TWI634886B - Compound composition for liver-free side effects with reduced liver fat for treating symptoms of non-alcoholic fatty liver disease (NAFLD) - Google Patents

Abstract

A safe non-side-effect compound composition for treating symptoms of Non-alcoholic Fatty Liver Disease (NAFLD), wherein the compound composition is selected from the group consisting of bioflavonoids At least one or any combination thereof: (-)-Epicetechin-3-gallate, paeoniflorin, isorhamnetin, ethyl eicosapentaenoate, quercetin, phlorizin, (-)-Epigallocetechin, alfalfa Peel, hesperetin, (-)-Epigallocetechin-3-gallate, gallic acid, kaempferol, hesperidin, puerarin, silybin, formononetin, baicalein, ortho-dihydroxy-acid , Astragalus, dihydroxycoumarin, daidzin, glycyrrhizin, 6-gingerol, rutin, glycyrrhizin, phloretin, daidzein, umbelliferone, genistein, (- )-Epicatechin, naringin, neohesperidin, wogonin, capillarazine, isoglycyrrhizin and ursolic acid, for the treatment of non-alcoholic fatty liver disease symptoms. Furthermore, the present invention also provides a use of a novel compound composition for treating non-alcoholic fatty liver disease for the manufacture of a medicament for reducing liver fat and blood fat symptoms.

Description

Compound composition for liver-free side effects with reduced liver fat for treating symptoms of non-alcoholic fatty liver disease (NAFLD)

The present invention relates to a safe non-side-effect compound composition for treating symptoms of non-alcoholic fatty liver disease (NAFLD), in particular to a combination of compounds containing at least one bioflavonoids. The composition of the compound is used to reduce liver fat and blood fat symptoms.

The liver is part of the animal's digestive system and is the main organ for the production and secretion of many digestive juices. The liver also has functions of absorption, metabolism, removal of toxic substances and immune protection. The liver is an important organ of fat metabolism and plays an extremely important role in the process of digestion, absorption, decomposition, synthesis and transportation of fatty foods. The liver is free fatty acid (FFA) in the blood, which will eventually synthesize triglyceride (TG) in the liver and store it, or transport TG out of the liver into the blood circulation in the form of VLDL. . Therefore, once the liver is damaged, it can cause abnormal metabolism and accumulation of lipids (especially triglyceride, TG) in liver cells.

Under normal circumstances, fat accounts for about 3% of the weight of the liver. Clinically, "fatty liver" means that the weight of fat in the liver exceeds 5% of the weight of the liver, or more than 10% of liver cells in the liver tissue section have fat vacuoles. Phenomenon (see Attachment 2 attached). Hepatic steatosis, especially non-alcohol fatty liver disease (non-alcohol fatty liver) Disease, NAFLD), in the past, is considered to be a more benign and reversible disease, so it is less valued, but in recent years, successive studies have found that it may cause liver fibrosis and cirrhosis, even liver cancer, followed by obesity Increase, there is also an increasing trend. Fatty liver can be distinguished from alcoholic fatty liver (AFLD), non-alcohol fatty liver disease (NAFLD) or other diseases such as obesity, diabetic, hyperlipidemia and insulin resistance. Appearance is characterized by fatty metamorphosis or steatosis, steatohepatitis, fibrosis and even cirrhosis. Mild fatty liver refers to less hepatocytes containing fatty degeneration. At 33%, the moderate is between 33-66%, while the severe is more than 66% (see references 3, 9 and 21 attached below).

The main liver diseases in Europe and the United States are due to excessive drinking, because most of the liver diseases are caused by alcohol damage. However, in the past 15-20 years, NAFLD has become the cause of liver function abnormalities in Europe and the United States (see Attached reference 2). Thaler described NAFLD in 1962. In 1980, Ludwig discovered "Non-alcoholic steatohepatitis" (NASH) in a group of obese women with diabetes and hyperlipidemia. In 1986, Schaffner re-emphasized that NASH plays an important role in the mechanism of degeneration of fibrosis in the course of NAFLD (see Attachment 21); until 1998, Day found that 15-50% of NASH patients were derived to varying degrees. The fibrosis (see reference 4 attached below), NAFLD began to receive the attention of clinicians. Today, NASH is not only a stage in the natural progression of NAFLD, but also because of its existence, NAFLD is no longer considered a benign liver disease.

The current NAFLD study in North America, South America, Japan, Northern Europe, Southern Europe, Australia and the Middle East has found a prevalence of 10-39%, and post-mortem pathological anatomical histopathology Among them, its prevalence rate is about 20%, and the accompanying rate of NASH is about 3-18%. Among them, the prevalence of NAFLD among obese people is as high as 57-74% (4.6 times that of normal people). Among them, 20-25% have NASH lesions, and those with cirrhosis also account for 2-3%; about 75% of type 2 diabetes suffer from NAFLD. In the past 30 years in Taiwan, due to the improvement of economic environment and diet, the prevalence of NAFLD has also increased year by year. In recent years, the prevalence rate is about 12.37%, which is not far from Japan's 9-13%, among which non-obese people The prevalence rate is about 10%, while the prevalence of morbidly obese people (BMI greater than 30) is as high as 80% (see references 15 and 23 below).

The pathogenesis of NAFLD is quite complicated, and the pathogenesis of NASH is still unclear. The Day and James in the United Kingdom proposed a two-hit hypothesis based on a large number of clinical and animal experiments. The first hit Fatty liver appears after the second hit, and steatohepatitis occurs after the second hit. The first hit is due to excessive accumulation of fat in the liver, due to obesity, hyperlipidemia, diabetes and insulin resistance (IR); the second hit is caused by oxidative stress and Reactive oxygen species (ROS) in the mitochondria cause lipid peroxidation on the membrane of the liver, releasing pro-inflammiatory cytokines and free radicals, and activating stellate cells to produce fibrosis, leading to liver cell disease. Necrosis (please refer to the attached references 4, 5 and 19) (first figure). The pathogenesis of NASH is mainly related to triglyceride lipid peroxidation, insulin resistance, oxidative stress, reactive oxygen species (ROS) reaction, increasing the peroxidation of lipids in hepatocytes, or cytokine and liver cytochrome P450 ( The enhancement of cytochrome P450, such as CYP2E1 and CYP4A, leads to a series of autoimmune interactions (see Attachment 12 below).

NAFLD virulence factors are (1) insulin resistance: 98% NAFLD patients With insulin resistance, insulin inhibits lipolysis. When peripheral tissues are resistant to insulin, it promotes the decomposition of triglycerides in hepatocytes and inhibits the esterification of free fatty acids, resulting in increased free fatty acids and liver fatty lesions. (2) Obesity: 70-100% NAFLD patients have obesity. Adipocytokines (such as TNF-α, leptin, adiponectin, resistin and interleukin-6) have functions of regulating energy metabolism and immunity. Many reports indicate that abnormal secretion of adipocytokines will promote liver inflammation and fibrosis in patients with NAFLD, aggravating liver disease. (3)Oxidative stress: mitochondria is the main source of ROS in cells. Due to insulin resistance, NAFLD patients have high free fatty acids, leading to the production of free radicals, destroying the mitochondrial respiratory chain and damaging the mitochondria. , causing hepatotoxicity (please refer to the attached references 5 and 7), so oxidative damage has been considered as a secondary hit that causes NAFLD to deteriorate into NASH and cirrhosis.

The formation of fatty liver is mostly the long-term intake of excessive animal fat, protein, carbohydrates, excess calories in the body converted into fat accumulation, leading to obesity and fatty liver. The GOT/GPT values in the blood of patients with fatty liver may be normal. To correctly diagnose fatty liver, it is necessary to pass the ultrasound examination of the abdomen. The current accuracy rate is over 97%.

According to the 2009 FDA announcement, there is currently no ideal therapeutic drug for NAFLD. The treatment policy is mainly to improve the potential risk factors or use drugs to control the progress of chronic diseases. It is recommended to treat the disease according to the cause of fatty liver, such as: overweight Fatty liver caused by moderate weight loss; alcoholic fatty liver, need to rely on alcohol and a balanced diet to improve; long-term exposure to liver damage to chemicals or drugs, fatty liver, you must stop using these drugs immediately; Fatty liver caused, such as hepatitis C, diabetes, high blood fat, etc., must start at the source, treat C liver, blood sugar, control blood lipids; if the body factors cause triglyceride (TG) is too high, you must take hypolipidemic Drugs can improve fatty liver. Medical research shows that liver and blood Too high TG is a risk factor for NAFLD, and it is also an important risk factor for chronic diseases such as coronary heart disease and hypertension. Therefore, how to reduce TG in liver and blood has become a chronic disease such as prevention of NAFLD, heart disease and hypertension. Important topic.

However, in the clinic, serum TG and cholesterol drugs are often accompanied by side effects such as hepatotoxicity, myalgia, myositis, rhabdomyolysis and other myopathy. Among the hypolipidemic drugs, muscle toxicity is the most important side effect. In particular, Statins has the highest rate of muscle toxicity, followed by Fibric acid. Current research confirms: (1) Most of the Statin drugs such as Atorvastatin (Lipitor®), Rosuvastatin (Crestor®), Pravastatin, Fluvastatin (Lescol XL®) and Simvastatin (Zocor®) do have hepatotoxicity. Risk, leading to cholestasis in the liver, causing jaundice or drug-induced liver injury, even cirrhosis and liver failure; (2) lipid-lowering drugs have a "lip-repellent" effect, can lipids in the blood "Driven" to the liver, and there is already fat accumulation in the liver, so it is difficult to treat a large amount of influx of fat, fat will accumulate in the liver, making fatty liver more serious, such as: Fenofibrate (Lipanthyl®), Fibric acid derivatives such as Gemfibrozil (Lopid®), it is known that hypolipidemic drugs are not suitable for the treatment of NAFLD.

Bioflavonoids are low-molecular natural plant phenolic compounds that exhibit a wide variety of pharmacological activities with low toxic side effects and are widely found in fruits, vegetables, grains, rhizomes, flowers, teas and red wines. Bioflavonoids have antioxidant, anti-inflammatory, lower blood cholesterol and blood pressure, prevent cardiovascular diseases, and fight cancer.

It can be seen that the above-mentioned conventional hypolipidemic and cholesterol drugs have their defects, and are not suitable for patients with NAFLD accompanied by hyperlipidemia, which is not a good designer, and needs to be improved. In view of the above-mentioned conventional use of hypolipidemic and cholesterol drugs to treat patients with improved NAFLD, the inventors of the present invention have Many areas for improvement are thoughts of improvement and innovation, painstaking research, bioflavonoids as a screening group, and oleic acids to induce fat accumulation in human liver cell line Hep G2 as a NAFLD hepatocyte model. Successfully developed and completed a combination of compounds that can reduce the fat in the liver, blood and improve the symptoms of non-alcoholic fatty liver disease (NAFLD), and understand the health and well-being of the world to provide natural compound compounds. .

references

1. Brunt EM, Janney CG, Di Bisceglie AM, Neuschwander-Tetri BA and Bacon BR. (1999) Nonalcoholic steatohepatitis: a proposal for grading and staging the histological lesions. Am J Gastroenterol 94: 2467-2674.

2. Clark JM and Diehl AM. (2003) Nonalcoholic fatty liver disease: an underrecognized cause of cryptogenic cirrhosis. JAMA 289: 300-304.

3. Carlisle R, Galambos JT, Warren WD. (1979) The relationship between conventional liver tests, quantitative function tests, and histopathology in cirrhosis. Dig Dis Sci 24: 358-362.

3. Cua IH. & George J. (2005) Non-alcoholic fatty liver disease. Hosp Med 66: 106-111.

4. Day CP and James OFW. (1998) Steatohepatitis: a tale of two ‘hits’? Gastroenterology 114: 842-845.

5. Dowman JK, Tomlinson JW and Newsome PN. (2010) Pathogenesis of non-alcoholic fatty liver disease. Q J Med 103: 71-83.

6. Food and Drug Administration (2003) Guidance for Industry: Pharmacokinetics in Patients with Impaired Hepatic Function: Study Design, Data Analysis, and Impact on Dosing and Labeling. US Department of Health and Human Services, FDA, Center for Drug Evaluation and Research, Center for Biologics Evaluation and Research.

7. French SW. (2001) Intragastric ethanol infusion model for cellular and molecular studies alcoholic liver disease. J Biomed Sci 8:20-27.

8. Gauthier MS, Favier R and Lavoie JM. (2006) Time course of the development of non-alcoholic hepatic steatosis in response to high-fat diet-induced obesity in rats. Br J Nutr 95: 273-281.

9. Guo HX, Liu DH, Ma Y, Liu JF, Wang Y, Du ZY, Wang X, Shen JK and Peng HL. (2009) Long-term baicalin administration ameliorates metabolic disorders and hepatic steatosis in rats given a high-fat Act. Pharmacol Sin 30: 1505-1512.

10. Herold C, Heinz R, Niedobitek G, Schneider T, Hahn EG, Schuppan D. (2001) Quantitative testing of liver function in relation to fibrosis in patients with chronic hepatitis B and C. Liver 21: 260-265.

11. Hu OY, Tang HS, Chang CL. (1994) The influence of chronic lobular hepatitis on pharmacokinetics of cefoperazone--a novel galactose single-point method as a measure of residual liver function. Biopharm Drug Dispos 15: 563-576.

12. Hu OY, Hu TM and Tang HS. (1995) Determination of galactose in human blood by high-performance liquid chromatography: comparison with an enzymatic method and application to the pharmacokinetic study of galactose in patients with liver dysfunction. J Pharm Sci 84 : 231-235.

13. Hu OY, Tang HS, Sheeng TY, Chen TC and Curry SH. (1995) Pharmacokinetics of promazine in patients with hepatic cirrhosis--correlation with a novel galactose single point method. J Pharm Sci 84: 111-114.

14. Keiding S, Johansen S, Tonnesen K. (1977) Kinetics of ethanol inhibition of galactose elimination in perfused pig liver. Scand J Clin Lab Invest 37: 487-494.

15. Keiding S, Johansen S, Winkler K. (1982) Hepatic galactose elimination kinetics in the intact pig. Scand J Clin Lab Invest 42: 253-259.

16. Knodell RG, Ishak KG, Black WC, Chen TS, Craig R, Kaplowitz N, Kiernan TW and Wollman J. (1981) Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. Hepatology 1: 431-435.

17. Leclercq IA, Farrell GC and Field J. (2000) CYP2E1 and CYP4A as microsomal catalysts of lipid peroxides in murine nonalcoholic steatohepatitis. J Clin Invest 105: 1067-1075.

18. Lindskov J. (1982) The quantitative liver function as measured by the galactose elimination capacity. I. Diagnostic value and relations to clinical, biochemical, and histological findings in patients with steatosis and patients with cirrhosis. Acta Med Scand 212: 295- 302.

19. Lin SC, Lin YH, Chen CF, Chung CY and Hsu SH. (1997) The hepatoprotective and therapeutic effects of propolis ethanol extract on chronic alcohol-induced liver injuries. Am J Chin Med 25: 325-332.

20. Liu CH, Huang MT and Huang PC. (1995) Sources of triacylglycerol accumulation in livers of rats fed a cholesterol-supplemented diet. Lipids 30: 527-531.

21. Mulhall BP, One JP and Younossi ZM. (2002) Non-alcoholic fatty liver disease: an overview. J Gastroenterol Hepatol 17: 1130-1143.

22. Nanji AA. (2004) Another animal model for nonalcoholic steatohepatitis: how close to the human condition? Am J Clin Nutr 79: 350-351.

23. Nistor A, Bulla A, Filip DA and Radu A. (1987) The hyperlipidemic hamster as a model of experimental atherosclerosis. Atherosclerosis 68: 159-173.

24. Sanyal AJ, Campbell-Sargent C and Mirshahi F. (2001) Nonalcoholic steatohepatitis: association of insulin resistance and mitochondrial abnormalities. Gastroenterology 120: 1183-1192.

25. Sass DA, Chang P and Chopra, KB. (2005) Nonalcoholic fatty liver disease: a clinical review. Dig Dis Sci 50: 171-180.

26. Schaefer EJ. (1997) Effects of dietary fatty acids on lipoproteins and cardiovascular disease risk: summary. Am J Clin Nutr 65: 1655S-1656S.

27. Schaffner F and Thaler H. (1986) Nonalcoholic fatty liver disease. Prog Liver Dis 8: 283-298.

28. Sullivan MP, Cerda JJ, Robbins FL, Burgin CW and Beatty RJ. (1993) The gerbil, hamster, and guinea pig as rodent models for hyperlipidemia. Lab Anim Sci 43: 575-578.

29. Tang HS and Hu OY. (1992) Assessment of liver function using a novel galactose single point method. Digestion 52: 222-231.

30. Tominaga K, Kurata JH and Chen YK. (1995) Prevalence of fatty liver in Japanese children and relationship to obesity. Dig Dis Sci 40: 2002-2009.

31. Zhang Z, Wang H, Jiao R, Peng C, Wong YM, Yeung VS, Huang Y and Chen ZYW (2009) Choosing hamsters but not rats as a model for studying plasma cholesterol lowering activity of functional foods. Mol Nutr Food Res 53: 921-930.

32. Zou Y, Li J, Lu C, Wang J, Ge J, Huang Y, Zhang L and Wang Y. (2006) High-fat emulsion-induced rat model of nonalcoholic steatohepatitis. Life Sci 79: 1100-1107.

33. Zhang D, Xie L, Jia G, Cai S, Ji B, Liu Y, Wu W, Zhou F, Wang A, Chu L, Wei Y, Liu J and Gao F. (2011) Comparative study on antioxidant capacity of Flavonoids and their inhibitory effects on oleic acid-induced hepatic steatosis in vitro. Eur J Med Chem 46: 4548-4558.

It is an object of the present invention to provide a composition for treating a symptom of Non-alcoholic Fatty Liver Disease (NAFLD), wherein the compound composition is for reducing liver fat and blood fat symptoms.

Wherein the compound composition is selected from the group consisting of bioflavonoids, which can reduce liver fat, blood lipids and treat non-alcoholic fatty liver disease (NAFLD). symptom.

In order to achieve the above object of the invention, a compound composition having reduced liver fat for treating symptoms of Non-alcoholic Fatty Liver Disease (NAFLD), the present invention first (1) screening with HepG2 Cell can effectively reduce three Glycerol, cholesterol bioflavonoids, and study the effect of bioflavonoids on the hypolipidemic effect of HepG2 Cell; (2) further according to the Department of Health's "Health Food Regulation Method for Regulating Blood Lipid Function" The specification was carried out, and the Syrian hamster (Syrian hamster), which is most consistent with the human liver lipid metabolism, was tested and induced by Syrian hamster to induce non-alcoholic fatty liver animal models, and then fed with bioflavonoids with hypolipidemic and blood lipid functions. Syrian hamster of alcoholic fatty liver, and after a 12-week trial, blood was taken from the liver to analyze the lipid content of the non-alcoholic fatty liver Syner hamster, and bioflavonoids were observed to reduce liver fat and blood fat for the treatment of fatty liver. influence; in addition to the use of conventional liver function markers glutamic grass Acid aminotransferase (AST), glutamate pyruvate amino turn enzyme (ALT), the present inventors further single point method using galactose (Galactose single point method, GSP) for rat, hamster remaining liver function Quantitative analysis, GSP is a simple and quantitative method for the determination of liver function, which is contained in the US Food and Drug Administration (FDA) Guidance for Industry, the Ministry of Health of the Republic of China, the Department of Health, "Pharmacokinetics of patients with hepatic insufficiency Test Benchmarks, and textbook "Applied Biopharmaceutics &Pharmacokinetics".

A liver-free side effect compound composition for treating the symptoms of Non-alcoholic Fatty Liver Disease (NAFLD), wherein the compound composition is selected from the following bioflavonoid compounds (bioflavonoids) group consisting of: (-)-Epicetechin-3-gallate, Isorhamnetin, Phloridzin, (-)-Epigallocetechin, Quercitrin, Hesperetin ( Hesperetin), Gallic Acid, Kaempferol, Hesperidin, Formononetin, Baicalein, Nordichydroguaiaretic acid, Oleanolic Acid, Daidzin, Glycyrrhizin (Glycyrrhizin), 6-Gingerol, Liquiritin, Phloetin, Daidzein, Umbelliferone, Genistein (Genistein) ), (-)-Epicatechin, Naringin, Neohesperidin, Wongonin, Capillarisin and Isoliquritigenin.

Wherein the compound composition is selected from the group consisting of the following bioflavonoid compounds and defines an effective dosage of the composition: (-)-Epicetechin-3-gallate is 15 to 150 mg, isoflavone The content of Isorhamnetin is 8~80 mg, the content of Phloridzin is 15-140 mg, the content of (-)-Epigallocetechin is 10~100 mg, and the content of Quercitrin is 15-150 mg. Hesperetin is 10~100 mg, Gallic Acid is 5~60 mg, Kaempferol is 10~100 mg, Hesperidin The content of 20~200 mg, Formononetin is 10~100 mg, Baicalein is 10~90 mg, and Nordichydroguaiaretic acid is 10 ~100 mg, the content of Oleanolic Acid is 15~150 mg, the content of daidzin is 10~100 mg, and the content of glycyrrhizin is 30~270 mg, 6- The content of 6-Gingerol is 10~100 mg, and the content of Liquiritin is 14~140 mg. The content of Phloretin is 10~100 mg, the content of daidzein is 10~100 mg, the content of Umbelliferone is 5~50 mg, and genistein (Genistein) The content of 10~100 mg, (-)-Epicatechin is 10~100 mg, the content of Naringin is 16-160 mg, and the content of Neohesperidin is 2-20 mg. The content of Wongonin is 10~90 mg, the content of Capillarisin is 10~100 mg and the content of Isoliquritigenin is 10~100 mg. Wherein the compound composition is for reducing liver fat and blood fat symptoms; wherein the compound composition is administered in the form of a gel, a spray, a pasty, a lozenge or a dispersible tablet; wherein the compound composition It is included in medical kits, kits or patient packs.

A composition of the above composition for use in the preparation of a non-alcoholic fatty liver disease (NAFLD) disease.

Wherein the medicament is administered in the form of a gel, a spray, a pastille, a lozenge or a dispersible tablet; wherein the medicament is included in a pharmaceutical pack, kit or patient pack.

The first picture shows the second strike hypothesis of NAFLD pathogenesis.

The second picture shows the hamster feeding (HUCHE025, HUCHE033 and HUCHE086 test group test group) / no feeding (blank control, high fat control, blood lipid control group) test substance after 12 weeks, liver weight / body weight ratio ( mg / g hamster) The amount of change. All data were expressed as Mean±SD (n=15), and *** indicates that each test group was compared with the high-fat control group and p < 0.005.

The third picture shows the change of triglyceride and cholesterol in the liver after 12 weeks of test substance feeding by hamsters (HUCHE025, HUCHE033 and HUCHE086 test group)/no feeding (blank control, high fat control, blood lipid control group) . All data were expressed by Mean±SD (n=15). ***, **, and * indicated that each test group was compared with the high-fat control group and the hypolipidemic control group, p <0.005, p <0.01, and p <0.05. .

The fourth panel shows the amount of change in the galactose single point method (GSP) measured after 12 weeks of test substance feeding by hamsters (HUCHE025, HUCHE033 and HUCHE086 test groups)/no feeding (blank control, high fat control, blood lipid control group). All data were expressed as Mean±SD (n=15), *** indicates that the high-fat control group and the blank control, the hypolipidemic control were compared with the test groups, p <0.005, ** indicates that the test group was compared with the hypolipidemic control group. After p <0.01.

The fifth picture shows the hamster feeding (HUCHE025, HUCHE033 and HUCHE086 test group) / no feeding (blank control, high fat control, blood lipid control group) liver tissue sections after 12 weeks of test substance.

The invention is further illustrated by the following examples, which are intended to be illustrative only and not to be construed as limiting.

Example 1 Screening of bioflavonoids effective to reduce triglycerides by using HepG2 Cell

Test cell strain

1.1 Test cell strain: The test cell strain is Hep G2 cells of human hepatoma cells, and the oleic acid-induced Hep G2 cell line is used to form fatty liver cells, and then screening for compounds which can effectively reduce TG in hepatocytes and culture medium. .

1.2 Cell culture technology

Preparation of cell culture medium: liquid medium Dulbecco's Modified Eagle's Medium [DMEM]-(high glucose cat No.12800017), dissolved in DMEM medium dissolved in 1400mL milli-Q water, stirred to dissolve and added 2g HEPES, and weighed 4g soudium bicarbonate powder In 400 mL milli-Q water, stir to dissolve and add to the mixed dissolved liquid medium, add milli-Q water to 2000 mL, and adjust the pH value to 7.3 ± 0.05 with 5N HCl. In a sterile dust station, dispense 0.2 μm sterile filter membrane medium, dispense 500ml sterile serum bottle per 450mL sterile medium, store at 4 ° C (called Medium A); Medium B is 450mL Medium A add 50mL to deactivate the tire Fetal bovine serum (FBS), 5mL sodium pyruvate 100mM, 5mL penicillin (100U/mL) & streptomycin (100U/mL) and 5mL MEM non-essential amino acids solution; Medium C is 450mL Medium A added 5mL sodium pyruvate 100mM 5mL penicillin (100U/mL) & streptomycin (100U/mL) and 5mL MEM non-essential amino acids solution 100x; Medium D is added to oleate/albumin complex for Medium B, and the preparation of oleate/albumin complex is based on Van Harken245 et al. The method published in 1989. Bovine serum albumin (5 g) was dissolved in 25 mL of Medium A, and the pH was adjusted to 7.4 with 1 N NaOH. After completion, the solution was placed in an ice bath at 0 °C. The oleic acid was dissolved in 95% ethanol 50 mL, titrated with 1 N NaOH to the end of the phenolphthalein titration, and the ethanol was blown dry with nitrogen. The sodium oleate formed in this step was dissolved in 37 ° C Medium A. Finally, the oleate/albumin complex solution was prepared by stirring the BSA solution into the sodium oleate solution; Medium E was dissolved in Medium C by silymarin; and Medium F was dissolved in Medium C as a test drug. Medium A~F was stored at 2~8 °C and used in a 37 °C water bath before warming up.

Deactivation of fetal calf serum: 500 mL of commercially available fetal bovine serum is placed in a water bath that has been heated to 56 ° C for 30 minutes. Immediately after the sputum time, the serum is placed in crushed ice to cool down, and after a few minutes, it is Transfer to a sterile clean room and dispense in a 50mL centrifuge tube and store in a -20 °C refrigerator.

Frozen cells: When cells are cultured, the cells are attached to the bottom of the culture dish. When the cells are almost full, the cells must be collected, and then the cells are frozen or colonized into new culture flasks for subculture and screening experiments. Cell freezing is performed in a 75T flask full of cells. The cells are first dispersed by trypsin-EDTA. Trypsin is a trypsin, which acts to decompose the attached protein of the cell and the bottle wall. EDTA is a chelating agent, which acts to remove Ca2+. The Mg2+ ion, EDTA and trypsin work together to cause the attached cells to fall off the wall of the bottle. Leave 2 mL to continue to multiply. The remaining cells are centrifuged at 1200-1500 rpm for 5 minutes to remove the supernatant. Then add 4 mL of freezing medium (10% DMSO + 90% FBS) and dispense into 3 tubes of antifreeze tubes. Put the antifreeze tube into the freezer operation box (with 2-propanol), then put the freeze operation box into the -80 °C refrigerator for at least 24 hours; then quickly move the antifreeze tube to the liquid nitrogen tank or -130 °C refrigerator in.

Thaw the cells: Remove the frozen cells on a floating pad and return to the temperature in a 37 ° C water bath. The temperature should not be more than 3 minutes. After adding 11 mL of medium B to the 75T flask and mixing with the warmed frozen cells, the frozen cells were cultured in a 75T flask. After thawing, change the medium every other day, because 1% DMSO will affect the growth of the cells, and then change the medium every 2 to 3 days depending on the cell growth.

Subculture of cells: When subcultured each culture flask, the old culture solution should be aspirated first, and the cells should be washed twice with 0.1 M phosphate buffer (PBS buffer, PBS, pH=7.4). 0.1 M PBS buffer is It is prepared by mixing 190mL soln'A plus 810mL soln'B and 1000mL milli-Q water. Soln'A is made up of 31.2g NaH2PO4.2H2O dissolved in 1000mL milli-Q water; son'B is 35.6g NaHPO4.2H2O dissolved in 1000mLmilli -Q water is blended. After adding 2mL trypsin-EDTA solution, put the culture flask into the 37 ° C incubator for about 3 minutes. After the cells are detached from the bottle wall, add appropriate amount of medium B to stop trypsin (because medium B contains fetal bovine serum, and serum Contains trypsin inhibitor, which can terminate trypsin). After mixing, extract 100 μl of cell culture medium, calculate the cell number with 0.4% trypan blue, then add fresh medium and transfer to new culture according to the dilution ratio (1:6). The flask was incubated in a 5% CO2 incubator at 37 °C.

Cell Count and Survival Test: The step of the survival test is dye exclusion, which uses 0.4% trypan blue to infiltrate into dead cells to develop color. The living cells are not able to penetrate because the cell membrane is intact, and the color does not appear. Mix 100 μl of cell suspension with an equal volume of 100 μl 0.4% trypan blue. A small mixture (about 20 μl ) was added from the groove above the hemocytometer tray, and the cover plate was covered under a 100-fold inverted microscope. The living cells were not stained, and the dead cells were blue.

2. Test screening method

2.1 Cell group: The HepG2 cell line was divided into 6 groups: the Blank group did not do any treatment, the Blank+DMSO group did not do any treatment plus DMSO (the solvent for screening drugs), and the Control group induced the formation of fat with oleic acid. Hepatocytes, Vehicle group, fatty liver cells plus DMSO, Positive control group, fatty liver cells plus silymarin as positive control group, Treatment group, fatty liver cells plus screening drugs.

2.2 Oleic acid-induced HepG2 cell line to form fatty liver cells: 15×106 HepG2 cells were cultured in 25T with medium B, cultured in an incubator for 24 hours, cells were attached to the bottom of 25T flask, and then used with medium C (serum-free) Culture medium) culture for 24 hours, and finally replace the medium D containing oleate/albumin complex culture medium for 48 hours to successfully induce the formation of fatty liver cells. After the formation of fatty liver cells, add silymarin or screening drugs to screen for effective reduction of fatty liver cells. And an effective therapeutic compound of TG in the culture solution.

3. Sample analysis

The test and analysis items include: measuring the content of TG in the cells and the culture medium, quantifying the intracellular protein, and calculating the relative content of intracellular TG.

3.1 Measurement of TG content in cells and culture medium: 15×106 cells were cultured in 25 T with medium B, and the pure components of different doses (1, 5, 10, 25 and 100 μM ) were treated. After 72 hours, the cells were treated with PBS. washed twice, after adding Trypsin-EDTA 0.5mL for 3 minutes, then add 2mL of PBS, the cells were scraped, move 15mL centrifuge tubes, then the cells will be shattered with ultrasonic, draw 20 μ L cells was measured Intracellular protein content. Transfer the remaining cell culture medium to a 15 mL centrifuge tube, add 9 mL of extract (chloroform:methanol=2:1), mix it evenly with shaking, add 0.73% NaCL 1 mL, and centrifuge at 3000 rpm for 5 minutes at 4 °C. The layer was taken up to another small glass test tube, and the solution was blown dry with nitrogen until the TG content was measured. Add 18 mL of the extract (chloroform:methanol=2:1) to the culture solution, mix it evenly into the chyle, add 0.73% NaCL 1 mL, and centrifuge at 3,000 rpm for 5 minutes at 4 °C to separate the layers and absorb organic The layer portion of the solution was transferred to another glass test tube, and the solution was blown dry with nitrogen until the subsequent measurement of the TG content.

3.2 Determination of TG content: The TG assay was performed using a commercially available combination reagent (Randox). Diluted with 5 μ L were collected standard sequences (10,20,30,40,60 and concentrations of 80 μ g / mL), the sequence of diluted standards and samples were completely dry with nitrogen are added 500 μ L The enzyme reagent was mixed well and reacted at room temperature for 30 minutes. TG in standards and samples produces H2O2 through the action of enzymes such as lipase, Glycerol-kinase and Glycerol-3-phosphate oxidase; H2O2 reacts with 4-aminophenazone and 4-chlorophenol to produce pink quinoneimine via peroxidase. The absorbance of the standard and the sample at OD525 was measured using a spectrophotometer within 60 minutes, and a standard curve was drawn. The sample can be used to derive the sample concentration using the standard curve.

Quantitative protein content of 3.3 cells: Cells were commercially available Bio-rad protein assay kit quantifying formulated to BSA (bovine serum albumin) standards, concentrations include 50, 200, 400, and 600 μ g / mL. Take 20 μ L above cell suspension (n = 3), places the control group cells was 20 μ L, 0.85% sodium chloride solution substituents, each tube was added 0.55mL Biuret reagent test article (containing 0.75mmol / L Cupric sulfate, 94mmole/L sodium hydroxide, tartrate, iodide carbonate, etc.), uniformly mixed and allowed to stand at room temperature for 10 minutes. Add 25 μL of Folin reagent (Ciocalten's phenol reagent) to each tube, mix well and let stand for 30 minutes at room temperature. The purple blue color is formed, and the absorbance of OD525 is measured by a spectrophotometer. The absorbance of the sample is compared with the standard concentration, and the standard curve is interpolated to obtain the protein concentration of the test product. Calculation of the relative content of intracellular triglyceride The ratio of the TG content obtained above divided by the protein content represents the relative content of TG in the cells.

4. Statistical analysis

All data are expressed as mean ± standard deviation (SD), and the test results are calculated by the single factor analysis of variance (ANOVA) test to determine whether there is a statistically significant difference. Using the Statistical Package of the Social Science program (Version 13, SPSS) Inc.) software was used to calculate; then multiple comparisons were made using the post hoc test for the least significant difference method to confirm significant differences between ethnic groups; the significant difference in ethnic mean was p < 0.05.

5. Test results

5.1 Effects of test substances on the cell fat effect of HepG2 cell

Positive control group

Positive control group (Silymarin) the measured results of TG levels reduced fat HepG2 cells such as Table II, Table III, it is seen from the Table II, when the concentration of Silymarin 100 μ M, HepG2 adipocytes reduced TG levels up to 78 ± 5%, using 100 μM as a screening test to reduce the TG content of HepG2 fat cells, bioflavonoids test substance test concentration.

Screening of test substances can reduce TG content in HepG2 fat cells

At 100 μ M bioflavonoids as compound (Bioflavonoids) screening test substance concentrations tested reduced the measured results of TG levels HepG2 adipocytes as shown in Table III. From the results at test conditions of 100 μ M concentration, compared with the control group The bioflavonoids had different effects on HepG2 adipocytes in different degrees of hepatic TG content, and the best effect was improved by (-)-Epicetechin-3-gallate (95±6%).

Screening of test substances can reduce TG content in HepG2 adipocytes and culture medium

According to the preliminary screening results of 100μM, the suitable bioflavonoids were selected for different concentration tests. The results of reducing the TG content in HepG2 adipocytes and culture medium were as shown in Table 4. The results showed that the test substances were at different concentrations (10 μM). , 5μM, 1μM), compared with the control group, each bioflavonoid compound has different effects on HepG2 adipocytes to reduce TG content in hepatocytes and hepatocyte culture, including quercetin with a concentration of 10 μM. (Quercetin) improved best (95 ± 5%), followed by hesperidin (92 ± 3%).

Example 2 Regulation of blood lipid function - Syrian hamster (Syrian rat)

The experimental design is based on the revised draft of the Regulation of Blood Lipid Assessment for Healthy Foods of the Taiwan Department of Health (amended by the Department of Health, No. 0960403114). The purpose of the study is to investigate the effects of bioflavonoids on the improvement of fatty liver. Lipids with 20% fat and 0.2% cholesterol (Cholesterol, TCHO) account for about 45% of the total calorie diet (High-Fat diet) for 8-12 weeks (see references 8, 9), induce simple fat production Liver animal model, and at the same time take different bioflavonoids (bioflavonoids), evaluate its hypolipidemic effect, observe whether it can improve the clinical symptoms of fatty liver, analyze blood TG, TCHO, AST, ALT, low density Cholesterol (LDL-C) and high density cholesterol (HDL-C) At the end of the experiment, the liver was analyzed for TG, TCHO and pathological tissue sections at the time of sacrifice.

1. Test materials and methods

1.1 Test animals: Select Syrian rats (Syrian hamster), which is the most compatible with human lipid metabolism in the liver, and recommend the test animals recommended by the Department of Health for the "Method for the evaluation of blood lipids in healthy foods". Counting 12 pieces.

1.2 Animal groups: The test animals were randomized into groups. [12 male Sprague-Dawley rats, 6 weeks old, weighing 95 g each] were divided into blank control group (Blank), high fat control group (HFD), hypolipidemic lipid. Control group (PS), bioflavonoids hesperetin (HUCHE025), quercetin (HUCHE033) and three groups of low, medium and high doses of ethyl icosapentate (HUCHE086) test group (L-HUCHE086, M-HUCHE086, H-HUCHE086) The dose intake range of the test product is 0.2-1 times of the daily recommended maximum intake of the human body. The Blank group was normal feed; the HFD group was given high fat diet; the blood lipid lower control group was high fat feed and tube fed Fluvastatin 10 mg/kg/day; the bioflavonoid test group was high fat feed and individual tube fed 100 mg/kg/ Day hesperetin, quercetin, and two different doses of ethyl icosapentate at 24.6, 123, and 246 mg/kg/day, equivalent to 1000 mg of hesperetin, quercetin, and 200, 1000, and 2000 mg of ethyl icosapentate per day for 60 kg of healthy adults.

1.3 Test methods: Blank group normal feed, HFD, PS and different bioflavonoid test group high-fat feed for food, Blank and HFD group once a day to feed the tube ddH2O, PS group once a day feeding tube to feed Fluvastatin, In the test group of different bioflavonoids, hesperetin, quercetin and 2 4.6, 123 and 246 mg/kg/day of three different doses of ethyl icosapentate were administered once a day for 12 weeks. Before the start of the experiment, the biochemical functions of liver injury were detected by eye socket or cardiac blood sampling at 4 and 8 weeks after the start of the test: AST, ALT, TG, TCHO, LDL-C, HDL-C. Finally, at the end of the 12th week, all the sacrifices were made after weighing. The liver biochemical function was examined by eye socket or cardiac blood sampling, and the liver specimens were taken by caesarean section. The hepatic weight/body weight ratio was compared after weighing, and the maximum right lobe liver was cut into two pieces. The tissue block of about 1 cm cube was fixed in 10% neutral formalin solution, and the paraffin-encapsulated sections were subjected to H&E staining for histopathological observation. In addition, the remaining livers were frozen and tested for TG and TCHO levels in the liver. Using a FDA and Taiwan Department of Health, it is recommended to measure the liver function of each group of animals by the Galactose single point (GSP) method for clinical use. At the end of the 12-week trial, the animal weight per kilogram. 0.5 g of galactose solution (GSP® 0.4 g/mL) was administered via intravenous administration, and about 0.5 ml of whole blood was taken as "Ganeng filter paper" 60 minutes after the administration to evaluate the liver function of the hamster. The higher the GSP value, the worse the residual function of the liver.

2. Test and analysis project:

2.1 Serum: Determination of biochemical functions related to liver injury

(1) AST

(2) ALT

(3) Triglyceride (TG)

(4) Cholesterol (TCHO)

(5) LDL-C

(6) HDL-C

(7) Liver weight/weight (%)

2.2 Detection of liver function biochemical index and blood lipids: All hamster blood samples were allowed to stand at room temperature for 1 hour to cause coagulation. Serum 13 was separated by centrifugation at 15,700 xg for 5 minutes at 4 °C in a refrigerated centrifuge. The liver biochemical index, such as AST, ALT, TG, Cholesterol, etc., is measured by an automatic blood biochemical analyzer, and the principles of AST and ALT detection are based on the standard methods of international federal clinical chemistry ^9,10 .

2.3 Histopathological tissue section: At the end of the 12-week trial, all hamsters were sacrificed. A piece of tissue of about 1 cm cube was cut from the largest right lobe liver and placed in 10% neutral fumarin for fixation. Ethanol (30, 50, 70, 95, 99.5%) and xylene were subjected to a dehydration and transparency step, then xylene was replaced with a hot paraffin solution, and finally the tissue was embedded in a paraffin solution. The completed paraffin specimens were cut into 5 μm paraffin sections using a microtome, and the sections were adhered to a clean glass slide and dried at 37 ° C for further H&E staining.

2.4 H&E (hematoxylin and eosin stain) staining method: The liver tissue sections were placed in xylene for 30 minutes for dewaxing, and then placed in 99.5, 95, 70, 50 and 30% ethanol for 30 minutes each time for rehydration, and then soaked. It can be dyed after 10 minutes of distilled water. The hematoxylin was first soaked for 30 seconds, and then washed with distilled water for several minutes, then stained with eosin for 2-5 minutes, and washed with distilled water for several minutes. After the dyeing process is completed, the dehydration process is carried out, and placed in 50, 70, 95 and 100% alcohol for 30 seconds, respectively, and then transparently treated with xylene twice, and finally sealed with a sealing rubber.

2.5 Histopathological observation: In order to observe the liver damage, liver cell damage, fat accumulation, necrosis or fibrosis, the liver tissue was H&E stained to assess the degree of liver fat accumulation. In order to avoid observing subjective deviations, all histopathological sections were cut from the same position of the largest right lobe liver and then pathologically stained. As for the semi-quantitative analysis of pathology, a double-blind analysis should be performed by a human or veterinary pathologist. All the sections are scored and compared without knowing the design of the experiment (Total HAI-score, see Knodell RG, Ishak KG, Black WC, Chen TS, Craig R, Kaplowitz N, Kiernan TW and Wollman J. (1981) Formulation and application of a numerical scoring system for assessing histological activity in asymptomatic chronic active hepatitis. Hepatology 1:431-435) Finally, the statistical analysis method was used to analyze the differences of each group.

3. Statistical analysis: All experimental data were expressed as mean±SD. Statistical analysis was performed using ANOVA (analysis of variance). The liver biochemical values of the three groups of different doses before and after the test were compared. Is it statistically significant? Significant differences (p < 0.05) were evaluated to determine whether the test sample had the function of protecting the liver or reducing one of the risk factors.

4. Test results

4.1 Effects of test substances on animal body weight and feed intake

The results in Table 5 show that after 12 weeks of testing, blank control group, high fat control group (HFD), hypolipidemic control group (PS), bioflavonoid test group (HUCHE025, HUCHE033, H-HUCHE086, M- HUCHE086 and LHUCHE086), there was no statistically significant difference in animal weight, weight gain and daily feed intake between the groups (p>0.05).

^a : All data is expressed in Mean ± SD (n = 15).

The results of the second graph showed that after 12 weeks of testing, the high-fat control group was significantly higher than the blank control (p<0.005) in the liver weight/weight ratio (mg/g hamster) analysis, except HUCHE033, L-HUCHE086 test. Outside the group, the ratios of the other three groups of HUCHE025, H-HUCHE086 and M-HUCHE086 were significantly lower than those of the high-fat control group (p<0.005).

4.2 Safety information of test substances

The results in Table 6 show that after 12 weeks of testing, blank control group (Blank), high fat control group (HFD), hypolipidemic control group (PS), bioflavonoid test group (HUCHE025, HUCHE033, H-HUCHE086, M- HUCHE086 and LHUCHE086), at week 0, during the 4th, 8th, and 12th week of the test substance, except for the H-HUCHE086 group (dose 246mg/kg/day) during the 8th week of the test, due to the galactose single point method (GSP) analysis caused 1 death, the other did not have any abnormal symptoms; 12 weeks of trial, anatomical examination of all animals did not observe any visual changes caused by the test substance, in the administration of different bioflavonoid test substances No animal death occurred during the test period. After the test, the animal was sacrificed and no lesions were observed due to the test substance. The clinical symptoms are present, so the test substance is safe.

4.3 Effect of test substances on serum lipids

n/n: Total number of abnormal or dead animals observed/Total number of animals examined n'/n': total number of animals observed to be abnormal or dead Number of abnormal or dead animals observed/Number of animals that survived the study period

The results in Table 7 show that after 12 weeks of testing, in the comparative analysis of serum lipid-related biochemical values, after 12 weeks of test, the results showed that bioflavonoids HUCHE025, HUCHE033 and three groups of different doses of HUCHE086 test group, blood TG, TCHO and The LDL-C content was higher in the lipid control group (p<0.005), and the HDL-C was significantly higher in the M-HUCHE086 and H-HUCHE086 groups than in the other experimental groups (p<0.005); the TCHO/HDL-C ratio was compared. HUCHE025, HUCHE033 and three groups of HUCHE086 test groups were significantly lower than the HFD group (13.5±5.9, p<0.005); the ratio of LDL-C/HDL-C was the lowest (0.5±0.1) in the H-HUCHE086 test group. The HUCHE025 (0.8±0.3) and M-HUCHE086 (0.9±0.4) test groups were significantly lower than the high-fat control group (3.5±1.5, p<0.005) and the hypolipidemic control group (1.7±0.5, p<0.005). ).

All data are expressed in Mean ± SD (n = 15).

4.4 Effect of test substances on liver lipids

The results of the third graph show that after the 12-week test period, the liver TG, TCHO content analysis, The HUCHE025, M-HUCHE086 and H-HUCHE086 test groups were significantly lower than the high-fat control group and the hypolipidemic control group (p<0.005), followed by the HUCHE033 test group (p<0.01), while the L-HUCHE086 test group was slightly lower. The high-fat control group (p<0.05) was not statistically different from the hypolipidemic control group.

4.5 Effects of test substances on liver residual function and pathological tissue analysis

The results of the third graph showed that after the 12-week test period, the results of residual liver function analysis showed that the GSP of the high-fat control group (603±75) was significantly higher than that of the blank control group (300±47) and the hypolipidemic control group ( 411±99), HUCHE025 test group (289±75), HUCHE033 test group (366±63) and H-HUCHE086 test group (311±41) (p<0.005), while HUCHE025 and HUCHE086 test group were also significantly lower than drop. The blood lipid control group (p<0.01) was no different from the normal control group, indicating that the liver function of HUCHE025, HUCHE033 and H-HUCHE086 hamsters was better than that of the high-fat control and hypolipidemic control group, and also indicated that the selected bioflavonoids had The effect of improving fatty liver or protecting liver function; the improvement by HUCHE086 is also reflected in the corresponding liver tissue. The higher the dose of HUCHE086, the better the improvement effect, and it is closer to the normal hamster liver tissue (as shown in the fifth figure).

4.6 Summary

According to the above test data, the hamster of fatty liver induced by high-fat diet has abnormal blood lipid, liver fat content and TCHO/HDL-C ratio, while HUCHE025, HUCHE033 and HUCHE086 can effectively reduce fatty liver hamsters. Liver TG, TCHO accumulation and improvement of HDL-C, LDL-C distribution ratio, among which HUCHE025 and dose 246mg/kg/day HUCHE086 improved best, and animal experiments confirmed that HUCHE025, HUCHE033 and HUCHE086 were safe.

It has been confirmed by animal experiments that HUCHE025, HUCHE033 and HUCHE086 are indeed effective in reducing triglyceride and total cholesterol in blood, and effectively improving fat accumulation in hepatocytes. With the phenomenon of liver function, the intake of bioflavonoids such as HUCHE025, HUCHE033 and HUCHE086 can effectively reduce blood fat and improve the symptoms of non-alcoholic fatty liver disease (NAFLD).

The invention provides a safe and non-side-effect compound composition for reducing liver fat and blood fat and for treating symptoms of non-alcoholic fatty liver disease (NAFLD), and the same as the above-mentioned hypolipidemic or fat-reducing When liver and other related drugs are compared with each other, they have the following advantages:

1. The invention provides a safe and non-side-effect compound composition for reducing liver fat, reducing blood fat and improving symptoms of non-alcoholic fatty liver disease (NAFLD), belonging to a low molecular natural plant phenolic compound. The phenolic compound is widely present in fruits and vegetables, grains, rhizomes, flowers, tea leaves and red wines, and has little toxic side effects, and after the animal test of the second embodiment of the present invention, the same result is obtained, and it is safe and flawless.

2. The invention provides a safe and non-side-effect compound composition for reducing liver fat and blood fat, which is used for non-alcoholic fatty liver disease (NAFLD) symptoms, and belongs to a low molecular natural plant phenolic compound. It does not need to be combined with other prescriptions to reduce liver fat and blood fat. Compared with common hypolipidemic drugs, the compound composition can effectively reduce the fat content in the liver, in addition to lowering blood fat. , the effect of lowering blood fat and improving the symptoms of non-alcoholic fatty liver disease (NAFLD), so it has developed into hypolipidemic, hypolipidemic, and nonalcoholic fatty liver disease (Non-alcoholic Fatty Liver Disease, NAFLD) The potential of a health food or drug for symptoms.

The above detailed description is specific to the possible embodiments of the present invention, but The examples are not intended to limit the scope of the invention, and equivalents or modifications, such as bioflavonoids, bioflavonoid derivatives, concentrations and ratios of bioflavonoids applied, and natural Equivalent examples of changes in the types of hypolipidemic compound combinations, etc., should be included in the scope of the patent.

In summary, this case is not only innovative, but also has a safe, no side effects, hypolipidemic, hypolipidemic, and non-alcoholic fatty liver disease (NAFLD) symptoms. The utility of the symptoms of Non-alcoholic Fatty Liver Disease (NAFLD) should be fully in line with the statutory invention patent requirements of novelty and progressiveness, and the application should be filed according to law, and you are requested to approve the invention patent application. In order to invent, to the sense of virtue.

Claims (12)

A pharmaceutical composition for reducing liver fat, wherein the pharmaceutical composition is selected from the group consisting of (-)-Epicetechin-3-gallate, Isorhamnetin, phloridzin (Phloridzin), (-)-Epigallocetechin, Quercitrin, Gallic Acid, Kaempferol, Formononetin, Baicalein, n-Dihydroxyl Nordic hydroguaiaretic acid, Oleanolic Acid, Daidzin, Glycyrrhizin, 6-Gingerol, Liquiritin, phloretin (Phloretin), daidzein, Umbelliferone, Genistein, (-)-Epicatechin, Naringin, Neohesperidin, Han Wongonin, Capillarisin and Isoliquritigenin, all of which are bioflavonoids for the treatment of nonalcoholic fatty liver disease, the treatment refers to lowering the liver Fat and no liver side effects. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is selected from the group consisting of the following bioflavonoid compounds and defines an effective dosage of the composition: (-)-Epicetechin- The content of 3-gallate is 15~150 mg, the content of isorhamnetin is 8~80 mg, the content of Phloridzin is 15-140 mg, and the content of (-)-Epigallocetechin is 10~ 100 mg, Quercitrin content of 15 to 150 mg, Gallic Acid content of 5 to 60 mg, Kaempferol content of 10 to 100 mg, formononetin ( Formononetin) is 10~100 mg, Baicalein is 10~90 mg, and dihydrogen is sore (Nordihydroguaiaretic acid) is 10~100 mg, the content of oleicolic acid is 15~150 mg, the content of daidzin is 10-100 mg, and the content of glycyrrhizin is Glycyrrhizin. It is 30~270 mg, 6-Gingerol is 10~100 mg, Liquiritin is 14~140 mg, and Phloretin is 10~100 mg. The content of daidzein is 10~100 mg, the content of Umbelliferone is 5~50 mg, the content of genistein is 10~100 mg, (-)- Epicatechin is 10~100 mg, Naringin is 16-160 mg, Neohesperidin is 2-20 mg, and Wongonin is 10-90. The content of milligrams and capillarisin is 10 to 100 mg and the content of isoliquritigenin is 10 to 100 mg. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition further reduces blood fat symptoms. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is administered in the form of a gel, a spray, a pastille, a lozenge or a dispersible tablet. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is contained in a medical kit, a kit or a patient pack. A pharmaceutical composition for the preparation of a medicament for lowering liver fat, the pharmaceutical composition comprising: (-)-Epicetechin-3-gallate, Isorhamnetin, Phloridzin, (-)-Epigallocetechin, Quercitrin, Hesperetin, Gallic Acid, Kaempferol, Hesperidin, Formononetin, Baicalein, normal dihydroxy Nordic hydroguaiaretic acid, Oleanolic Acid, Daidzin, Glycyrrhizin, 6-Gingerol, Liquiritin, phloretin (Phloretin), daidzein, Umbelliferone, Genistein, (-)-Epicatechin, Naringin, Neohesperidin, Han Wongonin, Capillarisin and Isoliquritigenin, all of which are bioflavonoids. The use according to claim 6, wherein the pharmaceutical composition is selected from the group consisting of the following compounds and defines an effective dosage of the composition: (-)-Epicetechin-3-gallate It is 15~150 mg, the content of isorhamnetin is 8~80 mg, the content of Phloridzin is 15-140 mg, the content of (-)-Epigallocetechin is 10~100 mg, suede (Quercitrin) content of 15 ~ 150 mg, Hesperetin content of 10 ~ 100 mg, Gallic Acid content of 5 ~ 60 mg, Kaempferol content of 10 ~ 100 mg, Hesperidin content of 20-200 mg, Formononetin content of 10~100 mg, Baicalein content of 10-90 mg, ortho-dihydroxy acne The content of acid (Nordihydroguaiaretic acid) is 10~100 mg, the content of oleicolic acid is 15~150 mg, the content of daidzin is 10-100 mg, and Glycyrrhizin The content is 30~270 mg, 6-Gingerol is 10~100 mg, Liquiritin The content of 14~140 mg, the content of Phloretin is 10~100 mg, the content of daidzein is 10~100 mg, and the content of Umbelliferone is 5~ 50 mg, gold Genistein content is 10~100mg, (-)-Epicatechin is 10~100mg, Naringin is 16~160mg, Neohesperidin is content. It is 2~20 mg, the content of Wongonin is 10~90 mg, the content of Capillarisin is 10~100 mg and the content of Isoliquritigenin is 10-100 mg. The use according to claim 6, wherein the pharmaceutical composition can reduce liver fat accumulation caused by Non-alcoholic Fatty Liver Disease (NAFLD). The use of claim 6, wherein the agent reduces liver fat by lowering liver triglyceride (TG). The use of claim 6, wherein the agent is for further reducing blood fat symptoms. The use according to claim 6, wherein the medicament is administered in the form of a gel, a spray, a pastille, a lozenge or a dispersible tablet. The use of claim 6, wherein the medicament is included in a medical kit, a kit or a patient pack.