B: 0.1 M triethanolamine, HCl / 0.02 M 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, placed in a centrifuge tube and reacted at 37 ° C. for 30 minutes, and then treated with 20 μl of 0.01 M sodium arsenite solution for 1 minute, followed by 100 μl of Protein was removed by reaction with citrate buffer (2M citrate / 3% sodium tungsten, pH 3.5) at 37 ° C. for 10 minutes and high speed centrifugation (25,000 × g, 15 minutes). 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. The reaction was then well mixed with 20 μl of DTNB buffer (3 mM DTNB / 0.1M triethanolamine / 0.2M EDTA, pH 7.4) and the absorbance at 412 nm wavelength was measured with a spectrophotometer to determine the amount of CoA-SH formed (HMG -CoA reductase activity) was calculated. Based on this, inhibition of HMG-CoA reductase activity of the control and administration groups was measured. The results are shown in Table 5 below.

Expression Lee HMG CoA Reductase Inhibition Rate (%) Control 0 Routine administration group 40 Quercetin-administered group 30

Example 5 Effect of Routine Human Administration on Blood Lipid Metabolism

Rutin was orally administered to humans (mid 50s, 2) in capsule form at a rate of 10 mg / kg per day. After 60 days, blood cholesterol and triglyceride (triglyceride) were measured and decreased by 25% and 20%, respectively. Thus, it can be seen that rutin has a therapeutic effect on human hyperlipidemia.

Example 6: Atherosclerosis Inhibitory Effect

Step 1: Animal Experiment

The experimental animals were healthy male rabbits of New Zealand White, 3 months old, weighing 2.5-2.6 Kg, and were used for the experiment. Animals were used in the experiments under conditions controlled by a temperature of 22 ± 2 ° C., a humidity of 55 ± 10% and a 12 hour light / dark cycle. The diet is a rabbit solid food (RC4 diet) consisting of water (7.6%), crude protein (22.8%), crude fat (2.8%), crude ash (8.8%), crude fiber (14.4%) and soluble nitrogen free (43.7%). , Oriental Yeast Co., Japan) were divided into four groups, including one control group that consumed only feed with cholesterol 1% and three test groups that mixed and administered each test substance at a certain ratio. Water was allowed to be taken freely. Detailed conditions are as shown in Table 6. Control drug lovastatin and test substance rutin and quercetin were purchased from Sigma Co. (St. Louis, MO.)

Experimental group Laboratory animals Marisu Duration of administration Dosing method 1234 Rabbit (♂) "" " 6 heads "" " 6 weeks "" " 1% Cholesterol (Control) Diet 1% Cholesterol + lovastatin (1 mg / kg) (Control) 1% Cholesterol + 0.1% Rutin Diet 1% Cholesterol + 0.1% Quercetin Diet

Step 2: Blood Chemistry Analysis

After breeding for 6 weeks, ketamine (50 mg / kg) was anesthetized by intramuscular injection in the thigh, and blood was collected from the heart at the expense of rabbits 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 blood chemistry analyzer (CIBA Corning 550 Express, USA) to measure changes in GOT, GPT, γ-GT, total cholesterol, triglycerides, etc. and then analyzed the differences between the experimental groups. The results are shown in Table 7.

Step 3: Intra-aortic Fat Gland Analysis

After the end of Step 2, the rabbits were incised to cut the chest and cut about 5 cm downward along the aorta. Then, the fats around the blood vessels were removed well and the central part of the aorta was cut along the longitudinal axis. Pinned and photographed. Fat gland staining was performed using Esper et al. (Esper E, et. Al., J. Lab. Clin. Med., 121: 103-110, 1993). The resultant was washed with water and transferred to an oil red saturated solution dissolved in propylene glycol, followed by dyeing for 30 minutes. Wash the stained aorta with 85% propylene glycol twice for 3 minutes each time to remove excess dye solution, wash with physiological saline, take a picture, and then tracing the image analyzer (LEICA, Q-600, Germany) By calculating the ratio (%) of the staining area (fatty gland area) to the total aortic area, the difference in the degree of atherosclerotic plaque formation between the animal groups was analyzed. The results are shown in Table 7.

1A, 1B and 1C are photographs showing arterial endothelial status of a control group, a lovastatin-administered group, and a routine-administered group, respectively. Referring to Figure 1a (control), it can be seen that a large number of macrophage-lipid layers accumulate on the endothelial surface of the arteries to form a thick layer of atherosclerotic plaques. In FIG. 1B (lovavadin-administered group) and FIG. 1C (routine-administered group), lipid layers are hardly observed in the arterial endothelium. Thus, it can be seen that the rutin-containing composition of the present invention effectively prevents macrophage-lipid layer deposition.

Stage 4: Histological Observation of Each Organ

In stage 3, the rabbit was incised at the time of necropsy and the aorta was cut and used for analysis of the fatty gland. Immediately, a portion of the aorta and heart, lung, liver, kidney, and muscle were excised to visually check for specific findings. 1/2 of was rapidly frozen and 1/2 was placed in 10% neutral formalin and fixed for at least 24 hours. After fixation, each tissue was rinsed sufficiently with running water, gradually dehydrated with 70%, 80%, 90%, and 100% alcohol, and then paraffin-permeated and embedded with a embedding device (SHANDON, Histocentre 2, USA). Tissue sections were prepared with a thickness of about 4 μm with a knitting machine (LEICA, RM2045, Germany), stained with H & E (Hematoxylin & eosin), invisible with xylene, and then encapsulated with permount to optical microscopy. Observed. As a result, there were no gross lesions in the tissues of each organ.

Example 7: Fatty liver formation inhibitory effect

Step 1: Measure the Morphology of Liver Tissue

Other investigators' methods (Fogt F. and Nanji A., Toxicology and Applied Pharmacology, 136: 87-93, 1996; and Keegan A. to determine the effect of high cholesterol diet and the administration of each test substance on liver tissue). , et al., Journal of Hepatology 23: 591-600, 1995), containing microscopic observations of hepatic tissue samples of each experimental group treated in step 1 of Example 6, based on the central vein, based on the central vein. Depending on the degree of distribution of cells, they are classified into 1+ (0-25%), 2+ (26-50%), 3+ (51-75%), and 4+ (75-100%), and the scores are analyzed. It was. The results are shown in Table 7. Figures 2a, 2b and 2c is a photograph showing the fatty liver formation state and liver tissue state of the control group, lovastatin administration group, routine administration group, respectively. In Figures 2a and 2b a number of cells with excess fat are observed in the periphery of the central vein. On the other hand, Figure 2c shows that almost all liver cells show normal morphology, and thus, rutin strongly inhibits fatty liver formation.

As described above, the results of blood analysis for demonstrating the improvement of lipid metabolism, liver function, and atherosclerosis suppression effect of rabbits by rutin and quercetin administration, and the results of plaque fat line area accumulated on arterial endothelial surface, fatty liver inhibitory effect, etc. It is shown in Table 7 below, using a statistical program (Microsoft excel, version 7.0) was analyzed and tested (student t-test) the difference of significance between each experimental group.

Analysis result Total Cholesterol (mg / dl) Trigly-seride (mg / dl) GOT (IU / ℓ) GPT (IU / ℓ) γGTP (IU / ℓ) Area of fat line measurement in artery (%) Abnormal Fatty Liver Cell Ratio Control (1% Cholesterol Diet) 1143.2 ± 260.6 55.8 ± 38.2 76.8 ± 8.2 65.2 ± 9 4.2 ± 1 35 ± 14 3.2+ (± 0.3) Pharmaceutical control group (1mg / kg lovastatin administration) 1209.5 ± 263.3 65.6 ± 16.9 125.6 ± 18 72.5 ± 9.3 12.3 ± 0.8 5 ± 4 3.4+ (± 0.5) Routine 0.1% administration group 789.2 ± 430.6 35.3 ± 18.4 87.0 ± 19.7 33.9 ± 10 1.92 ± 1.3 10 ± 5 2.75+ (± 0.3) Quercetin 0.1% dose 820.0 ± 270 30.0 ± 12 80 ± 15 30 ± 8.1 2.5 ± 1 8 ± 3 2.6+ (± 0.4)

In Table 7, the rutin and quercetin showed a very good result of 30% in total cholesterol lowering effect and 44 to 45% in neutral fat lowering effect, respectively, compared to the control group. In particular, it shows better total cholesterol and triglyceride lowering effects than lovastatin.

Example 8 Human Administration of Routine

Two men in their mid-50's received 10 mg / kg of rutin per day for 2 months. After 2 months, cholesterol and cholesterol decreased by 25% and 20%, respectively.

Example 9 Extraction of Routines

The buckwheat was left overnight under basic conditions (pH10-12) using calcium hydroxide (CaOH ₂ ) and neutralized to pH6-7.0 to recover a precipitate obtained to obtain a crude routine having a purity of 40 to 50%. Alternatively, the mixture was extracted with 20-97% alcohol or extracted with water at high pressure and high temperature (60-121 ° C) to obtain a concentrate.

The crude extract was obtained by repeating the same procedure using leaves, stems and flowers of buckwheat. From this, it was confirmed that the leaves, stems, etc. contain about 0.6% rutin and about 3% in the flowers.

Buckwheat, leaves, stems and flowers were dried and then ground to obtain a powder form.

In addition, buckwheat berries were soaked in water, soaked in a suitable container, and sometimes watered to maintain sufficient moisture to grow the shoots to about 1-10 cm, dried and powdered or extracted.

Example 10

Two people in their mid-fifties were given 10 g of buckwheat flower powder per day for 60 days in water, which lowered cholesterol by 25% and triglyceride by 20%.

Therefore, it can be seen that even when the buckwheat flower is taken as it is, it suppresses arteriosclerosis and hyperlipidemia.

Pharmaceutical compositions and food compositions of the present invention can be prepared as follows.

Formulation Example 1 Preparation of Pharmaceutical Composition

The following ingredients were mixed to prepare hard gelatin capsules by conventional methods.

Amount (mg / capsule)

Rutin, Quercetin or a mixture thereof (active ingredient) 200 mg

Vitamin C 50mg

Lactose 150mg

400 mg total

Formulation Example 2 Preparation of Functional Food

(1) Preparation of Tomato Ketchup, Sauce and Juice

Functional ketchup, sauce and juice were prepared by adding buckwheat powder pulverized to a size of 100 to 200 μm so that the amount of rutin is 0.01 to 10% by weight to conventional tomato ketchup, sauce and juice.

Alternatively, the buckwheat extract obtained in the same manner as in Example 9 was added to the food so that the amount of rutin is 0.01 to 10% by weight to prepare a functional food.

(2) Manufacture of flour foods such as ramen, noodles, bread, cakes, cookies, and crackers

In order to manufacture the flour food, the final food was prepared after adding buckwheat powder to flour as a raw material so that the amount of rutin is 0.01 to 10% by weight.

Alternatively, the buckwheat extract obtained in the same manner as in Example 9 was added to the food so that the amount of rutin is 0.01 to 10% by weight to prepare a functional food.

(3) Preparation of soups and gravy

Buckwheat powder was added to the raw materials of soup or broth so that the amount of rutin was 0.01 to 10% by weight to prepare a final food.

Alternatively, the buckwheat extract obtained in the same manner as in Example 9 was added to the food so that the amount of rutin is 0.01 to 10% by weight to prepare a functional food.

(4) Manufacture of Ground Beef

Buckwheat powder was added to the ground beef raw material so that the amount of rutin was 0.01 to 10% by weight to prepare a final food.

Alternatively, the buckwheat extract obtained in the same manner as in Example 9 was added to the food so that the amount of rutin is 0.01 to 10% by weight to prepare a functional food.

(5) Manufacture of dairy products (milk, ice cream, cheese, yogurt, etc.)

The final food was prepared after adding the cereal or fruit vegetable powder containing rutin or quercetin to the milk so that the amount of rutin was 0.01 to 10% by weight.

Alternatively, the buckwheat extract obtained in the same manner as in Example 9 was added to the food so that the amount of rutin is 0.01 to 10% by weight to prepare a functional food.

At this time, in particular, during the manufacture of cheese, the milk protein was coagulated and recovered, and then the citrus peel component was added.

Formulation Example 3 Preparation of Supplements

The following ingredients were mixed to prepare a dietary supplement by conventional methods.

Ginseng Powder or Extract 50%

Rutin, Quercetin or any mixture thereof 30%

Other sweeteners and flavors 20%

100% total

Compositions of the present invention containing rutin and quercetin inhibit the activity of 3-hydroxy-3-methyl glutaryl CoA reductase (HMG CoA), a cholesterol biosynthetic enzyme in liver tissue of animals when administered to animals, It inhibits acyl CoA: cholesterol acyltransferase (ACAT) activity involved in the absorption, inhibits the formation of macrophage-lipid layers or atherosclerotic plaques on the inner walls of blood vessels of animals, and inhibits hepatocellular protective action and fatty liver formation. Therefore, hyperlipidemia, arteriosclerosis, it can be usefully used as a medicine and food for the prevention, treatment of liver disease.

Claims (5)

A functional food or beverage composition prepared by adding rutin, quercetin or a mixture thereof in an amount of 0.01 to 10% by weight. The method of claim 1, Wherein said routine is in the form of a powder or extract of buckwheat. The method of claim 1, The routine is in the form of a powder or extract of buckwheat flowers, leaves or stems. The method of claim 1, Wherein said food is tomato ketchup, sauce, juice, soup, gravy, ground beef, ramen, noodles, bread, cake, cookie, cracker, milk, ice cream, cheese or yogurt. The method of claim 1, The beverage is a composition characterized in that the vegetable juice, fruit juice or carbonated beverage.