RU2201228C2 - Naringin and naringenin as agents for prophylaxis or treatment of liver disease - Google Patents

Abstract

FIELD: medicine, hepatology, pharmacy. SUBSTANCE: invention proposes novel component of pharmaceutical composition, food composition or beverage used for prophylaxis or treatment of hepatic lipid dystrophy or hepatic cirrhosis in mammals, among them, humans. As such component the known flavonoids naringin or naringenin are used. EFFECT: expanded assortment of agents of designation claimed. 9 cl, 6 dwg, 6 tbl, 10 ex

Description

Field of Invention
The present invention relates to the use of naringin or naringenin for inhibiting the activity of acyl CoA-cholesterol-o-acyltransferase (ACAT), inhibiting the accumulation of macrophage-lipid complexes on arterial endothelium, and for preventing or treating liver diseases in mammals.

BACKGROUND OF THE INVENTION
In recent years, coronary cardio-circulatory diseases, such as atherosclerosis and hypercholesterolemia, are increasingly becoming the leading cause of death. It has been reported that elevated plasma cholesterol levels cause the deposition of fat, macrophages and foam cells on the walls of blood vessels and that such deposits lead to plaque formation and then to atherosclerosis (Ross, R., Nature, 362, 801-809, 1993) . One way to lower plasma cholesterol levels is through alimentotherapy to reduce cholesterol and lipid absorption. Another way is to inhibit cholesterol absorption by inhibiting the involved enzymes.

 Acyl CoA-cholesterol-o-acyltransferase (ACAT) promotes the formation of cholesterol esters in the blood. Foam cells are formed as a result of the action of ACAT and contain a large amount of cholesterol ester, which is transferred by low density lipoproteins (LDL). The formation of foam cells on the walls of the artery grows with an increase in ACAT activity, and accordingly, an ACAT inhibitor can also serve as a means for the prevention of atherosclerosis. It was further reported that blood levels of LDL cholesterol can be lowered by inhibiting ACAT activity (Witiak, D.T. and DR Feller (eds.), Anti-Lipidemic Drugs: Medicinal, Chemical and Biochemical Aspects, Elsevier, pp. 159- 195, 1991).

 On the other hand, impaired liver function can occur due to excessive consumption of alcohol or high lipid foods, or as a result of infection with hepatitis B or C viruses, and this can develop into hepatitis, lead to liver cirrhosis or liver cancer. In particular, overconsumption of foods high in fat and alcohol causes liver obesity when large amounts of lipids are deposited in the liver tissue and serum GOT (glutamate oxaloacetate transaminase), GPT (glutamate pyruvate transaminase), and γ-GTP ( γ-glutamyl transpeptidase) (T. Banciu et al., Med. International., 20, 69-71, 1982; and A. Par et al., Acta. Med. Acad. Sci. Hung., 33, 309 -319, 1976).

 Numerous attempts have been made to develop drugs that inhibit ACAT activity; and as a result, there were reports of the isolation of several compounds from cultures of various microorganisms. Examples of such compounds include pyripyropenes isolated from a culture of Aspergillus fumigatus (S. Omura et al., J. Antibiotics, 46, 1168-1169, (993) and acaterin isolated from Pseudomonas sp. (S. Nagamura et al., J. Antibiotics, 45, 1216-1221, 1992).

Further, as an agent for treating hypercholesterolemia, an HMG-CoA reductase inhibitor called lovastatin (Lovastatin ^® ) was developed, which is marketed by Merck Co., USA

 However, it is known that this medicine causes a harmful side effect - it increases the content of creatine kinase in the liver.

 Accordingly, there remains a need to create non-toxic ACAT inhibitors and inhibitors of the accumulation of macrophage-lipid complexes on the epithelium of arteries and agents for the prevention or treatment of liver diseases.

 The authors of the present invention attempted to develop a new and effective ACAT inhibitor, an inhibitor of the accumulation of macrophaglipid complexes and an agent for the prophylaxis and treatment of liver diseases from natural materials, and as a result, naringin or naringenin have potential ACAT-inhibiting activity that inhibits the accumulation of macrophage-lipid complexes, and activity against the prevention or treatment of liver diseases.

Naringin (C ₂₇ H ₃₂ O ₁₄ , M.V.: 580.53) and naringin aglycon, naringenin (C ₁₅ H ₁₂ O ₅ , M. V.: 272.25), are flavonoids found in lemons, grapefruits, tangerines, citrons and oranges (Citrus sinensis) (Horowitz, Gentili, Tetrahedron, 19, 773, 1963).

 Naringin or naringenin has been reported to have anticancer, antiviral, and cholesterol lowering activities (Monforte, M.T., et al., Farmaco., 50 (9), 595-599, 1995, Sep .; JP 95-86929; JP 95-86930; Felica, V., et al., J. Med. Virol., 15, 71-79, 1985; EP 0352147 A2, 1990.1.24 and Martin MJ, et al., Pharmacal. 49, 144-150, 1994).

 Naringin was then used as a bitterness agent, sweetener, or as a base for chewing gum.

 However, neither ACAT inhibitory activity, nor inhibitory activity against the accumulation of the macrophage-lipid complex, nor the prophylactic or therapeutic activity of naringin or naringenin has been reported so far.

 Summary of the invention.

 Accordingly, the main objective of the present invention is a new use of naringin or naringenin for the inhibition of ACAT activity in mammals.

 Another objective of the present invention is a new use of naringin or naringenin for inhibiting the accumulation of macrophage-lipid complexes on the endothelial walls of arteries in mammals.

 The next objective of the present invention is a new use of naringin or naringenin for the prevention or treatment of liver diseases in mammals.

 A brief description of the drawings.

The above and other objects and features of the present invention will become clearer from the following description of the invention together with the accompanying drawings, where:
on figa, 1B and 1C presents the arteries of rabbits, which were injected with 1% cholesterol; 1% cholesterol plus 1 mg / kg Lovastatin ^® and 1% cholesterol plus 0.1% naringin, respectively, and
on Ffg. 2A, 2B and 2C show the microscopic features of the rabbit liver, which was injected with 1% cholesterol; 1% cholesterol plus 1 mg / kg Lovastatin ^® and 1% cholesterol plus 0.1% naringin, respectively.

DETAILED DESCRIPTION OF THE INVENTION
In accordance with one aspect of the present invention, the use of naringin or naringenin for inhibiting the activity of acyl CoA cholesterol-o-acyltransferase (ACAT) in mammals is provided.

 In accordance with another aspect of the present invention, the use of naringin or naringenin for inhibiting the accumulation of macrophage-lipid complexes on endothelial walls of arteries in mammals is provided.

 In accordance with a further aspect of the present invention, there is provided the use of naringin or naringenin for the prophylaxis or treatment of liver diseases in mammals.

 Naringin or naringenin can be extracted from citrus peel or synthesized according to the method disclosed by Zemplen, Bognar, Ber., 75, 1043, 1943, and Seka, Prosche, Monatsh., 69, 284, 1936. Further, naringenin can be obtained by hydrolysis of naringin.

 Naringin or naringenin exhibit an inhibitory effect on ACAT activity and inhibitory accumulation of macrophage-lipid complexes on the endothelial walls of arteries, as well as a prophylactic or therapeutic effect on liver diseases in doses of 0.1 mg / kg / day or more, and the inhibitory effect increases with increasing doses.

 Moreover, despite the potential efficacy of naringin or naringenin, they exhibit low toxicity or mitogenicity in mouse tests. More specifically, naringin or naringenin is generally non-toxic when administered orally to mice at a dose of 1000 mg / kg, which corresponds to an oral dose of 50 to 100 g / kg body weight of naringin or naringenin for patients weighing 50 kg. In addition, naringin and naringenin have no harmful effect on liver function.

 The present invention also provides a pharmaceutical composition for inhibiting ACAT activity and inhibiting the accumulation of macrophage-lipid complexes on the endothelial walls of arteries and for preventing or treating liver diseases, which comprises naringin or naringenin as an active ingredient and pharmaceutically acceptable excipients, carriers or diluents.

 The pharmaceutical composition can be prepared in accordance with any of the usual procedures. In preparing the composition, the active ingredient is preferably mixed with a carrier, or diluted with a carrier, or enclosed in a carrier, which may be in the form of a capsule, cachet, or any other container. If the carrier serves as a diluent, it may be a solid, semi-solid or liquid material and functions as a carrier, excipient or medium for the active ingredient. Thus, the composition may be in the form of tablets, pills, powders, wafers, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injectable solutions, powders in sterile packaging, and the like.

 Examples of suitable carriers, excipients and diluents are lactose, dextrose, sucrose, sorbitol, mannitol, starches, acacia gum, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidobenzene, hydroxybenzenes magnesium stearate and mineral oil. The compositions may further include excipients, antiagglutination agents, lubricants, wetting agents, flavorings, emulsifiers, preservatives, and the like. The compositions of the present invention can be prepared so as to provide quick, delayed or delayed release of the active ingredient after their administration to mammals, using any method known to those skilled in the art.

 The pharmaceutical compositions of the present invention can be administered in various ways, including oral, transdermal, subcutaneous, intravenous and intramuscular administration. For humans, a typical daily dose of naringin or naringenin can be in the range of about 0.1 to 100 mg / kg body weight, preferably 3 to 10 mg / kg body weight, and can be administered as a single dose or as divided doses.

 However, it should be understood that the amount of the active ingredient that is actually administered should be determined taking into account various factors, including the condition to be treated, the chosen route of administration, the age, gender and weight of the patient, and the severity of the symptoms; and therefore, the above doses should in no way limit the scope of the invention.

 Moreover, naringin or naringenin can be included in foods or drinks as supplements or dietary supplements for inhibiting ACAT activity, inhibiting the accumulation of macrophage-lipid complexes on the endothelial walls of arteries and / or for preventing or treating liver diseases. Food or drink may include various types of meat; juices such as vegetable juices (e.g., carrot juice and tomato juice) and fruit juices (e.g., orange juice, grape juice, pineapple juice, apple juice and banana juice); chocolates; snacks candy; Pizza products made from cereal flour, such as bread, muffins, crackers, cookies, biscuits, noodles, etc. chewing gum; dairy products such as milk, cheeses, yogurt and ice cream; soups; broths; pastes; ketchups and sauces; teas alcoholic drinks; carbonated drinks such as Coca-Cola and Pepsi-Cola; vitamin complexes and various healthy foods.

 In this case, the content of naringin or naringenin in foods or drinks may be in the range from 0.01 to 5 wt.%. In particular, a beverage according to the present invention may contain from 200 to 10,000 mg of naringin or naringenin per 1000 ml of beverage.

 As indicated above, naringin or naringenin can be used as an effective non-toxic pharmaceutical agent for inhibiting ACAT activity, inhibiting the accumulation of macrophage-lipid complexes on arterial endothelium and / or for preventing or treating liver diseases.

 The following examples are intended to further illustrate the present invention without limiting its scope.

 The following percentages for mixtures of solid in solid, liquid in liquid and solid in liquid are based on weight / weight, volume / volume and weight / volume, respectively; and all reactions are carried out at room temperature, unless otherwise indicated.

 Example 1. Extraction of naringin from citrus peel.

The peel of tangerines (Cheju Island, Korea), citrons (Jeollanamdo, Korea) and oranges, grapefruits and lemons (California, CA, USA) are dried at room temperature and ground into a powder with a particle size of 100 to 200 microns. 50 ml of methanol is added to 500 mg of each of the citrus peel powders and extracted in a water bath at 50 ^{° C.} for 6 hours. The extract thus obtained is cooled and filtered, and then methanol is added to the filtrate to a volume of 50 ml.

To determine the naringin content in the obtained extracts, 5.0 μl of the obtained extract was processed using high performance liquid chromatography (HPLC) using a Lichrosorb RP-8 column (5 μm, 4x250 mm), which was pre-equilibrated with 37% methanol and maintained at a temperature of 30 ^o C. The extract is eluted with 37% methanol at a flow rate of 1.0 ml / min. Standard solutions are prepared by dissolving naringin (Sigma Chemical Co. USA) in methanol to final concentrations of 0.1, 0.2, 0.3, 0.4, and 0.5 mg / ml and processed by HPLC under the same conditions. as indicated above. The eluates are detected at 280 nm using a UV-visible spectrophotometer, and the naringin content is calculated by comparing the areas under the HPLC curves of citrus peel extracts and a standard solution. The content (%) of naringin in various extracts of citrus peel is presented in table. I.

 Example 2. Toxicity by oral administration of naringin or naringenin.

7-8 week old specific non-pathogenic ICR female mice (6 pieces), each weighing from about 25 to 29 g, and male mice (6 pieces), each weighing about 34 to 38 g, are grown at a temperature of 22 ± 1 ^o С , humidity 55 ± 5% and photoperiod 12 hours light / 12 hours darkness. Food (Cheiljedang Co., food for mice and rats) and water are sterilized and given to mice.

 Naringin and naringenin are dissolved in 0.5% Tween 80 to a concentration of 100 mg / ml, and this solution is administered orally to mice in an amount of 0.2 ml per 20 g of body weight of mice. The solution is administered once, and the mice are monitored for 10 days for signs of harmful effects or death in accordance with the following scheme: 1, 4, 8 and 12 hours after injection and every 12 hours after that. Weight changes in mice are recorded every day to determine the effect of naringin or naringenin. Then, on day 10, the mice were sacrificed, and visually examined internal organs.

 After 10 days, all mice were alive, and naringin or naringenin showed no toxicity at a dose of 1000 mg / kg. An autopsy showed that the mice did not develop any abnormalities and there was no weight loss in 10 days of the test period. Accordingly, it was concluded that naringin or naringenin is non-toxic when administered orally to animals.

 Example 3. The introduction of naringin or naringenin to animals.

 30 four-week-old Sprague-Dawley rats (Tainan laboratory animal center, Korea), each weighing from 90 to 110 g, were randomly divided into three dietary groups. Rats in these three groups are given three different foods with a high cholesterol content, i.e. AIN-76 laboratory animal feed (ICN, Biochemicals, Cleveland, OH, USA) containing 1% cholesterol (control group) and 1% cholesterol plus 0 , 1% naringin or naringenin, respectively. The composition of the feed that gave these three groups is presented in table. II.

 Rats, if desired, were fed special food along with water for 6 weeks, the absorbed amount was recorded daily, and the rats were weighed every 7 days, and then the results were analyzed. All rats showed normal growth rates and no noticeable differences between the three groups in terms of feed intake and weight gain were observed.

 Example 4. Determination of total cholesterol, HDL cholesterol and neutral lipids in plasma.

 The effect of rat administration of naringin or naringenin on plasma cholesterol and neutral lipids is determined as follows.

 Blood samples were taken from rats of three different dietary groups, and plasma HDL fractions were isolated from them using HDL-cholesterol reagent (Sigma Chemical Co., Cat. 352-3) containing dextran sulfate. Total cholesterol and HDL cholesterol levels are determined using the Sigma Diagnostic Kit Cat. 352-100 (Sigma Chemical Co., U.S.A.) (Allain et al., Clin. Chem., 20, 470-475, 1974). Neutral lipid levels are determined using a Sigma Diagnostic Kit Cat. 339-50 (Bucolo G. and David H., Clin. Chem., 19, 476-482, 1973). The results are presented in table. III, where the levels of total plasma cholesterol in rat groups in the feed of which was naringin or naringenin were reduced by 32% and 18%, respectively, compared with the control group.

 Example 5. The activity of naringin and naringenin in relation to the inhibition of ACAT.

 (Step 1) Preparation of microsomes.

 To determine the effect on rats of food with naringin or naringenin on ACAT activity, microsomes are isolated from liver tissues to be used as a source of enzymes.

First, the rats of the three groups obtained in Example 3 are euthanized by cutting off the head and the liver is excised. 1 g from each liver is homogenized in 5 ml of homogenization medium (0.1 M KH ₂ PO ₄ , pH 7.4, 0.1 mM EDTA and 10 mM β-mercaptoethanol). The homogenate is centrifuged at 3000 g for 10 minutes at 4 ^{° C.} and the supernatant thus obtained is centrifuged at 15,000 g for 15 minutes at 4 ^{° C.} to obtain a supernatant. This supernatant was placed in an ultracentrifuge ampoule (Beckman) and centrifuged at 100,000 g for 1 h at 4 ^° C, obtaining a precipitate of microsomes, which was then suspended in 3 ml of homogenization medium, and centrifuged at 100,000 g for 1 h at 4 ^{° C.} The precipitate thus obtained is suspended in 1 ml of homogenization medium. The concentration of proteins in the resulting suspension is determined by the Lowry method, and then adjusted to a value of from 4 to 8 mg / ml. The resulting suspension was stored at a very low temperature (Biofreezer, Forma Scientific Inc.).

 (Stage 2) ASAT analysis.

 6.67 μl of a 1 mg / ml solution of cholesterol in acetone was mixed with 6 μl of 10% Triton WR-1339 (Sigma Co.) in acetone, and then acetone was removed from the mixture by evaporation using nitrogen gas. To the resulting mixture was added distilled water in such an amount as to bring the concentration of cholesterol to 30 mg / ml.

To 10 μl of the resulting aqueous cholesterol solution, add 10 μl of 1 M KN ₂ PO ₄ (pH 7.4), 5 μl of 0.6 mm bovine serum albumin (BSA), 10 μl of the microsome solution obtained in stage 1, and 55 μl of distilled water (total 90 μl). The two mixtures were preincubated in a water bath at 37 ^° C for 30 minutes.

10 μl of a solution of (l- ¹⁴ C) oleoyl-CoA (0.05 μCi, final concentration: 10 μM) was added to the pre-incubated mixture, and the resulting mixture was incubated in a water bath at 37 ^° C for 30 minutes. To this mixture was added 500 μl of a mixture of isopropanol: heptane (4: 1 (v / v)), 300 μl of heptane and 200 μl of 0.1 M KH ₂ PO ₄ (pH 7.4), and the resulting mixture was stirred vigorously, using a centrifuge and then left to stand at room temperature for 2 minutes.

 200 μl of the resulting supernatant was placed in a scintillation vial, and 4 ml of scintillation fluid (Lumac) was added thereto. The resulting mixture was analyzed for radioactivity using a 1450 Microbeta scintillation counter (Wallacoy, Finland). ACAT activity is calculated as picomoles of cholesterol oleate synthesized per minute per mg of protein (pmol / min / mg protein). The results are presented in table. IV.

 As can be seen from the data table. IV, ACAT activity observed for groups of rats in whose feed naringin or naringenin was added is lower than for the control group by 20.2% and 17.4%, respectively.

 Example 6. Inhibition of plaque formation caused by macrophage-lipid complexes in animals fed with naringin or naringenin.

 (Stage 1). The introduction of animals naringin or naringenin.

24 three-month-old New Zealand white rabbits (Yeonam Horticulture and Animal Husbandry College, Korea), each weighing about 2.5 to 2.6 kg, are raised under conditions of 20 ± 2 ^o C and relative humidity 55 ± 5%, and a photoperiod of 12 hours light / 12 h darkness. Rabbits are divided into groups of 6 rabbits, and rabbits of four groups are fed with four different compositions of feed, i.e. RC4 food (Oriental Yeast Co., Japan) containing 1% cholesterol (control group); 1% cholesterol plus 1 mg / kg Lovastatin ^® (Merck, USA) (comparative group); 1% cholesterol plus 0.1% naringin and 1% cholesterol plus 0.1% naringenin, respectively. RC4 feed contains 7.6% moisture, 22/8% crude protein, 2.8% crude fat, 8.8% crude ash, 14.4% crude pulp and 43.6% soluble nitrogen-free substances. Rabbits are fed this way for 6 weeks with free access to food and water.

 (Stage 2). Analysis of strips of fat in the main artery.

 Rabbits that were fed according to the scheme of stage 1, kill and cut the chest. The main artery is cut from it about 5 cm in the forward direction from the point 1 cm above the aortic valve, and the fat surrounding the main artery is removed. The main artery is cut in the middle along the long axis and placed in a cup. The main artery is photographed, and then stains of fat are stained according to the method of Esper, E., et al., (J. Lab. Clin. Med., 121, 103-110, 1993) as follows.

 Part of the excised main artery is washed three times for 2 min with anhydrous propylene glycol and stained for 30 min with a saturated solution of Oil Red O (ORO, Sigma Co.) in propylene glycol. After this, the artery is washed twice for 3 minutes with 85% propylene glycol to remove residual staining solution, and then washed with saline. The artery is photographed and the photograph is examined. The area of the stained area (area with strips of fat) is determined using an image analyzer (LEICA, Q-600, Germany) and its fraction (%) of the total artery area is calculated.

 On the other hand, another part of the main artery is stained in accordance with the hematoxylin-eosin (H & E) method and Masson tri-color staining method and observed under a microscope to confirm that macrophage-lipid complexes accumulate in the inner membrane, Internus, elastic membrane and medium.

 Next, blood samples were taken from rabbits and the levels of total cholesterol and triglycerides were determined according to the method of Example 4.

 The results are presented in table. V.

As can be seen from the results of table. V, the accumulation area of the macrophage-lipid complex on the arterial endothelium is significantly reduced for groups with 1 mg / kg Lovastatin ^® , 0.1% naringin and 0.1% naringenin compared with the control group. Accordingly, it was confirmed that naringin and naringenin inhibit the accumulation of macrophage-lipid complexes on arterial endothelium. In particular, it is noteworthy that the activity of naringin or naringenin with respect to inhibiting the accumulation of macrophage-lipid complexes appears at blood cholesterol levels of more than 1100 mg / dl, which is much higher than in healthy rabbits, i.e. about 50 mg / dl. This result suggests the possibility of a new mechanism for preventing the onset of atherosclerosis, which differs from blocking cholesterol synthesis due to an HMG-CoA reductase inhibitor, blocking cholesterol absorption due to an ACAT inhibitor, or blocking cholesterol transfer due to a CETP inhibitor.

In FIG. 1A, 1B and 1C show the arteries of rabbits, which were injected with 1% cholesterol (control group); 1% cholesterol plus 1 mg / kg Lovastatine ^® comparison group); 1% cholesterol plus 0.1% naringin, respectively. As can be seen in Figs. 1A, 1B and 1C, a thick layer of the macrophage-lipid complex is observed on the endothelium of rabbit arteries injected with 1% cholesterol, while very thin layers or no macrophage-lipid complexes are observed on the endothelium of the rabbit arteries, which were administered 1 % cholesterol plus 1 mg / kg Lovastatine ^® and 1% cholesterol plus 0.1% naringin, respectively.

 Accordingly, it was concluded that naringin or naringenin strongly inhibit the accumulation of macrophage-lipid complexes on the endothelium of arteries.

 Example 7. Prevention of liver disease using naringin.

 (Stage 1). The introduction of rats naringin.

 20 four-week-old Sprague-Dawley rats (Taihan laboratory animal center, Korea), each weighing from 90 to 110 g, are randomly divided into two dietary groups. Rats in these two groups are given two different foods with a high cholesterol content, that is, AIN-76 laboratory animal feed (ICN, Biochemicals, Cleveland, OH, USA) containing 1% cholesterol (control group) and 1% cholesterol plus 0.02% of naringin, respectively. The composition of the feed that was given to these two groups is presented in table. VI.

 Rats, if desired, were fed special food along with water for 6 weeks, the absorbed amount was recorded daily, and the rats were weighed every 7 days, and then the results were analyzed. All rats showed normal growth rates, and no noticeable differences between the two groups in terms of feed intake and weight gain were observed.

 (Stage 2). Determination of serum GOT and GPT levels.

 The effect of rat administration of naringin on liver function is investigated as follows.

 Blood samples were taken from rats of two dietary groups and serum levels of GOT (glutamate-oxaloacetate transaminase) and GPT (glutamate-pyruvate transaminase) were determined by the method of Reitman and Frankel (Reitman, S. and JS Frankel, Am.J. Clin. Pathol., 28, 56, 1956). GOT and GPT are synthesized in the liver and heart, and are released into the blood stream when these organs are damaged. Accordingly, GOT and GPT are representative markers of liver function, and high serum levels of GOT and GPT indicate serious liver damage.

 The results show that the GOT and GPT levels for the naringin group were lower than the levels for the control group by about 30 and 10%, respectively.

 (Stage 3). Experiment on rabbits.

The procedure of step 1 is repeated, except that 30 three-month-old New Zealand white rabbits (Yeonam Horticulture and Animal Husbandry College, Korea), each weighing about 2.5 to 2.6 kg, are used instead of rats, and the rabbits are fed for six weeks with three various feed compositions, i.e. RC4 feed containing 1% cholesterol (control group); 1% cholesterol plus 1 mg / kg Lovastatine ^® (comparative group) and 1% cholesterol plus 0.1% naringin, respectively.

 After that, the liver is removed from rabbits and histopathological observations are carried out as follows.

 Rabbits are anesthetized with an intramuscular injection of ketamine (75 mg / kg) and the abdominal cavity is cut. The color and degree of liver sclerosis is determined by eye, and the liver removed from the rabbit is fixed in 10% neutral buffered formalin for more than 24 hours. The fixed liver is washed with plenty of water, dehydrated step by step with 70, 80, 90 and 100% ethanol, and then enclosed in paraffin. The liver enclosed in paraffin is cut into 4 μm layers using a microtome and stained with hematoxicillin and eosin. Stained liver samples are made transparent with xylene, placed with permount and observed under a microscope to confirm the presence of lesions.

In FIG. 2A, 2B and 2C show the microscopic features of the liver of rabbits, which were injected with 1% cholesterol (control group); 1% cholesterol plus 1 mg / kg Lovastatine ^® (comparative group) and 1% cholesterol plus 0.1% naringin, respectively. As can be seen in FIGS. 2A and 2B, the liver cells of the control group and the comparative group are irregular and enlarged, and a large amount of fat is deposited on them. On the contrary, as can be seen in FIG. 2C, the liver cells of the naringin group remained normal and no fat deposition was observed on them. This result shows that naringin strongly inhibits liver obesity without any harmful effects on liver cells.

 (Stage 4). Human experiment.

 Naringin is orally administered to a 55-year-old person at a daily dose of 10 mcg / kg for 68 days and GOT, GPT and γGTP levels are determined immediately before administration (day 0) and 45 and 68 days after administration (days 45 and 68), respectively. Serum GOT levels on day 45 and 68 decreased by 17%, respectively, compared to day 0. Serum GPT levels on day 45 and 68 decreased by 15 and 19%, respectively, compared to day 0. Further, serum γGTP levels were 45. and 68 days decreased by 25 and 51%, respectively, compared with day 0. Unexpectedly, a decrease in serum γGTP was found to be more than 50%, and this result suggests that naringin or naringenin has high liver protective activity, as well as prophylactic activity in regarding liver diseases such as hepatitis Ita, fatty liver and alcoholic fatty liver.

In another case, naringin was orally administered 56-year old man, who always used the alcohol in an amount of 100 cm ³ per day, in a daily dose of 6 mg / kg for 30 days, and the level γGTP serum was determined just before the administration (day 0) and 30 days after administration (day 30). Accordingly, the initial serum γGTP level on day 0 was 129 international. u / l, whereas on the 30th day it decreased to 69 intern. u / l, which corresponds to the normal value. This result shows that naringin or naringenin is highly active in the prevention of alcoholic liver obesity and hepatocirrhosis.

 Example 9. Products containing naringin or naringenin.

 Products containing naringin or naringenin are prepared as follows.

 (1). Cooking tomato ketchup and sauce.

 Naringin or naringenin is added to tomato ketchup or sauce in an amount in the range of 0.01 to 5% by weight to obtain a health improving tomato ketchup or sauce.

 (2). Preparation of wheat flour products.

 Naringin or naringenin is added to wheat flour in an amount in the range of 0.01 to 5% by weight, and bread, muffins, cookies, crackers and noodles are prepared using this mixture to produce health-improving products.

 (3). Cooking soups and gravy.

 Naringin or naringenin is added to soups and gravy in an amount in the range from 0.01 to 5 weight. % to get health-improving soups and gravy.

 (4). Cooking ground beef.

 Naringin or naringenin is added to ground beef in an amount in the range of 0.01 to 5% by weight to obtain a health improving ground beef.

 (5). Cooking dairy products.

 Naringin or naringenin is added to milk in an amount in the range from 0.01 to 5% by weight, and various dairy products, such as butter and ice cream, are prepared from this milk.

 However, in the preparation of cheeses, naringin or naringenin is added to the coagulated milk protein; and in the case of yogurt, naringin or naringenin is added to the coagulated milk protein after fermentation.

 Example 10. Drinks containing naringin or naringenin.

 (1). Cooking vegetable juice.

 200 to 10,000 mg of naringin or naringenin is added to 1000 ml of tomato or carrot juice to produce health-improving vegetable juices.

 (2). Making fruit juice.

 200 to 10,000 mg of naringin or naringenin is added to 1000 ml of apple or grape juice to produce health-improving fruit juices.

 (3). Preparation of carbonated drinks.

 200 to 10,000 mg of naringin or naringenin is added to 1000 ml of Coca-Cola or Pepsi-Cola to produce health-improving carbonated drinks.

 Although the present invention has been described in relation to the above examples, it should be understood that those skilled in the art can make various modifications and changes to the invention that fall within the scope of the invention and are defined by the appended claims.

Claims (9)

 1. The use of naringin or naringenin as an active ingredient in a composition for the prevention or treatment of liver lipodystrophy or cirrhosis in mammals.  2. The use of claim 1, wherein the mammal is a human.  3. The use according to claim 1, wherein naringin or naringenin is administered to the mammal in the form of a composition comprising naringin or naringenin, said composition being selected from the group consisting of a pharmaceutical composition, a food composition, and a drink.  4. The use according to claim 3, where the effective amount of naringin or naringenin contained in the pharmaceutical composition is in the range from 0.1 to 100 mg / kg body weight / day.  5. The use according to claim 3, where the content of naringin or naringenin in the food composition is 0.01-5 weight. %  6. The use according to claim 3, where the food composition is meat, chocolate, snacks, sweets, pizza, products made from cereal flour, chewing gum, dairy products, soups, broths, pastes, ketchups, sauces, vitamin complexes or products for health.  7. The use according to claim 6, where the products made from cereal flour are bread, muffins, crackers, cookies, biscuits or noodles.  8. The use of claim 3, wherein the beverage compositions are dairy products, vegetable juices, fruit juices, teas, spirits, or carbonated drinks.  9. The use according to claim 3, where the content of naringin or naringenin in the beverage compositions is in the range from 200 to 10,000 mg per 1000 ml of beverage.

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