Hesperidin and hesperetin as an inhibitor of acyl coa - cholesterol-o-acyltransferase, inhibitors accumulation of complexes of macrophage-lipid on the walls of the arteries, and as a means for the prevention or treatment of liver diseases

 

(57) Abstract:

A new active ingredient of the pharmaceutical composition, a food composition or beverage for prevention or treatment of lipodystrophy liver or cirrhosis of the liver in mammals, including man. As such ingredients are known flavonoids hesperidin or hesperetin. The invention expands the Arsenal of the declared destination. 8 C.p. f-crystals, 2 ill., 6 table.

The invention relates to the use of hesperidin or hesperetin for inhibiting the activity Adil COA-cholesterol-o-acyltransferase (AST), inhibiting the accumulation of complexes of macrophage-lipid on the endothelium of arteries and for the prevention or treatment of liver diseases in mammals.

Background of the invention

In recent years, coronary cardio-circulatory diseases, such as atherosclerosis and hypercholesterolemia, are increasingly becoming a major cause of mortality. It was reported that elevated levels of cholesterol in the plasma cause the deposition of fat, macrophages and foam cells in the walls of blood vessels, and that these deposits lead to the formation of plaques, and then to atherosclerosis (Ross, R., Nature, 362, 801-809 (1993)). One of the ins and lilydev. Another way is the inhibition of cholesterol absorption by inhibiting participating in this enzyme.

Acyl COA-cholesterol-o-acyltransferase (ACAT) promotiom the formation of ester cholesterol in the blood. Foam cells are formed as a result of AST and contain a large number of ester cholesterol, which carry low-density lipoprotein (LDL). The formation of foam cells in the artery walls increases with the increase of AST activity, and accordingly, the ACAT inhibitor may also serve as a means for the prevention of atherosclerosis. Further, it was reported that the level of blood LPN-cholesterol can be lowered by inhibiting ACAT activity (Witiak, D. T. and D. R. Feller (eds. ), Anti-Lipidemic Drugs; Medicinal, Chemical and Biochemical Aspects, Elsevier, pp 159-195 (1991)).

On the other hand, the deterioration of the liver may be due to excessive absorption of alcohol or food with a high content of lipids, or as a result of infection with hepatitis b or C, and it can develop in hepatitis can lead to cirrhosis or liver cancer. In particular, excessive consumption of food high in fat and alcohol causes fatty liver, when a large number of LIPI alamat-pyruvate transaminase) and GTP (-glutamyl of transpeptidase) (T. Banciu et al., Med. Interne., 20, 69-71 (1982); and A. Par et al., Acta. Med. Acad. Sci. Hung., 33, 309-319 (1976)).

There have been many attempts to develop drugs that would be inhibited ACAT activity; and the result is reported that the selection of multiple compounds from cultures of different microorganisms. Examples of such compounds include peribiliary isolated from the culture of Aspergillus fumigatus (S. Omura et al., J. Antibiotics, 46, 1168-1169 (1993)) and Ekaterin isolated from Pseudomonas sp. (S. Nagamura et al., J. Antibiotics. 45, 1216-1221 (1992)).

Further, as means for treatment of hypercholesterolemia was developed inhibitor of HMG-CoA reductase, called lovastatin (Lovastatin), which is marketed by Merck Co., U. S. A. However, it is known that this medicine causes harmful side effect - increases the content of creatine kinase in the liver.

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

The authors of the present invention have attempted to develop new and effective ACAT inhibitor, an inhibitor of accumulation of complexes of macrophage-lipid and means one or hesperetin have the potential ACAT inhibitory activity, inhibiting the accumulation of complexes of macrophage-lipid activity and activity in the prevention or treatment of liver diseases.

Hesperidin (C28H34O15M. B.: 610,55) and the aglycone of hesperidin, hesperetin (C16H14O6M. B. : 302,27) are flavonoids found in lemons, grapefruits, tangerines, nitrong and oranges (Citrus sinensis)(Horowitz, Gentili, Tetrahedron, 19, 773 (1943)).

It was reported that hesperidin or hesperetin possess strengthens capillaries, reduces permeability, inhibiting platelet aggregation, anti-inflammatory, antiviral, lowering blood pressure and reducing cholesterol activities (Meuag, O. C., Angiology, 45, 579-584 (1994); Struckmann, J. R., et al., Angiol, 45, 419-428 (1994); Matsubara, Y., et al. , Japan Organic Synthesis Chem. Association Journal, 52, 318-327 (1994. Mar. ); Galati, E. M., et al., Farmaco., 51(3), 219-221 (1996, Mar.); Monforte, M. T., et al., Farmaco., 50(9), 595-599 (1995, Sep.); JP 95-86929; JP 95-86930; Chung, M. I., et al., Chin. Pharm. J. (Taipei)., 46, 429-437 (1994, Nov.); Galati, E. M., et al., Farmaco., 40(11), 709-712 (1994, Nov.); and Emim, J. A., et al., J. Pharm. Pharmacol., 46.(2). 118-122 (1994)).

In addition, hesperidin used for the prevention and treatment of cerebral anemia, retina (retinal) hemorrhage and pelioma.

However, still do not report about ACAT inhibitory actively therapeutic activity against liver disease of hesperidin or hesperetin.

Summary of the invention

Accordingly, the present invention is a new use of hesperidin or hesperetin for inhibition of ACAT activity in mammals.

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

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

Brief description of 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, in which:

in Fig. 1A, 1B and 1C presents artery of rabbits, which were administered with 1% cholesterol; 1% cholesterol plus 1 mg/kg Lovastatinand 1% cholesterol plus 0.1% hesperidin, respectively;

in Fig. 2A, 2B and 2C presents the microscopic features of the disease of rabbits that were administered with 1% cholesterol; 1% cholesterol plus 1 mg/kg Lovastatinand 1% cholesterol plus 0.1% hesperidin, respectively.

Pedroia of hesperidin or hesperetin for inhibiting the activity of acyl COA-cholesterol-o-acyltransferase (AST) in mammals.

In accordance with another aspect of the present invention proposed the use of hesperidin or hesperetin for inhibiting the accumulation of complexes of macrophage-lipid on the endothelial lining of the arteries in mammals.

In accordance with the following aspect of the present invention proposed the use of hesperidin or hesperetin for the prevention or treatment of liver diseases in mammals.

Hesperidin or hesperetin can be extracted from the peel of citrus fruit or to synthesize the way, opened Zemplen, Bognar, Ber., 75, 1043 (1943) and Seka, Prosche, Monatsh., 69, 284 (1936). Next, hesperetin can be obtained by hydrolysis of hesperidin.

Hesperidin or hesperetin demonstrate inhibitory effect on AST activity and accumulation of complexes of macrophage-lipid on the endothelial lining of the arteries, as well as preventive or therapeutic effect against diseases of the liver at doses of 0.1 mg/kg/day or more, and the inhibitory effect increases with increasing dose.

Moreover, despite the high efficacy of hesperidin or hesperetin, they demonstrate low toxicity or mitogenome in tests on mice. More specifically, hesperidin or gasp the second introduction from 3 to 10 g/kg of body weight of hesperidin or hesperetin for patients weighing 50 kg In addition, hesperidin and hesperetin no harmful effects on the liver.

The present invention also proposed a pharmaceutical composition for the inhibition of ACAT activity and inhibiting the accumulation of complexes of macrophage-lipid on the endothelial lining of the arteries, and for the prevention or treatment of liver diseases, which includes hesperidin or hesperetin 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 the preparation of the composition the active ingredient is preferably mixed with a carrier, or diluted by a carrier, or enclosed in a carrier, which may be in the form of a capsule, cachet, or any other container. When the carrier serves as a diluent, it may be 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 and hard gelatin capsules, sterile solutions for injectible are lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, alginates, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. The composition may additionally include fillers, anti-agglutination agents, lubricating agents, wetting agents, flavoring agents, emulsifiers, preservatives, etc. of the Composition of the present invention can be prepared so as to provide quick, slow or delay the release of active ingredient after their introduction mammals, using any well-known specialists of the way.

The pharmaceutical compositions of the present invention can be administered in a number of ways, including oral, transcutaneous, subcutaneous, intravenous and intramuscular introduction. For men the typical daily dose of hesperidin or hesperetin may be in the range of from about 0.1 to 100 mg/kg body weight, preferably from 3 to 10 mg/kg of body weight, and can be entered as a single dose or in divided doses.

However, it should be understood that the amount of the active ingredient, which in the state, the selected route of administration, age, sex and weight of the patient, and the severity of the symptoms; and therefore, the above dose in no way should limit the scope of the invention.

Moreover, hesperidin or hesperetin can be included in foods or drinks as supplements or dietary supplements for inhibition of ACAT activity, inhibiting the accumulation of complexes of macrophage-lipid on the endothelial lining of the arteries and/or for the prevention or treatment of liver diseases. Food or drinks may include various meats; juices, such as vegetable juices (for example, carrot juice and tomato juice) and fruit juices (such as 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 and so on; chewing gum; dairy products such as milk, cheese, yogurt and ice cream; soups; bouillons; pastes, ketchups and sauces; tea; alcoholic beverages; soft drinks such as Coca-Cola and Pepsi-Cola; complexes of vitamins and a variety of healthy foods.

In this case, the content of hesperidin or hesperetin in food or written in order to contain from 200 to 10,000 mg of hesperidin or hesperetin per 1000 ml of the drink.

As mentioned above, hesperidin or hesperetin can be used as an effective non-toxic pharmaceutical agents for the inhibition of ACAT activity, inhibiting the accumulation of complexes of macrophage-lipid on the endothelium of arteries and/or for the prevention or treatment of liver diseases.

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

Next, the following percent for mixtures of solid in solid, liquid in liquid and solid in a liquid is given based on the weight/weight, volume/volume and weight/volume, respectively; and all reactions are conducted at room temperature, if no other special instructions.

Example 1: Extraction of hesperidin from citrus peel

Peel tangerines (Cheju Island, Korea), dei Cedri (Jeollanamdo, Korea), and oranges, grapefruits and lemons (California, CA, U. S. A.), dried at room temperature and pulverized into a powder with a particle size of from 100 to 200 μm. 50 ml of methanol is added to 500 mg of each powder citrus peel and extracted in a water bath at 50oC for 6 hours. Thus obtained extract was cooled and filtered, and then methanol is added to the filtrate to a volume of the same process using vysokoeffektivnoi liquid chromatography (HPLC), using column Lichrosorb RP-8 (5 ám, 4250 mm), which is pre-balance 37% methanol and support at a temperature of 30oC. Extract elute 37% methanol at a flow rate of 1.0 ml/min Standard solutions prepared by dissolving hesperidin (Sigma Chemical Co., U. S. A.) in methanol to final concentrations of 0.1, 0,2, 0,3, 0,4 and 0,5 mg/ml and processing using HPLC under the same conditions as described above. Eluate detected at 280 nm with a spectrophotometer operating in the UV-visible range, and the content of hesperidin calculated by comparing the area under the curves HPLC of extracts of citrus peel and a standard solution. Content (%) of hesperidin in various citrus peel extracts are presented in table I.

Example 2: Toxicity by oral administration of hesperidin or hesperetin

7-8 weeks without specific pathogens ICR female mice (6 pieces), each weighing from about 25 to 29 g, and male mice (6 pieces), each weighing from about 34 to 38 g, grown in conditions 221oC, humidity 555% and photoperiod of 12 h light/12 h darkness. Food (Cheiljedang Co., food for mice and rats) and sterilized water and allow mice.

Hesperidin and hesperetin dissolved in 0.5% tween 80 to a concentration of 100 mg/ml and this rastvoryenii 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 thereafter. Weight change of mice register every day to determine the effect of hesperidin or hesperetin. Next, on day 10 the mice kill and visually examine the internal organs.

After 10 days, all mice were alive, and hesperidin or hesperetin has shown no toxicity at a dose of 1000 mg/kg Autopsy showed that mice did not develop any pathological deviations, and within 10 days of the test period there was no loss of weight. Accordingly, it was concluded that hesperidin or hesperetin not toxic when administered orally to animals.

Example 3: Introduction of hesperidin or hesperetin animals. 30 four-week rat strain Sprague-Dawley (Taihan laboratory animal center, Korea), weighing from 90 to 110 g uniformly randomly divided into three dietary groups. Rats in the three groups give three different foods with high cholesterol content, that is, AIN-76 laboratory animal feed (ICN, Biochemicals, Cleveland, HE, U. S. A.) containing 1% cholesterol (control group) and 1% cholesterol plus 0.1% hesperidin or hesperetin respectively. The composition of the feed, which gave these three groups are presented in table II.

Rats who were daily and the rats were weighed every 7 days, and then analysed the results. All rats showed a normal growth rate, and no significant differences between the three groups from the point of view of the feed consumed and weight gain were observed.

Example 4: Determination of full cholesterol, HDL-cholesterol and neutral lipids in the plasma.

The impact of the introduction of rats of hesperidin or hesperetin on the plasma cholesterol and neutral lipids is determined as follows:

Blood samples taken from rats of three different dietary groups and allocate one fraction of plasma HDL using HDL-cholesterol reagent (Sigma Chemical Co., Cat. 352-3) containing dextran-sulfate. The levels are full of cholesterol and IDPs-cholesterol determined using Sigma Diagnostic Kit Cat. 352-100 (Sigma Chemical Co., U. S. A.) (Allain et al., Clin. Chem. , 20, 470-475 (1974)). The content of neutral lipids determined using 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 rats, the stern of which was hesperidin or hesperetin reduced by 11% and 15% respectively compared with the control group.

Example 5: the Activity of hesperidin and hesperis feed with hesperidin or hesperetin on the activity of AST microsome assay isolated from the tissues of the liver, to use as a source of enzymes.

Initially, the rats of the three groups obtained in example 3, killed by decapitation and cut out the liver. 1 g of each liver homogenized in 5 ml of homogenization medium (0.1 M KN2RHO4, pH 7.4, 0.1 mm EDTA and 10 mm-mercaptoethanol). The homogenate was centrifuged at 3000 g for 10 minutes at 4oWith and the resulting supernatant centrifuged at 15000 g for 15 minutes at 4oC to obtain the supernatant. This supernatant is placed in ultracentrifuge tube (Beckman) and centrifuged at 100000 g for 1 h at 4oWith getting the precipitate of microsomes, which is then suspended in 3 ml of homogenization medium, and centrifuged at 100000 g for 1 h at 4oC. the thus Obtained residue is suspended in 1 ml of the homogenization medium. The concentration of protein in the resulting suspension is determined by the method of Lowry, and then adjusted to a value of from 4 to 8 mg/ml of the Obtained suspension stored at very low temperature (Biofreezer, Forma Scientific Inc.).

(Stage 2) AST analysis

to 6.67 μl of 1 mg/ml cholesterol in acetone mixed with 6 μl of 10% Triton WR-1339 (Sigma Co.) in acetone, and then the acetone is removed from the mixture by evaporation, use the cholesterol concentration up to 30 mg/ml

To 10 μl of the resulting aqueous solution of cholesterol add 10 ál of 1 M KH2PO4(pH of 7.4), 5 μl of 0.6 mm bovine serum albumin (BSA), 10 μl of a solution of microsomes obtained in stage 1, and 55 µl of distilled water (90 μl). Two mixture is pre-incubated in a water bath at 37oC for 30 minutes.

10 μl of the solution (114C) oleoyl-COA (0,05 MK Curie, final concentration: 10 μm) are added to a pre-incubated mixture and the resulting mixture was incubated in a water bath at 37oC for 30 minutes. To this mixture is added 500 μl of a mixture of isopropanol:heptane (4:1 (V/V)), 300 μl of heptane and 200 μl of 0.1 M KN2RHO4(pH 7,4) and the resulting mixture is intensively stirred using a centrifuge, and then left to stand at room temperature for 2 minutes.

200 μl of the obtained supernatant is placed in a scintillation vial and to this add 4 ml of scintillation fluid (Lumac). The resulting mixture was analyzed for radioactivity using a scintillation counter 1450 Microbeta (Wallacoy, Finland). ACAT activity is calculated as picomole of cholesterolaemia synthesized per minute per mg protein (pmol/min/mg protein). The results are presented in t who was b added hesperidin or hesperetin, lower than for the control group by 19.2% and 23.5%, respectively.

Example 6: Inhibition of plaque formation caused by complexes of macrophage-lipid, animals, food which added hesperidin or hesperetin

(Stage 1) Introduction to the animals of hesperidin or hesperetin

24 three-month new Zealand white rabbit (Yeonam Horticulture and Animal Husbandry College, Korea), each weighing from about 2.5 to 2.6 kg, grown in conditions 202oC and relative humidity 555%, and photoperiod of 12 h light/12 h darkness. Rabbits are divided into groups of 6 rabbits and rabbits four groups fed four different composition of feed, i.e. RC4 feed (Oriental Yeast Co., Japan) containing 1% cholesterol (control group); 1% cholesterol plus 1 mg/kg Lovastatine(Merck, U. S. A.) (comparative group); 1% cholesterol plus 0.1% hesperidin; and 1% cholesterol plus 0.1% of hesperetin respectively. Food RC4 contains 7.6 percent moisture, 22,8% crude protein, 2.8% of crude fat, 8,8% crude ash, to 14.4% crude cellulose and 43.6% of the soluble compounds that do not contain nitrogen. Rabbits fed this way for 6 weeks with free access to food and water.

(Stage 2) Analysis of strips of fat into the main artery

Rabbits fed scheme stage 1, wordplaying 1 cm above the aortic valve, and the fat that surrounds the main artery is removed. The main artery cut in the middle along the long axis and placed in a Cup. Wet artery photographed, and then produce staining strips of fat by way Esper, E., et al., (J. Lab. Clin. Med., 121, pp. 103 -- 110 (1993)), as follows.

Part isecheno main artery washed three times for 2 min anhydrous propylene glycol and stained for 30 min with saturated solution of Oil Red O (ORO, Sigma Co.) in the propylene glycol. After this artery washed twice for 3 min in 85% propylene glycol to remove residual dye solution, and then washed with saline solution. Artery photograph and photography to explore. The area of the colored section (section with strips of fat) is determined using the image analyzer (LEICA Q-600), Germany) and calculate its proportion (%) of the total area of the artery.

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

Next in rabbits take blood samples and determined the go in table V.

As can be seen from the results of table V, the area of the accumulation of complexes of macrophage-lipid on the endothelium of the arteries is significantly reduced for groups with 1 mg/kg Lovastatinand groups with 0.1% hesperidin and 0.1% hesperetin compared with the control group. Accordingly, it was confirmed that hesperidin and hesperetin inhibit the accumulation of complexes of macrophage-lipid on the endothelium of the arteries. In particular, it is noteworthy that the activity of hesperidin or hesperetin in respect of inhibiting the accumulation of complexes of macrophage-lipid occurs when cholesterol levels in the blood 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 to prevent the onset of atherosclerosis, which is different from blocking the synthesis of cholesterol due to an inhibitor of HMG-CoA reductase, blocking the absorption of cholesterol by ACAT inhibitor, or block transfer of cholesterol due to SETR inhibitor.

In Fig.1A, 1B and 1C presents artery of rabbits, which were administered with 1% cholesterol (control group); 1% cholesterol plus 1 mg/kg Lovastatin(comparative group); 1% cholesterol plus 0.1% hesperidin, respectively. As the output was administered with 1% cholesterol, while very thin layers or even the absence of complexes of macrophage-lipid observed in the endothelium of arteries of rabbits, which were administered with 1% cholesterol plus 1 mg/kg Lovastatinand 1% cholesterol plus 0.1% hesperidin, respectively.

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

Example 7: Prevention of liver diseases using hesperidin

(Stage 1) administration to rats of hesperidin

20 four-week rat strain Sprague-Dawley (Taihan laboratory animal center, Korea), weighing from 90 to 110 g uniformly randomly divided into two dietary groups. Rats in these two groups give two different foods with high cholesterol content, that is, AIN-76 laboratory animal feed (ICN, Biochemicals, Cleveland, HE, U. S. A.) containing 1% cholesterol (control group) and 1% cholesterol plus 0.04% hesperidin, respectively. The composition of the feed, which gave these two groups are presented in table VI.

Rats were fed on request special food together with water for 6 weeks, absorbed amount was recorded daily and the rats were weighed every 7 days, and then analyzed the results. Su the Ki of view, amount of feed consumed and weight gain were observed.

(Stage 2) determine the levels of GOT and GPT in serum

The impact of the introduction of rats of hesperidin on liver function are examined as follows.

Blood samples taken from rats of the two diet groups and determine the levels of serum GOT (glutamate-oxaloacetate transaminase levels) and GPT (glutamate-pyruvate transaminase levels) according to the method of Reitman and Frankel (Reitman, S and J. S. Frankel, Am. J. Clin. Pathol., 28, 56 (1956)). GOT and GPT are synthesized in the liver and heart and secreted into the blood stream if the damage these organs. Accordingly GOT and GPT are representative markers of liver function, and high levels of GOT and GPT in serum indicate serious liver damage.

The results show that the GOT and GPT levels for a group of hesperidin were below the levels for the control group by about 30% and 10%, respectively.

(Stage 3) Experiment on rabbits

Repeat the procedure stage 1, except that instead of rats use 30 three-month new Zealand white rabbits (Yeonam Horticulture and Animal Husbandry College, Korea), each weighing from about 2.5 to 2.6 kg, and in rabbits fed for six weeks in three different composition of feed, i.e. RC4 feed containing 1% cholesterol (control group); is responsible.

Then in rabbit liver extract and conduct histopathological observations as follows.

Rabbits anaesthetize intramuscular injection of ketamine (75 mg/kg) and cut the abdominal cavity. The color and degree of sclerosing liver is determined on the eyes and livers removed from the rabbit, fixed in 10% neutral bateriafina formalin for more than 24 hours. Otfiksirovat the liver was washed with a sufficient amount of water, gradually dehydrated with 70%, 80%, 90% and 100% ethanol, and then conclude in paraffin. Enclosed in paraffin-embedded liver is cut into layers with a thickness of 4 μm by using a microtome and stained with hematoxylin and eosin. Painted samples of the liver make transparent xylene, placed in the focus and observe under a microscope to confirm the presence of lesions.

In Fig. 2A, 2B and 2C presents the microscopic features of the disease of rabbits that were administered with 1% cholesterol (control group); 1% cholesterol plus 1 mg/kg Lovastatin(comparative group) and 1% cholesterol plus 0.1% hesperidin, respectively. As can be seen in Fig.2A and 2B, the liver cells of control group and comparative group irregular and enlarged, and has accumulated a large amount of fat. In contrast,deposits of fat. This result shows that hesperidin strongly inhibits fatty liver without any harmful effects on the liver cells.

(Stage 4) Experiment on humans

Hesperidin orally administered 55-year-old man in a daily dose of 10 mg/kg over 68 days and determine the levels of serum GOT and GPT and GTP directly before administration (day 0) and after 45 and 68 days after injection (days 45 and 68, respectively. The levels GOT in the serum for 45 day 68 day decreased by 17%, respectively, compared to day 0. The levels of GPT in the serum of 45 and 68 day decreased by 15% and 19%, respectively, compared to day 0. Further, the GTP levels in serum for 45 day 68 day decreased by 25% and 51%, respectively, compared to day 0. Unexpectedly, the reduction of the level of GTP in the serum on day 68 was more than 50%, and this result suggests that hesperidin or hesperetin has a high protecting the liver activity, and preventive activity against liver diseases such as hepatitis, fatty liver and alcoholic fatty liver.

In another case, the oral hesperidin has introduced a 56-year-old man who constantly consumed alcoholic beverages in quantities of 100 cm3in day to day ez 30 days after administration (day 30). Accordingly, the initial GTP level in serum at day 0 was 129 international. u/l, whereas the 30 day he decreased to 69 international. units/l, which corresponds to the normal value. This result shows that hesperidin or hesperetin have high activity in the prevention of alcoholic fatty liver disease and hepatocirrhosis.

Example 8: Products containing hesperidin or hesperetin

Products containing hesperidin or hesperetin, prepare as follows:

(1) Preparation of tomato ketchup and sauce

Hesperidin or hesperetin add to tomato ketchup or sauce in a quantity in the range of from 0.01 to 5 wt.% to obtain a health-improving tomato ketchup or sauce.

(2) Preparation of products made from wheat flour

Hesperidin or hesperetin added to wheat flour in an amount in the range of from 0.01 to 5 wt.% and prepare bread, muffins, cookies, crackers and noodles using this mixture, to obtain health-improving products.

(3) Preparation of soups and sauces

Hesperidin or hesperetin add to soups and gravies in amount in the range of from 0.01 to 5 weight. % to get a health-boosting soups and gravies.

the e in the interval from 0.01 to 5 wt.% to obtain a health-improving beef.

(5) Preparation of dairy products

Hesperidin or hesperetin add to milk in quantities in the range of from 0.01 to 5 weight. % of this milk is prepared various dairy products such as butter and ice cream.

However, in the preparation of cheeses hesperidin or hesperetin added to the coagulated milk protein; and in the case of making yogurt hesperidin or hesperetin added to the coagulated milk protein after fermentation.

Example 9: Drinks containing hesperidin or hesperetin

(1) Preparation of vegetable juice

From 200 to 10,000 mg of hesperidin or hesperetin add to 1000 ml of tomato or carrot juice to obtain health-improving vegetable juices.

(2) Preparation of fruit juice

From 200 to 10,000 mg of hesperidin or hesperetin added to 1,000 ml of Apple or grape juice to obtain health-improving fruit juices.

(3) Preparation of carbonated beverages

From 200 to 10,000 mg of hesperidin or hesperetin add to 1000 ml of Coca-Cola or Pepsi-Cola to obtain health-improving carbonated drinks.

Although the present invention has been described in respect of the above concretamente, that fall within the scope of the invention as defined by the attached claims.

1. The use of hesperidin or hesperetin as an active ingredient of a composition for the prevention or treatment of lipodystrophy liver or cirrhosis of the liver in mammals.

2. Application under item 1 where the mammal is man.

3. Application under item 1, where the specified composition selected from the group consisting of pharmaceutical compositions, food compositions and drink.

4. Application under item 3, where an effective amount of hesperidin or hesperetin contained in the pharmaceutical composition is in the range from 0.1 to 100 mg/kg body weight/day.

5. Application under item 3, where the content of hesperidin or hesperetin in the food composition is 0.01 - 5 weight. %.

6. Application under item 3, where food is meat, chocolates, snacks, candy, pizza, foods prepared from flour of cereals, chewing gum, dairy products, soups, broths, pastes, ketchups, sauces, vitamins or health products.

7. Application under item 6, where the products are prepared from flour of cereals, are bread, muffins, crackers, cookies, biscuits or noodles.

9. Application under item 3, where the content of hesperidin or hesperetin in the compositions of the beverage is in the range from 200 to 10000 mg per 1000 ml of the drink.

 

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The invention relates to biology, and further can be used in medicine and for the stimulation of meiosis of germ cells in mammals

The invention relates to new compounds 2,4-xylidide and m-phenetidine 2-acetylaminofluorene-3-carboxylic acid of General formula 1

,

where Ia) R=2,4-(CH3)2WITH6H3; IB) R=m-C2H5OS6H4that possess anticoagulant activity and can be used in medicine
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