WO2017084234A1 - 预防或治疗脂肪肝的药物组合物 - Google Patents

预防或治疗脂肪肝的药物组合物 Download PDF

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Publication number
WO2017084234A1
WO2017084234A1 PCT/CN2016/078039 CN2016078039W WO2017084234A1 WO 2017084234 A1 WO2017084234 A1 WO 2017084234A1 CN 2016078039 W CN2016078039 W CN 2016078039W WO 2017084234 A1 WO2017084234 A1 WO 2017084234A1
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Prior art keywords
liver
mannitol
combination
acid
sucralose
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PCT/CN2016/078039
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English (en)
French (fr)
Inventor
胡幼圃
何欣恬
吴永恩
唐熙卉
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欣耀生医股份有限公司
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Priority to CN202310336366.5A priority Critical patent/CN116440143A/zh
Priority to CN201680021216.3A priority patent/CN107613968A/zh
Priority to JP2018526123A priority patent/JP2018534323A/ja
Priority to EP16865417.6A priority patent/EP3391881A4/en
Priority to US15/564,507 priority patent/US10925854B2/en
Priority to TW105131118A priority patent/TWI781912B/zh
Priority to CA3232521A priority patent/CA3232521A1/en
Priority to KR1020187011548A priority patent/KR20180058795A/ko
Priority to EA201890810A priority patent/EA201890810A1/ru
Priority to MX2018003700A priority patent/MX2018003700A/es
Priority to JP2018535221A priority patent/JP2018537517A/ja
Priority to AU2016327930A priority patent/AU2016327930B2/en
Priority to BR112018005905-6A priority patent/BR112018005905B1/pt
Priority to SG10202002573RA priority patent/SG10202002573RA/en
Priority to KR1020247008968A priority patent/KR20240042148A/ko
Priority to PCT/CN2016/100187 priority patent/WO2017050298A1/en
Priority to MYPI2018701141A priority patent/MY190647A/en
Priority to CN201680020530.XA priority patent/CN107614475A/zh
Priority to EP16848179.4A priority patent/EP3353144A4/en
Priority to EP21173210.2A priority patent/EP3991724A3/en
Priority to CA2999368A priority patent/CA2999368A1/en
Priority to US15/564,526 priority patent/US10456371B2/en
Publication of WO2017084234A1 publication Critical patent/WO2017084234A1/zh
Priority to HK18103734.5A priority patent/HK1244213A1/zh
Priority to HK18103735.4A priority patent/HK1244266A1/zh
Priority to MX2022007816A priority patent/MX2022007816A/es
Priority to PH12018500659A priority patent/PH12018500659A1/en
Priority to ZA2018/02373A priority patent/ZA201802373B/en
Priority to US16/573,506 priority patent/US11285123B2/en
Priority to AU2021205040A priority patent/AU2021205040B2/en
Priority to JP2021176691A priority patent/JP7261850B2/ja
Priority to US17/686,013 priority patent/US20220265587A1/en
Priority to AU2024200963A priority patent/AU2024200963A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
    • A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
    • A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
    • A23L3/3463—Organic compounds; Microorganisms; Enzymes
    • A23L3/3472—Compounds of undetermined constitution obtained from animals or plants
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/047—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates having two or more hydroxy groups, e.g. sorbitol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
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    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/428—Thiazoles condensed with carbocyclic rings
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7004—Monosaccharides having only carbon, hydrogen and oxygen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A—HUMAN NECESSITIES
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    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A—HUMAN NECESSITIES
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    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/75—Rutaceae (Rue family)
    • A61K36/752—Citrus, e.g. lime, orange or lemon
    • A—HUMAN NECESSITIES
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    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
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    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents

Definitions

  • the present invention relates to a composition and method for preventing or treating fatty liver, protecting liver function, or ameliorating liver disease caused by fatty liver or other related disorders.
  • 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 very low density lipoprotein (VLDL). In the form of TG transports the liver out of the bloodstream. Therefore, once the liver is damaged, it will cause abnormal metabolism and accumulation of lipids (especially TG) in the liver cells.
  • FFA free fatty acid
  • TG triglyceride
  • VLDL very low density lipoprotein
  • fatty liver disease means that the weight of fat in the liver exceeds 5% of the weight of the liver, or more than 10% of the liver tissue sections.
  • Hepatocytes have a phenomenon of fat vacuolization 2 .
  • Fatty liver can be distinguished according to the cause of alcoholic fatty liver (AFLD), non-alcohol fatty liver disease (NAFLD) or other factors, such as drugs, fatty liver disease, pathology Appearance includes characterization of fatty metamorphosis or steatosis, steatohepatitis, and the like.
  • AFLD alcoholic fatty liver
  • NAFLD non-alcohol fatty liver disease
  • Appearance includes characterization of fatty metamorphosis or steatosis, steatohepatitis, and the like.
  • Mild fatty liver refers to less than 33% of hepatocytes with steatosis, moderate to 33-66%, and severe 66% to 3,9,21 .
  • fatty liver was considered to be a more benign and reversible disease, so it was less valued.
  • studies have found that it will cause liver fibrosis and cirrhosis, even liver cancer, and with the increase of obese population. There is an increasing trend.
  • NAFLD Non-alcoholic steatohepatitis
  • 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%
  • the prevalence rate of morbidly obese people (BMI greater than 30) is as high as 80% 15,23 .
  • Fatty liver appeared after the first strike, and appeared after the second strike.
  • Fatty hepatitis (steatohepatitis).
  • the first strike was caused by excessive accumulation of fat in the liver, due to obesity, hyperlipidemia, etc.
  • the second strike was caused by oxidative stress and reactive oxygen species in the mitochondria.
  • ROS causes lipid peroxidation on the liver cell membrane, releasing primary inflammatory cytokines and free radicals, and activating stellate cells to produce fibrosis, leading to necrosis of hepatocytes 4,5,19 .
  • the pathogenesis of NASH is mainly related to triglyceride lipid peroxidation, oxidative stress, ROS reaction, increased peroxidation of lipids in hepatocytes, or enhancement of cytokines and liver enzymes, resulting in a series of autoimmune Reciprocal reactions 12 .
  • 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.
  • GOT/GPT in the blood of patients with fatty liver The values may be normal. To correctly diagnose fatty liver, it is necessary to pass the abdominal ultrasound examination. The current accuracy rate is over 97%.
  • FLD has no specific therapeutic effect for specific curative effect.
  • the treatment policy is mainly to improve the potential risk factors or to use drugs to control the progress of chronic diseases. It is recommended to treat the symptoms according to the formation of fatty liver, such as: fatty liver caused by overweight, 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 immediately stop using these drugs; fatty liver caused by disease, Such as hepatitis C, high blood fat, etc., must start at the source, treat C liver, control blood lipids; if the body factors cause triglyceride too high, you can not improve fatty liver by weight loss.
  • lipid-lowering drugs have a "lip-repellent" effect, which can "catch" the lipids in the blood to the liver, and the liver has already accumulated fat, so it is difficult to treat a large amount of influx of fat, and fat will be Accumulation in the liver makes the fatty liver more serious. It can be seen that hypolipidemic drugs are not suitable for the treatment of FLD.
  • the present invention provides that one or more excipients (including flavonoid compounds, etc.) have the effect of preventing or treating fatty liver, protecting liver function, or improving liver disease caused by fatty liver or other related diseases, and the compound is selected from the following Group of components: sodium lauryl sulfate, menthol, sucralose, mannitol, sorbitol, saccharin, glycerin, benzene Sodium benzoate, oxide red, pregelatinized starch, sodium cyclamate, sorbic acid, lemon oil, lemon Citric acid, and butylated hydroxyanisole, poncirin, isovitexin, eriodictyol, Ergosterol, ⁇ -myrcene, hyperoside, catechin ((+)-catechin), galangin, morin, ginseng Flavonoids (sciadopitysin), didymin, cotton cellulose (gossypin), luteolin-7-Glucoside, dihydroquercetin
  • the invention provides the use of the compounds for the preparation of a composition for preventing or treating fatty liver, protecting liver function, or ameliorating hepatic lesions or other related conditions caused by fatty liver.
  • the present invention also proposes a method of preventing or treating fatty liver, protecting liver function, or improving liver disease caused by fatty liver or other related conditions by administering the compound.
  • the compound is selected from the group consisting of sodium lauryl sulfate, menthol, sucralose, mannitol, sorbitol. (sorbitol), saccharin, glycerin, sodium benzoate, oxide red, pregelatinized starch, sodium cyclamate, Sorbic acid, lemon oil, citric acid, and butylated hydroxyanisole, and any combination thereof.
  • the compound is selected from the group consisting of poncirin, isovitexin, eriodictyol, ergosterol, and myrcene.
  • poncirin isovitexin
  • eriodictyol ergosterol
  • myrcene hyperoside
  • catechin ((+)-catechin)
  • galangin morin
  • sciadopitysin scented grass
  • cotton cellulose cellulose
  • luteolin-7-Glucoside (+)-taxifolin, trans-cinnamic acid, fragrant Diosmin, linarin, xylitol, luteolin, swertiamarin, and any combination thereof.
  • the compound is selected from the group consisting of: eriodictyol, mannitol, menthol, sucralose, saccharin, And any combination thereof.
  • the compound is selected from the group consisting of: (1) a combination of saccharin and mannitol, (2) a combination of menthol and mannitol, and (3) sucralose and mannitol. Combination, (4) a combination of eriodictyol and mannitol, (5) a combination of eriodictyol and sucralose, (6) menthol, mannitol, eriodictyol, or (7) sucralose, mannitol , a combination of ervain.
  • one or more of the compounds described herein are used in combination with one or more selected from the group consisting of puerarin, phloridzin, and sinensetin.
  • the compounds of the invention reduce the liver fat content of an individual.
  • the compounds of the invention reduce the fat content of hepatocytes in an individual.
  • the compounds of the invention may reduce liver damage in an individual, for example, liver tissue damage or liver function damage.
  • the compounds of the invention increase the liver antioxidant activity of an individual.
  • the compounds of the invention may be used to ameliorate related conditions caused by accumulation of hepatic fat for a variety of reasons including, but not limited to, fatty liver, acute and chronic alcoholic fatty liver, acute and chronic nonalcoholic fatty liver disease. , acute and chronic alcoholic hepatitis, acute and chronic nonalcoholic steatohepatitis, non-alcoholic cirrhosis, alcoholic cirrhosis (ICD-9-CM diagnostic code 571.8, 571.0, 571.1, 571.2, 571.3, 571.4, 571.5, 571.9 ).
  • the individual suitable for the compound of the invention is a patient with fatty liver disease or an obese person.
  • the compounds of the invention may be formulated into pharmaceuticals, food supplements or health foods.
  • the present invention provides a composition comprising any two or more compounds selected from the above.
  • composition of the present invention comprises any two or more compounds selected from the group consisting of: eriodictyol, mannitol, menthol, Sucralose and saccharin.
  • compositions of the present invention comprise a combination selected from the group consisting of: (1) a combination of saccharin and mannitol, (2) a combination of menthol and mannitol, and (3) three. a combination of sucralose and mannitol, (4) a combination of eriodictyol and mannitol, (5) a combination of eriodicty and sucralose, (6) menthol, mannitol, eriodictol, or (7) A combination of sucralose, mannitol, and eriodictyol.
  • Figure 1 shows liver tissue sections after 4 weeks of treatment with different test substances in mice after induction of fatty liver production by mice.
  • the present invention discloses that one or more of the compounds described above have the effect of reducing liver fat content and ameliorating related conditions. Accordingly, the present invention provides the use of a compound according to the composition for the preparation of a composition for preventing or treating fatty liver, protecting liver function, or ameliorating liver disease caused by fatty liver or other related disorders.
  • the invention also provides a method for preventing or treating fatty liver, protecting liver function, or improving liver disease caused by fatty liver Or a method of other related disorders which comprises administering to a subject in need thereof an effective amount of the compound.
  • the invention also provides compositions for preventing or treating fatty liver, protecting liver function, or ameliorating hepatic lesions or other related conditions caused by fatty liver.
  • liver fat content refers to the amount of fat accumulated in the liver in an individual, including lipids in a broad sense, such as triglyceride (TG) and cholesterol.
  • reducing liver fat content generally refers to a reduction in abnormal liver fat content in an individual, that is, a reduction in abnormal liver fat content, and more specifically, a reduction to a normal level. For example, under normal circumstances, fat accounts for about 3% of the liver's weight. When the weight of fat in the liver exceeds 5% of the weight of the liver, it is abnormal accumulation of fat. (The above-mentioned liver fat content is a relative value and is an example, may be due to individual ethnic groups and Other factors have changed).
  • reducing liver fat content may mean lowering the abnormal liver fat content in an individual, for example, from 5% or more of the liver weight to 3% of the liver weight.
  • Standard methods of analysis can be used to assess liver fat content, including but not limited to, ultrasound analysis, magnetic resonance imaging MRI, magnetic resonance spectrum MRS, computed tomography CT, and liver pathology.
  • liver function refers to one or more of the many physiological functions performed by the liver and can be analyzed by a number of routine tests, for example, alanine aminotransferase (ALT) analysis or aspartic Aspartate transaminase (AST) analysis.
  • ALT alanine aminotransferase
  • AST aspartic Aspartate transaminase
  • the compounds can be used to protect liver function, including improving liver function or avoiding damage to liver function.
  • liver lesions may refer to liver cells that are injured or destroyed by certain factors and may cause liver function to be affected. According to the invention, the compounds can be used to improve liver lesions caused by fatty liver. More specifically, “hepatic injury” as used herein refers to a condition in which the liver is damaged in tissue or biochemical function compared to normal liver. In the specific aspect, “hepatic injury” as used herein refers to liver damage caused by alcohol or non-alcoholic factors such as a high-fat diet or obesity. In a specific aspect, “liver injury” may damage the liver tissue, and may be selected from one or more of the following characteristics: steatosis, lobular inflammation, hepatocyte ballooning.
  • liver injury may be an impaired biochemical function of the liver, which may be determined by serum alanine aminotransferase (ALT) or aspartate transaminase (AST) activity. Judging, the higher the activity, the more severe the damage to the biochemical function of the liver.
  • ALT serum alanine aminotransferase
  • AST aspartate transaminase
  • liver antioxidant activity refers to the activity or ability of the liver to combat oxidative stress.
  • the compound according to the present invention may increase the liver antioxidant activity of an individual, including, but not limited to, reducing the oxidative stress or increasing the enzyme activity or content of a member of the antioxidant system, and the member of the antioxidant system may be glutathione Glutathione peroxidase (GPx), glutathione (GSH), glutathione reductase (GRd), and/or superoxide dismutase (SOD).
  • GPx glutathione Glutathione peroxidase
  • GSH glutathione
  • GRd glutathione reductase
  • SOD superoxide dismutase
  • the compounds include conventional excipients and bioflavonoids, which are useful for lowering liver fat content and ameliorating related conditions.
  • the "related conditions” described herein include conditions caused by abnormal accumulation of liver fat, including but not limited to, fatty liver, acute and chronic alcoholic fatty liver, acute and chronic nonalcoholic fatty liver, acute and chronic alcoholic hepatitis.
  • prevention refers to a preventive or preventive measure for a disease, a symptom or condition of a disease, including but not limited to, the application or administration of one or more active agents to a possible unrecognized condition.
  • treatment refers to a treatment for a disease, a symptom or condition of a disease, including, but not limited to, the administration or administration of one or more active agents to have the disease, the symptoms or condition of the disease, or An individual whose disease has deteriorated, the purpose of which is to treat, cure, alleviate, alleviate, alter, remedy, improve, improve, or affect the disease, the symptoms or conditions of the disease, the disability caused by the disease, or the deterioration of the disease.
  • the terms "individual” or “individual” include human or non-human animals, in particular, mammals, such as companion animals (such as dogs, cats, etc.), farm animals (such as cattle, sheep, pigs). , horses, etc.), or experimental animals (such as rats, mice, guinea pigs, etc.).
  • mammals such as companion animals (such as dogs, cats, etc.), farm animals (such as cattle, sheep, pigs). , horses, etc.), or experimental animals (such as rats, mice, guinea pigs, etc.).
  • the term "effective amount” refers to the amount of active ingredient that produces a desired biological or medical effect on an individual being treated, for example, reducing the liver fat content of an individual or ameliorating a related condition.
  • a therapeutically effective amount of the active ingredient according to the invention may be formulated into a pharmaceutical composition in a suitable form with apharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present invention is preferably a package
  • the active ingredient is present in an amount of from about 0.1% by weight to about 100% by weight, based on the total weight of the composition.
  • the term "pharmaceutically acceptable” means that the carrier is compatible with the active ingredient of the composition (without affecting the action of the active ingredient), and is preferably stable to the active ingredient and is safe for the individual to be treated .
  • the carrier can be a diluent, carrier, excipient, or vehicle of the active ingredient.
  • suitable excipients include lactose, glucose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, poly Vinyl pyrrolidone, cellulose, sterile water, syrup, and methylcellulose.
  • the composition may additionally comprise a lubricant such as talc, magnesium stearate, and mineral oil; a wetting agent; an emulsifier and a suspending agent; a preservative such as methyl and propyl hydroxybenzoate; a sweetener; Agent.
  • a lubricant such as talc, magnesium stearate, and mineral oil
  • a wetting agent such as talc, magnesium stearate, and mineral oil
  • an emulsifier and a suspending agent such as methyl and propyl hydroxybenzoate
  • a preservative such as methyl and propyl hydroxybenzoate
  • the composition may be in any form, for example, as a tablet, a pill, a powder, a lozenge, a sachet, a cachet, an elixir, a suspension, an emulsion, a solution, a syrup, a soft and hard gelatin capsule. , suppositories, sterile injections, and packaging powders.
  • compositions of the invention may be delivered via any physiologically acceptable route, for example, orally, parenterally (e.g., intramuscular, intravenous, subcutaneous, and intraperitoneal), transdermal, suppository, and intranasal methods.
  • parenterally e.g., intramuscular, intravenous, subcutaneous, and intraperitoneal
  • transdermal e.g., transdermal
  • suppository e.g., transdermal
  • intranasal methods e.g., transdermal, suppository, and intranasal methods.
  • a sterile aqueous solution which may contain other substances such as salts or glucose sufficient to render the solution isotonic with blood.
  • the aqueous solution is suitably buffered (preferably having a pH of 3 to 9) depending on the requirements.
  • suitable parenteral compositions under sterile conditions by standard standard standard pharmacological techniques.
  • the human liver cancer cell line Hep G2 was used to analyze the activity of various compounds of the present invention to reduce fat content.
  • DMEM Dulbecco's Modified Eagle's Medium
  • the DMEM culture medium of No. A-F was stored at 2-8 ° C, and it was used after being warmed in a 37 ° C water bath before the experiment.
  • 0.4% trypan blue will infiltrate into dead cells and color, while living cells will not penetrate because of the integrity of the cell membrane. Take 100 ⁇ l of cell suspension and mix it with 100 ⁇ l of 0.4% trypan blue. uniform. A small amount of mixed solution (about 20 ⁇ l) was added from the groove above the blood cell counting disk chamber, and the cover plate was covered under a light microscope to observe that the living cells were not stained, and the dead cells were blue.
  • HepG2 cell line 15 ⁇ 10 6 HepG2 cell line was cultured in DMEM medium of No. B, cultured in a 37 ° C, 5% CO 2 incubator for 24 hours, and then serially numbered DMEM medium (serum-free medium)) After 24 hours of culture, the DMEM medium (oleic acid ester/albumin complex) of No. D was finally replaced and cultured for 48 hours to induce HepG2 cell line into fatty liver cells.
  • the HepG2 cell line was divided into 6 groups, including: (1) blank group: no treatment; (2) DMSO group: blank group cells were added with dimethyl sulfoxide (DMSO); (3) control group: Oleic acid induced the formation of fatty liver cells; (4) Carrier group: Fatty liver cells induced by oleic acid were added to DMSO; (5) Positive control group: Fatty liver cells were added with silymarin; and (6) Test group: Fatty liver cells were added A variety of compounds of the invention.
  • DMSO group blank group cells were added with dimethyl sulfoxide (DMSO)
  • control group Oleic acid induced the formation of fatty liver cells
  • Carrier group Fatty liver cells induced by oleic acid were added to DMSO
  • Positive control group Fatty liver cells were added with silymarin
  • Test group Fatty liver cells were added A variety of compounds of the invention.
  • the cells were washed twice with phosphate buffered saline (PBS), and then added with 0.5 mL of trypsin/ethylenediaminetetraacetic acid (trypsin/EDTA) for 3 minutes, and then 2 mL of PBS was added.
  • PBS phosphate buffered saline
  • trypsin/EDTA trypsin/ethylenediaminetetraacetic acid
  • 2 mL of PBS was added.
  • the cells were scraped off, transferred to a centrifuge tube, and the cells were disrupted by ultrasonic waves. 20 ⁇ L of the cell extract was taken to measure the protein content in the cells.
  • the TG assay was carried out using a commercially available combination reagent (Randox). The ratio obtained by dividing the TG content obtained above by the protein content represents the relative content of TG in the cells.
  • mice B6 mice recommended by the Department of Health's "Determination of Liver Health Benefits of Healthy Foods" were selected. The number of test animals in each group was only 4, and only 12 of each group were confirmed. Male mice were housed in the normal light and dark cycle (lighting period from 7:00 am to 7:00 pm, the rest is dark period), temperature 23 ⁇ 2 ° C, relative humidity 55 ⁇ 15% in the animal room, weight 18-23g It was purchased by Lesco (Taipei) into the National Defense Animal Center, and the Department of Animal Experiments was conducted in accordance with the National Health Center Experimental Guide. First, feed 3-5g/day with normal feed every day, and keep 1-2 weeks to observe the health status. The water is supplied indefinitely, and the body weight is recorded once a week.
  • test animals were randomly divided into a blank control group (Blank), a high-fat control group (HFD), a positive control group (PS), and a test group.
  • the blank control group was given normal feed; the high-fat control group was given high-fat diet; the positive control group was given high-fat diet and tube fed silymarin (5 mg/kg/day); and the experimental group was given high-fat feed and tube feeding.
  • Test compound was given normal feed; the high-fat control group was given high-fat diet; the positive control group was given high-fat diet and tube fed silymarin (5 mg/kg/day); and the experimental group was given high-fat feed and tube feeding. Test compound.
  • the blank control group was given normal feed for 12 weeks, and the high-fat control group, the positive control group and the test group were fed with high-fat diet for 12 weeks. After 8 weeks of feeding, the blank control group and the high-fat control group were given deionized water once a day; the control group was given a silymarin once a day; the test group was given a test compound once a day for 4 weeks or 8 weeks. .
  • AST aspartate transaminase
  • ALT Alanine aminotransferase
  • TG total cholesterol
  • TCHO total cholesterol
  • TC low density lipoprotein cholesterol
  • HDL-C high
  • mice were taken abdominal fat and liver specimens after laparotomy. After weighing, the fat weight, liver weight and liver weight/body weight ratio were compared, and the largest right lobe liver was cut into two pieces of about 1 cm cube. It 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 cryopreserved and the levels of triglycerides and total cholesterol in the liver were measured. In addition, the liver function of each group of animals was evaluated using the Galactose Single Point Method, which is recommended by the US Food and Drug Administration and the Taiwan Department of Health for the quantitative use of liver function in clinical use.
  • mice were sacrificed, and a piece of tissue of about 1 cm cube was cut from the largest right lobe liver and fixed in 10% neutral formalin, followed by ethanol at different concentrations (30, 50, 70, 95, 99.5%) and xylene were subjected to dehydration and transparency steps, then xylene was replaced by hot paraffin solution, and finally the tissue was embedded in a paraffin solution.
  • the completed paraffin specimens were cut into 5 ⁇ m paraffin sections by a microtome, and the sections were adhered to a clean glass slide and dried at 37 ° C for further H&E staining.
  • 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 to rehydrate, and then immersed in distilled water for 10 minutes to be dyed.
  • 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.
  • 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.
  • liver tissue was H&E stained to assess liver fat accumulation.
  • all histopathological sections were cut from the same position of the largest right lobe liver and then pathologically stained.
  • double-blind analysis should be performed by a human or veterinary pathologist. If the design of the experiment is not clear, all the sections should be scored (NAS score) 16 and finally statistical analysis. Methods The differences in each group were analyzed.
  • liver tissue of the sacrificial animal was taken, homogenized by a homogenizer (biomasher) for 10 minutes, and then 9 times hepatic weight (w/w) buffer (pH 7.4, 50 mmol/L Tris-HCl, 180 mmol/) was added. L KCl), then mix with a test tube shaker (Vortex).
  • the obtained liver tissue homogenate samples were analyzed for various components of the liver antioxidant system, including glutathione peroxidase (GPx), glutathione (GSH), and glutathione reduction.
  • GPx glutathione peroxidase
  • GSH glutathione
  • glutathione reduction glutathione reduction.
  • the enzyme glutthione reductase, GRd
  • SOD superoxide dismutase
  • the results of reducing the TG content of HepG2 adipocytes measured at the test concentrations of the immobilized test compounds are shown in Table 3.
  • the results showed that the test compound exhibited different effects on reducing the TG content of hepatocytes in the fatty liver cells induced by HepG2 cells compared with the control group.
  • the TG reduction rate (%) was calculated as: [1 - (test group TG content - blank group TG content) / (oleic acid induced group TG content - blank group TG content)] x 100%.
  • Table 3 Test compounds reduce TG content in fatty liver cells
  • Table 3-1 Some test compounds from Table 3 can reduce TG content in fatty liver cells
  • Table 3-2 Part of the test compound (bioflavonoids) from Table 3 reduces TG content in fatty liver cells
  • Table 3-3 Part of the test compound (excipient) from Table 3 reduces TG content in fatty liver cells
  • the other animals were given fatty liver treatment. After 8 weeks, the animals in each group were given different treatments for 4 weeks or 8 weeks, respectively.
  • the group and the high-fat control group were given deionized water, the silymarin was given to the control group, and the test group was given different test compounds, including puerarin, phlorizin, eriodicty, sucralose, mannitol, saccharin, orange peel, A combination of menthol, or a portion of the test compound.
  • Table 4-1 Results of liver weight and body fat weight analysis of test compounds.
  • test compounds are safe and sound.
  • test compound has the effect of reducing liver lipids
  • Figure 1 shows that in mice with induced fatty liver, liver cells in the hilar region (including bile duct, portal vein, and hepatic artery) are covered with many large vesicular fat droplets, and hepatocytes appear ballooning, which successfully induces fatty liver animal models.
  • Table 5-1 Test compounds reduce liver lipids in animals (during 4 weeks of dosing)
  • Table 5-2 Test compounds reduce liver lipids in animals (during 8 weeks of dosing)
  • orange peel, eriodictyol, phloridin, mannitol, menthol, sucralose, and saccharin can significantly reduce total cholesterol in the liver; in particular, saccharin treatment for 4 weeks, excellent results, can be reduced by about 56% Total liver cholesterol (p ⁇ 0.005).
  • a combination of saccharin and mannitol When a combination of two test compounds is administered, a combination of saccharin and mannitol, a combination of menthol and mannitol, a combination of sucralose and mannitol, a combination of eriodictyol and mannitol, or eriodictyol and three
  • the combination of sucralose can effectively reduce the triglyceride in the liver; in particular, the combination of menthol and mannitol for 4 weeks, the excellent effect, can reduce the liver triglyceride content of about 77% (p ⁇ 0.005)
  • the combination of eriodictin and sucralose for 8 weeks achieved excellent results, reducing the liver triglyceride content by about 78% (p ⁇ 0.005).
  • the combination of sucralose and mannitol, the combination of eriodictyol and mannitol, or the combination of eriodicty and sucralose can significantly reduce total cholesterol in the liver; among them, eriodictin and sucralose At 8 weeks of combined treatment, an excellent effect was achieved, which reduced the total cholesterol content of the liver by about 77% (p ⁇ 0.005).
  • a combination of menthol, mannitol, eriodictyol, sucralose, mannitol, and eriodictol can effectively reduce triglycerides in the liver; in particular, three When the combination of sucralose, mannitol, and eriodictin was treated for 8 weeks, the excellent effect was obtained, and the liver triglyceride content was reduced by about 79% (p ⁇ 0.005). In addition, the combination of sucralose, mannitol, and eriodictol can significantly reduce total cholesterol in the liver.
  • test compound has the effect of reducing liver damage
  • FIG. 1 shows liver tissue damage in fatty liver animals, including liver cells in the hilar region (including bile duct, portal vein, and hepatic artery), which are covered with many large vesicular fat droplets, and the hepatocytes appear ballooning.
  • liver tissue sections showed a significant reduction in large vesicular fat droplets in liver cells, of which mice treated with silymarin were still observed.
  • liver tissue type of mice treated with menthol, eriodictol, or mannitol was closer to the blank group, indicating that fatty liver disease was mild.
  • NAS score results are shown in Table 6.
  • Test compounds can reduce liver damage in animals
  • the NAS Nonalcoholic Fatty Liver Disease Activity Score
  • the NAS Nonalcoholic Fatty Liver Disease Activity Score
  • liver tissue damage was induced in mice with fatty liver (increased NAS score). Strugram and mannitol significantly reduced liver damage when administered to a single test compound. It is worth noting that the combination of menthol and mannitol gave excellent results when administered a combination of the two test compounds, and almost no liver damage was observed, and the NAS score was the same as that of the blank group.
  • Table 8-1 Test compounds reduce liver damage in animals (during 4 weeks of dosing)
  • Table 8-2 Test compounds reduce liver damage in animals (during 8 weeks of dosing)
  • ALT and AST are the most commonly used enzyme indicators to reflect liver biochemical damage. These enzymes are normally present in hepatocytes, and are leaked when the liver cells are destroyed. The rise in serum ALT and AST values usually reflects liver inflammation and liver function damage.
  • liver function was impaired in induced fatty liver animals (increased ALT and AST values).
  • mannitol was treated for 4 weeks, an excellent effect was obtained, which was reduced by about 64% ALT value (p ⁇ 0.005) and about 60% AST value (p ⁇ 0.005).
  • the combination of menthol and mannitol or the combination of eriodictin and sucralose can significantly reduce the ALT value; the combination of menthol and mannitol, the combination of sucralose and mannitol , or a combination of saccharin and mannitol, can significantly reduce the AST value.
  • the combination of menthol and mannitol was treated for 4 weeks, an excellent effect was obtained, and the ALT value (p ⁇ 0.005) and the AST value of about 62% (p ⁇ 0.005) were reduced by about 76%.
  • sucralose, mannitol, and eriodictol significantly reduced the ALT value (p ⁇ 0.005) when a combination of the three test compounds was administered.
  • Table 9-1 Test compounds increase liver antioxidant capacity (Gpx and GSH)
  • Gpx, GSH, Grd, and SOD are members of the common liver antioxidant system that reduces the oxidative stress in the liver and protects the liver from oxidative stress.
  • An increase in the values of Gpx, GSH, Grd, and SOD represents that the liver maintains better antioxidant activity.
  • mice induced by fatty liver were decreased.
  • orange peel, puerarin, eriodictyol, phlorizin, mannitol, and sucralose can significantly improve the antioxidant activity of the viscera.
  • mannitol was treated for 4 weeks, an excellent effect was obtained, and the levels of Gpx, GSH, Grd, and SOD were greatly increased (p ⁇ 0.005).
  • the compounds provided by the present invention can reduce liver fat content, reduce liver damage and increase liver antioxidant activity.
  • These compounds are low-molecular natural plant phenolic compounds, which are widely found in fruits and vegetables, grains, rhizomes, flowers, teas and red wines.
  • liver fat and improve related diseases such as Fatty liver, acute and chronic alcoholic fatty liver, acute and chronic non-alcoholic fatty liver disease (NAFLD), acute and chronic alcoholic hepatitis, acute and chronic nonalcoholic steatohepatitis, non-alcoholic liver
  • NAFLD non-alcoholic fatty liver disease
  • acute and chronic alcoholic hepatitis acute and chronic nonalcoholic steatohepatitis
  • non-alcoholic liver The potential of health foods or drugs for conditions such as cirrhosis, alcoholic cirrhosis (ICD-9-CM Diagnosis Code 571.8, 571.0, 571.1, 571.2, 571.3, 571.4, 571.5, 571.9).

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Abstract

本发明关于一种预防或治疗脂肪肝、保护肝功能、或改善脂肪肝引起的肝病变或其他相关病症之组合物及方法。

Description

[根据细则37.2由ISA制定的发明名称]　预防或治疗脂肪肝的药物组合物 技术领域
本发明关于一种预防或治疗脂肪肝、保护肝功能、或改善脂肪肝引起的肝病变或其他相关病症之组合物及方法。
背景技术
肝脏是动物体消化系统的一部分，也是许多消化液制造与分泌的主要器官，肝脏亦具有吸收、代谢、清除有毒物质及免疫保护等功能。肝脏是脂肪代谢的重要器官，在脂肪类食物上的消化、吸收、分解、合成以及运输的过程中扮演着极为重要的角色。肝脏由血液中所摄取的游离脂肪酸(free fatty acid，FFA)，最终会在肝脏中合成三酸甘油脂(triglyceride，TG)并储存起来，或以极低密度脂蛋白(very low density lipoprotein,VLDL)的形式将TG转运出肝脏进入血液循环中。因此，一旦肝脏受到损伤后，将导致脂质(尤指TG)在肝细胞内异常的代谢及堆积。
正常情况下脂肪约占肝脏重量的3％，临床上所谓「脂肪肝(fatty liver disease，FLD)」是指肝脏内的脂肪重量超过肝脏重量的5％，或肝组织切片中超过10％以上的肝细胞有脂肪空泡变现象2。脂肪肝依照病因可区分酒精性脂肪肝(alcoholic fatty liver，AFLD)、非酒精性脂肪肝疾病(non-alcohol fatty liver disease，NAFLD)或其他因素，如药物，所衍生的脂肪肝疾病，病理学外观上包括脂肪变性(fatty metamorphosis or steatosis)、脂肪性肝炎(steatohepatitis)等表征。轻度脂肪肝是指含脂肪变性的肝细胞少于33％，中度为介于33-66％，而重度则占66％以上3,9,21。过去大都被认为脂肪肝是较良性且可逆的病症，因此较不被重视，但近年来陆续的研究发现，其严重将引发肝脏纤维化及肝硬化，甚至是肝癌，且随着肥胖人口增加，有增加之趋势。
欧美国家主要肝脏疾病系因长期饮酒过量，因此，绝大部份肝脏疾病皆因酒精伤害所致。但近15-20年，NAFLD却成为欧美国家中，肝功能异常必先考虑的病因2。Thaler曾在1962年对NAFLD有所描述，1980年Ludwig在一群肥胖女性糖尿病及高脂血症患者中，发现伴随的NAFLD中提出「非酒精性脂性肝炎」(Non-alcoholic steatohepatitis，NASH)，随后在1986年Schaffner更再次强调NASH在NAFLD病程中衍生纤维化的机制中扮演着重要的角色21；直到1998年Day发现有15-50％的NASH患者衍生不同程度的纤维化4，NAFLD才开始受到临床医师的重视。如今，除了AFLD外，在临床上NASH不仅只是NAFLD自然病程发展中的一个阶段，也由于其存在使得NAFLD不再被视为一种良性的肝脏疾病。
目前在北美、南美、日本、北欧、南欧、澳洲及中东地区之FLD研究，发现至少有10-39％的盛行率，而死后病理解剖组织病理学检查中，NAFLD盛行率约在20％上下，其中所伴随的NASH之发现率约为3-18％不等；其中肥胖者中NAFLD的盛行率则高达57-74％(是正常人的4.6倍)，其中20-25％存有NASH病变，罹患肝硬化者亦占2-3％。台湾地区近三十年来，由于经济环境及饮食的改善，NAFLD盛行率亦有逐年增高趋势，近年来盛行率约12-37％，与日本的9-13％相较不远，其中非肥胖者盛行率约10％，而属病态肥胖者(BMI大于30者)盛行率则高达80％15,23。
有关NAFLD致病机转，英国Day与James根据大量临床及动物实验研究提出二次打击假说(Two-hit hypothesis)，第一次打击后出现脂肪肝(fatty liver)，第二次打击后则出现脂肪性肝炎(steatohepatitis)。第一次打击肇因于肝脏内脂肪的过度堆积，原因有肥胖、高血脂症等；第二次打击则是起因于氧化压力(oxidative stress)及粒线体中活性氧物种(Reactive oxygen species,ROS)作用，造成肝细胞膜上脂质过氧化(lipid peroxidation)，释放出原发炎细胞激素及自由基，活化星状细胞(stellate cells)产生纤维化，导致肝细胞患坏死4,5,19。而NASH的致病机转主要与三酸甘油脂过氧化作用、氧化压力、ROS反应、增加肝细胞本身脂质的过氧化反应、或与细胞激素及肝脏酵素的增进，导致一系列自体免疫的相互反应有关12。
脂肪肝的形成大多是长期摄取过多动物性脂肪、蛋白质、碳水化合物，过剩的热量在体内转化成脂肪囤积起来，导致肥胖及脂肪肝。脂肪肝患者血中的GOT/GPT 数值可能都正常，要正确诊断脂肪肝，必须透过腹部超音波检查，目前准确率达97％以上。
目前FLD尚无特定疗效的理想治疗药物，治疗方针主要是改善潜在危险因子或使用药物控制慢性疾病的进展为主，建议依形成脂肪肝原因对症加以治疗，如：体重过重导致的脂肪肝，须适度减轻体重；酒精性脂肪肝，需靠戒酒及摄取均衡饮食才能改善；长期接触伤害肝脏的化学物质或药物，引起的脂肪肝，则须立即停止使用这些药物；疾病引起的脂肪肝，如C型肝炎、血脂肪过高等，须针对源头着手，治疗C肝、控制血脂；若是体质因素导致三酸甘油脂太高，则无法由减肥方式改善脂肪肝。
然而，目前臨床常用降低血清中的三酸甘油脂、胆固醇之药品常伴随者肝毒性(hepatotoxicity)、肌痛、肌炎、横纹肌溶解等肌肉病变(myopathy)副作用，其中降血脂药物中，肌肉毒性是最值得关注的副作用，尤以舒脂锭(Statins)发生肌肉毒性比例最高，纤维酸(Fibric acid)次之。此外，降血脂药物具有「驱脂」作用，能将血液中的脂类「驱赶」到肝脏，而肝脏内本来就已有脂肪堆积，故对大量涌入的脂类难以进行处理，脂肪便会堆积在肝脏内，使得脂肪肝更加严重。由此可知降血脂用药并不适合用于治疗FLD。
因此，仍有需要寻求一种有效的预防及治疗脂肪肝、脂肪肝引起之肝病变、保护肝功能及改善相关病症之有效成分。
发明内容
本发明提出一或多种赋形剂(包括类黄酮化合物等)具有预防或治疗脂肪肝、保护肝功能、或改善脂肪肝引起的肝病变或其他相关病症之功效，该化合物系选自以下所组成之群：硫酸月桂酸钠(sodium lauryl sulfate)、薄荷醇(menthol)、三氯蔗糖(sucralose)、甘露醇(mannitol)、山梨醇(sorbitol)、糖精(saccharin)、甘油(glycerin)、苯甲酸钠(sodium benzoate)、氧化铁红(oxide red)、预糊化淀粉(pregelatinized starch)、环己烷氨基磺酸钠(sodium cyclamate)、山梨酸(sorbic acid)、柠檬油(lemon oil)、柠檬酸(citric acid)、及丁基羟基茴香醚(butylated hydroxyanisole)、枸橘苷(poncirin)、异牡荆素(isovitexin)、圣草酚(eriodictyol)、 麦角固醇(ergosterol)、月桂烯(β-myrcene)、金丝桃苷(hyperoside)、儿茶素((+)-catechin)、高良姜素(galangin)、桑叶素(morin)、金松双黄酮(sciadopitysin)、香风草甙(didymin)、棉纤维素(gossypin)、木犀草素-7-葡萄糖甙(luteolin-7-Glucoside)、双氢槲皮素((+)-taxifolin)、肉桂酸(trans-cinnamic acid)、香叶木素(diosmin)、蒙花甙(linarin)、木糖醇(xylitol)、木犀草素(luteolin)、獐牙菜苦素(swertiamarin)、及其任何组合。
因此，在一方面，本发明提出所述化合物用于制备预防或治疗脂肪肝、保护肝功能、或改善脂肪肝引起的肝病变或其他相关病症之组合物的用途。本发明也提出藉由投用所述化合物以预防或治疗脂肪肝、保护肝功能、或改善脂肪肝引起的肝病变或其他相关病症之方法。
在部份具体实例中，该化合物系选自于以下所组成群组：硫酸月桂酸钠(sodium lauryl sulfate)、薄荷醇(menthol)、三氯蔗糖(sucralose)、甘露醇(mannitol)、山梨醇(sorbitol)、糖精(saccharin)、甘油(glycerin)、苯甲酸钠(sodium benzoate)、氧化铁红(oxide red)、预糊化淀粉(pregelatinized starch)、环己烷氨基磺酸钠(sodium cyclamate)、山梨酸(sorbic acid)、柠檬油(lemon oil)、柠檬酸(citric acid)、及丁基羟基茴香醚(butylated hydroxyanisole)、及其任何组合。
在部份具体实例中，该化合物系选自于以下所组成群组：枸橘苷(poncirin)、异牡荆素(isovitexin)、圣草酚(eriodictyol)、麦角固醇(ergosterol)、月桂烯(β-myrcene)、金丝桃苷(hyperoside)、儿茶素((+)-catechin)、高良姜素(galangin)、桑叶素(morin)、金松双黄酮(sciadopitysin)、香风草甙(didymin)、棉纤维素(gossypin)、木犀草素-7-葡萄糖甙(luteolin-7-Glucoside)、双氢槲皮素((+)-taxifolin)、肉桂酸(trans-cinnamic acid)、香叶木素(diosmin)、蒙花甙(linarin)、木糖醇(xylitol)、木犀草素(luteolin)、獐牙菜苦素(swertiamarin)、及其任何组合。
在部份具体实例中，该化合物系选自于以下所组成群组：圣草酚(eriodictyol)、甘露醇(mannitol)、薄荷醇(menthol)、三氯蔗糖(sucralose)、糖精(saccharin)、及其任何组合。
在部份具体实例中，该化合物系选自于以下所组成群组：(1)糖精与甘露醇的组合、(2)薄荷醇与甘露醇的组合、(3)三氯蔗糖与甘露醇的组合、(4)圣草酚与甘露醇的组合、(5)圣草酚与三氯蔗糖、(6)薄荷醇、甘露醇、圣草酚的组合、或(7)三氯蔗糖、甘露醇、圣草酚的组合。
在部份具体实例中，此处所述的一或多种化合物系与选自于以下化合物之ㄧ或多者并用：葛根素(puerarin)、根皮苷(phloridzin)、甜橙黄酮(sinensetin)、(-)-表没食子儿茶素((-)-epigallocatechin)、山柰(kaempferol)、熊果酸(ursolic acid)、水飞蓟素、(silymarin)、(+)-柠檬油精((+)-limonene)、橙皮甙(hesperidin)、(-)-表儿茶素-3-没食子酸酯((-)-epicatechin-3-gallate)、水飞蓟宾(silybin)、芒柄花黄素(formononetin)、十四烷酸乙酯(myristic acid ethyl ester)、二十碳五烯酸(eicosapentaenoic acid,EPA)、汉黄芩素(wongonin)、聚维酮K-30(povidone K-30)、原儿茶酸(protocatechuic acid)、伞形酮(umbelliferone)、橙皮(hesperitin)、去甲二氢愈创木酸(nordihydroguaiaretic acid)、新橙皮苷(neohesperidin)、柚皮苷(naringin)、(-)-表儿茶素((-)-epicatechin)、甘草甜素(glycyrrhizin)、黄芩苷(baicalin)、斛皮素(quercitrin)、及黄芩甙元(baicalein)。
在部份具体实例中，本发明的化合物可降低个体之肝脂肪含量。
在部份具体实例中，本发明的化合物可降低个体之肝细胞之脂肪含量。
在部份具体实例中，本发明的化合物可降低个体之肝损害，例如，肝组织损害或肝功能损害。
在部份具体实例中，本发明的化合物可提高个体之肝脏抗氧化活性。
在部份具体实例中，本发明的化合物可用于改善因各种原因肝脂肪累积引起的相关病症，包括但不限于，脂肪肝、急性与慢性酒精性脂肪肝、急性与慢性非酒精性脂肪肝、急性与慢性酒精性肝炎、急性与慢性非酒精性脂肪肝炎、非酒精性肝硬化、酒精性肝硬化(ICD-9-CM诊断码571.8,571.0,571.1,571.2,571.3,571.4,571.5,571.9)。
在部份具体实例中，适用于本发明的化合物的个体是脂肪肝疾病之患者或肥胖者。
在部份具体实例中，本发明的化合物可制成药物、食品添加物或健康食品。
在又一方面，本发明提供一种组合物，其包括选自于以上所述的任何两种或以上的化合物。
在部份具体实例中，本发明之组合物包括选自于以下所组成的群组的任何两种或以上的化合物：圣草酚(eriodictyol)、甘露醇(mannitol)、薄荷醇(menthol)、三氯蔗糖(sucralose)及糖精(saccharin)。
在部份具体实例中，本发明之组合物包括选自于以下所组成的群组的组合：(1)糖精与甘露醇的组合、(2)薄荷醇与甘露醇的组合、(3)三氯蔗糖与甘露醇的组合、(4)圣草酚与甘露醇的组合、(5)圣草酚与三氯蔗糖、(6)薄荷醇、甘露醇、圣草酚的组合、或(7)三氯蔗糖、甘露醇、圣草酚的组合。
无须进一步的阐述，咸相信本发明所属技术領域中具有通常知識者基于前述說明即可利用本发明至最广的程度。因此，可以理解以下的說明仅仅是作为例示說明之用，而非以任何方式限制其余的揭露内容。
附图说明
欲说明本发明，图式具体实施例如下。然而，应理解到，本发明未局限于所示之较佳具体实施例。在图式中：
图1显示小鼠诱导产生脂肪肝后，再依组别给予不同之试验物质治疗4周后之肝脏组织切片。
具体实施方式
除非另有指明，所有在此处使用的技术性和科学性术语具有如同本发明所属技艺中之通常技术者一般所了解的意义。
本文所使用的「一」乙词，如未特别指明，系指至少一个(一个或一个以上)之數量。
本发明系揭示一或多种如上所述的化合物具有降低肝脂肪含量及改善相关病症之作用。因此，本发明系提供一种所述之化合物在制备预防或治疗脂肪肝、保护肝功能、或改善脂肪肝引起的肝病变或其他相关病症之组合物之用途。本发明亦提供一种预防或治疗脂肪肝、保护肝功能、或改善脂肪肝引起的肝病变 或其他相关病症之方法，其包括对有需要的个体投予有效量之所述的化合物。本发明亦提供可供预防或治疗脂肪肝、保护肝功能、或改善脂肪肝引起的肝病变或其他相关病症之组合物。
本文所使用的「肝脂肪含量」是指个体内累积于肝脏的脂肪含量，包括广义的脂质，例如，三酸甘油脂(triglyceride，TG)及胆固醇等。本文所使用的「降低肝脂肪含量」通常是指使个体内的异常肝脏脂肪含量降低，也就是使异常肝脏脂肪含量减少，更特定而言，是减少至正常水平。例如，正常情况下脂肪约占肝脏重量的3％，当肝脏内的脂肪重量超过肝脏重量的5％时则属于脂肪异常累积(上述肝脏脂肪含量为相对数值且为举例说明，可能因个体族群及其他因素有所变动)。在具体态样中，本文所使用的「降低肝脂肪含量」可指使个体内的异常肝脏脂肪含量降低，例如，从肝脏重量的5％或更高降至肝脏重量的3％。标准的分析方式可用以评估肝脂肪含量，包括但不限于，超音波分析、磁振造影MRI、磁共振频谱MRS、计算机断层扫描CT、肝脏病理切片。
本文所使用的「肝功能」是指是指一个或一个以上的由肝脏执行的许多生理功能，可由许多常规的试验予以分析，例如，丙胺酸转胺酶(alanine aminotransferase，ALT)分析或天冬氨酸氨基转移酶(aspartate transaminase，AST)分析。根据本发明，所述化合物可用以保护肝功能，包括，改善肝功能或避免肝功能的损坏。
本文所使用的「肝病变」可指肝脏细胞因某些因素受伤或被破坏，因而可能导致肝脏机能受到影响。根据本发明，所述化合物可用以改善由脂肪肝所引起的肝病变。更特定而言，本文所使用的「肝损伤」是指肝脏与正常肝脏相比有组织或生化功能受损的情形。在具体态样中，本文所使用的「肝损伤」系由酒精或非酒精性因素，例如，高脂饮食或肥胖，引起的肝损伤。在具体态样中，「肝损伤」可为肝脏的组织受损，可选自以下之一或多种特征：脂肪变性(steatosis)、肝小叶发炎(lobular inflammation)、肝脏细胞气球化(hepatocyte ballooning)及肝脏细胞产生泡状脂肪油滴。在具体态样中，「肝损伤」可为肝脏的生化功能受损，可由血清中的丙胺酸转胺酶(alanine aminotransferase，ALT)或天冬氨酸氨基转移酶(aspartate transaminase，AST)活性予以判断，活性越高表示肝脏的生化功能受损越严重。
本文所使用的「肝脏抗氧化活性」是指肝脏中对抗氧化压力的活性或能力。根据本发明的化合物可提高个体之肝脏抗氧化活性系指，包括但不限于，降低氧化压力或提高抗氧化系统的成员之酵素活性或含量，该等抗氧化系统的成员可为麸胱甘肽过氧化酶(glutathione peroxidase，GPx)、榖胱甘肽(glutathione，GSH)、麸胱甘肽还原酶(glutathione reductase,GRd)、及/或超氧化物歧化酶(superoxide dismutase,SOD)。
根据本发明，所述的化合物包括常用的赋形剂及生物类黄酮，可用于降低肝脂肪含量及改善相关病症。此处所述的「相关病症」包括因肝脂肪累积异常导致的病症，包括但不限于，脂肪肝、急性与慢性酒精性脂肪肝、急性与慢性非酒精性脂肪肝、急性与慢性酒精性肝炎、急性与慢性非酒精性脂肪肝炎、非酒精性肝硬化、酒精性肝硬化(ICD-9-CM Diagnosis Code 571.8,571.0,571.1,571.2,571.3,571.4,571.5,571.9)。
本文所使用的「预防」乙词是指对疾病、疾病之症状或病况的预防的或防止措施，包括但不限于，施加或投予一或多种活性药剂的至可能是尚未被诊断为罹患此疾病、疾病之症状或病况，但对其易感受或有其倾向之个体，其目的在于避免、阻止或推迟此疾病、疾病之症状或病况的发生。
本文所使用的「治疗」乙词是指对疾病、疾病之症状或病况的治疗措施，包括但不限于，施加或投予一或多种活性药剂至具有此疾病、疾病之症状或病况、或疾病恶化之个体，其目的在于治疗、治愈、缓解、减轻、改变、补救、改善、改进、或影响此疾病、疾病之症状或病况、疾病引发之失能、或疾病恶化。
本文所使用的「个人」或「个体」等词包括人类或非人类动物，特地而言，为哺乳类动物，例如，陪伴动物(如狗、猫等)、农场动物(如牛、绵羊、猪、马等)、或实验动物(如大鼠、小鼠、天竺鼠等)。
本文所使用的「有效量」乙词是指于接受处理的个体产生所欲的生物功效或医疗效果的活性成分的量，例如，降低个体的肝脂肪含量或改善相关病症。
为达输送及吸收目的，根据本发明之治疗有效量之活性成分可与医药上可接受载体配制成适当形式之医药组合物。依据投予模式，本发明之医药组合物较佳为包 含约0.1％重至约100％重之活性成分，其中百分比重系以组合物之总重量为基准计算。
本文所使用的「医药上可接受」乙词是指载体与组合物之活性成分相容(不影响活性成分的作用)，且较佳为可稳定该活性成分且对于接受治疗之个体具安全性。该载体可为活性成分之稀释剂、载剂、赋形剂、或介质。适用之赋形剂的一些实例包括乳糖、葡萄糖、蔗糖、山梨糖醇、甘露醇、淀粉、阿拉伯胶、磷酸钙、藻酸盐、黄蓍胶、明胶、硅酸钙、微晶纤维素、聚乙烯吡咯烷酮、纤维素、无菌水、糖浆、及甲基纤维素。本组合物可额外包含润滑剂如滑石、硬脂酸镁、及矿物油；润湿剂；乳化剂及悬浮剂；防腐剂如甲基及丙基羟基苯甲酸酯；增甜剂；以及调味剂。在投予至病患后，本发明之组合物可提供活性成分快速、持续、或缓慢释放之效果
依据本发明，该组合物之形式可任何形式，例如，为片剂、丸剂、粉剂、锭剂、囊剂、扁囊剂、酏剂、悬剂、乳液、溶液、糖浆、软与硬明胶胶囊、栓剂、无菌注射液、及包装粉剂。
本发明之组合物可经由任何生理上可接受途径输送，例如，口服、非口服(如肌内、静脉、皮下、及腹腔)、经皮、栓剂、及鼻内方法。关于非口服投予，较佳为使用无菌水溶液，其可包含其他物质，如足以使溶液与血液等张的盐类或葡萄糖。水溶液可视需求适当地经缓冲(较佳为具pH值3至9)。本领域之技术人员可由习知之标准药理学技术于无菌条件下制备适合的非口服组合物。
本发明通过下面的实施例进一步的说明，下面的实施例仅提供作为示范目的，而非限制本发明。本领域的技术人员应能根据本发明了解，不脱离本发明的精神和范围，而对本发明所公开的特定具体实施例中进行许多改变，仍然能获得相同或相似的结果。
实例
1.材料方法
1.1细胞株及细胞培养基
使用人类肝癌细胞株Hep G2分析本发明的各种化合物降低脂肪含量的活性。
使用DMEM(达尔伯克氏改良伊格尔培养基，Dulbecco’s Modified Eagle’s Medium)进行后续实验，共配制编号A-F的DMEM培养液，如表1所示。
表1：编号A-F的DMEM培养液的配制
Figure PCTCN2016078039-appb-000001
编号A-F的DMEM培养液置于2-8℃保存，实验进行前放在37℃水浴槽中温热后使用。
1.2细胞计数及存活测试
0.4％台盼兰(trypan blue)会渗入死细胞中而呈色，活细胞则因细胞膜完整，染料无法渗入而不会呈色。取100μl细胞悬浮液与100μl 0.4％台盼兰等体积混合均 匀。取少许混合液(约20μl)由血球计数盘室上方凹槽中加入，盖上盖坡片于光学显微镜下观察，活细胞不染色，死细胞则为蓝色。
1.3油酸诱导HepG2细胞株形成脂肪肝细胞
将15×106的HepG2细胞株培养于编号B的DMEM培养液，在37℃，5％CO2的培养箱中培养24小时，然后再以编号C的DMEM培养液(无血清培养液))培养24小时，最后更换为编号D的DMEM培养液(含油酸酯/白蛋白复合物)继续培养48小时，使HepG2细胞株诱导为脂肪肝细胞。
1.4脂肪肝细胞之各组处理
将HepG2细胞株分成6组，包括：(1)空白组：未作任何处理；(2)DMSO组：空白组细胞加入二甲基亚砜(dimethyl sulfoxide，DMSO)；(3)对照组：以油酸诱导形成脂肪肝细胞；(4)载体组：以油酸诱导形成的脂肪肝细胞加入DMSO；(5)阳性对照组：脂肪肝细胞加入水飞蓟素；以及(6)试验组：脂肪肝细胞加入各种本发明的化合物。
1.5细胞的三酸甘油脂(TG)的测定
各组经处理的细胞于72小时培养后，以磷酸盐缓冲液(PBS)清洗两次，之后加入0.5mL胰蛋白酶/乙二胺四乙酸(trypsin/EDTA)培养3分钟，再加入2mL的PBS将细胞刮下，移到离心管中，以超音波将细胞震碎，取20μL细胞萃取液，测细胞内蛋白质含量。TG测定是用市售组合试剂(Randox)进行。将上述所得TG含量除以蛋白质含量所得比值，即代表细胞中TG之相对含量。
1.6试验动物
选择卫生署公告「健康食品之护肝保健功效评估方法」规范建议采用之试验动物B6小鼠，预试验每组试验动物只数≧4只，确认试验每组试验动物只数≧12只。雄性小鼠饲养于正常明暗周期(上午7:00到下午7:00为亮期，其余为暗期)，温度23±2℃，相对湿度55±15％的动物房内，体重为18-23g，由乐斯科(台北)购买入国防动物中心，动物实验系遵照国卫院实验指南进行。先以一般饲料每天喂食3-5g/日，饲养1-2周观察健康状态，水则无限供应，每周记录体重一次。
1.7动物组别
将试验动物随机分组，分为空白对照组(Blank)、高脂对照组(HFD)、正向对照组(Positive control，PS)与试验组。空白对照组系给予正常饲料；高脂对照组系给予高脂饲料；正向对照组系给予高脂饲料与管喂水飞蓟素(5mg/kg/day)；以及试验组系给予高脂饲料与管喂试验化合物。
1.8.试验方法
空白对照组以正常饲料任食12周，高脂对照组、正向对照组与试验组则以高脂饲料任食12周。开始喂食8周后，空白对照组与高脂对照组每天一次管喂给予去离子水；正向对照组每天一次管喂给予水飞蓟素；试验组每天一次管喂给予试验化合物，为期4周或8周。
试验开始前及试验开始后之第8、12或16周，以脸颊或心脏采血。最后于结束时秤重后全部牺牲，以脸颊或心脏采血。小鼠血液样品在室温下放置1小时以使其凝结。再以冷冻离心机于4℃下15,700x g离心5分钟，来分离血清，然后以血液自动生化分析仪检测肝功能生化指数，包括：天冬氨酸氨基转移酶(aspartate transaminase，AST)、丙胺酸转胺酶(alanine aminotransferase，ALT)、三酸甘油脂(TG)、总胆固醇(TCHO，TC)、低密度脂蛋白胆固醇(LDL-C)及高密度脂蛋白胆固醇(HDL-C)。
此外，牺牲后的小鼠剖腹取腹部脂肪及肝脏标本，秤重后比较其脂肪重、肝重及肝重/体重比值，并将最大右叶肝割取两块约l公分立方之组织块，固定于10％的中性福尔马林(formalin)液中，石蜡包封切片后分别作H&E染色来进行组织病理学观察。另外，将其余肝脏冷冻保，检测肝脏中三酸甘油脂及总胆固醇之含量。另，利用美国食品及药物管理局及台湾卫生署认可，推荐给临床使用之定量肝脏剩余功能的半乳糖单点测定法(Galactose Single Point Method)分析各组动物之肝功能，其系在试验结束时，依动物体重每公斤给于0.5g半乳糖溶液(G.S.P.
Figure PCTCN2016078039-appb-000002
0.4g/mL)，经由静脉给药，投药完毕后60分钟以滤纸取大约0.5ml全血，评估小鼠肝功能。GSP值愈高，表示肝脏剩余功能愈差(FDA:“Guidance for Industry:Pharmacokinetics in Patients with Impaired.Hepatic Function—Study Design,Data Analysis and Impact on Dosing and.Labeling.2003)。
1.9组织病理组织切片：
试验结束时，所有小鼠均予以牺牲，于最大右叶肝割取一块约l公分立方之组织块，放入10％的中性福尔马林中固定，接着以不同浓度之乙醇(30、50、70、95、99.5％)以及二甲苯(xylene)进行脱水与透明步骤，然后以热石腊溶液取代二甲苯，最后以石蜡溶液将组织进行包埋。完成的石蜡标本利用切片机切成5μm的石蜡切片，将切片沾黏在干净载玻片上，于37℃烘干后，用做进一步H&E染色。
1.10苏木紫-伊红染色(hematoxylin and eosin stain，H&E)染色法
将肝脏组织切片置入二甲苯30分钟脱蜡，再依序置于99.5、95、70、50及30％乙醇各30分钟以进行复水，再浸泡于蒸馏水10分钟后即可染色。首先浸泡苏木精30秒染细胞核，再用蒸馏水清洗数分钟，接着使用伊红染色2-5分钟，用蒸馏水清洗数分钟。完成染色过程后进行脱水流程，依序放置于50、70、95及100％酒精两次中各30秒，再以二甲苯进行透明化两次，最后以封片胶封存。
1.11组织病理学的观察
为观察肝损伤时，肝细胞的受损、脂肪堆积、坏死或是否有纤维化等变化，将肝组织做H&E染色以评估肝脂肪堆积程度。为避免观察主观上的偏差，所有的组织病理切片都是由最大右叶肝的同一位置切取下来，再去做病理染色。至于病理的半定量分析之评估，应由人医或兽医病理医师进行双盲分析确认，在不清楚本实验设计的情况下，对所有切片进行评分比较(NAS score)16，最后再以统计分析方法进行各组差异性的分析。
1.12肝脏抗氧化分析
取牺牲动物的肝组织约0.1克，以匀浆器(biomasher)离心均质10分钟，再加入9倍肝重(w/w)的缓冲液(pH 7.4，50mmol/L Tris–HCl，180mmol/L KCl)，再以试管震动机(Vortex)混匀备用。取所得之肝组织均质液样品进行各种肝抗氧化系统成员之分析，包括麸胱甘肽过氧化酶(glutathione peroxidase，GPx)、榖胱甘肽(glutathione，GSH)、麸胱甘肽还原酶(glutathione reductase,GRd)、及超氧化物歧化酶(superoxide dismutase,SOD)，相关分析方式可参见已知文献，如(台湾卫生福利部公布的健康食品之护肝保健功效评估方法草案)。
1.13统计分析
所有的数据皆以平均±标准偏差(SD)表示，试验结果以单因子变异数分析(ANOVA)测试法来计算是否具有统计上的显著差异，使用社会科学统计软件包(Statistical Package of the Social Science program，Version 13,SPSS Inc.)来计算；随后使用事后比较(post hoc test)最小差异显著性(least significant difference)方法做多重比较，以确认族群间的显著差异；族群平均之显著差异为p<0.05。
2.结果
2.1细胞实验
在细胞实验中，阳性对照组(水飞蓟素)所测出降低HepG2细胞TG含量结果如表2所示。
表2：阳性对照组之水飞蓟素降低HepG2脂肪细胞中TG含量之作用
Figure PCTCN2016078039-appb-000003
以固定试验化合物之测试浓度，所测出降低HepG2脂肪细胞TG含量之结果如表3所示。结果显示，在固定测试浓度条件下，与对照组相比，试验化合物对HepG2细胞经诱导形成之脂肪肝细胞展现不同程度之降低肝细胞TG含量之效果。TG降低率(％)计算公式为：[1-(试验组TG含量-空白组TG含量)/(油酸诱导组TG含量-空白组TG含量)]x 100％。
表3：试验化合物可降低脂肪肝细胞之TG含量
Figure PCTCN2016078039-appb-000004
Figure PCTCN2016078039-appb-000005
Figure PCTCN2016078039-appb-000006
表3-1：来自表3的部分试验化合物可降低脂肪肝细胞之TG含量
Figure PCTCN2016078039-appb-000007
Figure PCTCN2016078039-appb-000008
表3-2：来自表3的部分试验化合物(生物类黄酮)可降低脂肪肝细胞之TG含量
Figure PCTCN2016078039-appb-000009
Figure PCTCN2016078039-appb-000010
表3-3：来自表3的部分试验化合物(赋形剂)可降低脂肪肝细胞之TG含量
Figure PCTCN2016078039-appb-000011
Figure PCTCN2016078039-appb-000012
2.2动物实验
在动物实验中，除空白组给予正常饲料外，其余动物给予诱导脂肪肝处理，8周后，各组动物除维持给予原饲料外，再给予不同处理达4周或8周，其中，空白对照组及高脂对照组给予去离子水，正向对照组给予水飞蓟素，以及试验组给予不同试验化合物，包括葛根素、根皮苷、圣草酚、三氯蔗糖、甘露醇、糖精、橙皮、薄荷醇、或部分试验化合物之组合。
2.2.1试验化合物对动物体重、肝重、体脂肪重的影响及安全性评估
动物实验的结果显示，各组间动物之肝重、体脂肪重与体重增加量结果如表4-1与4-2。
表4-1：试验化合物之肝重、体脂肪重分析结果。
Figure PCTCN2016078039-appb-000013
Figure PCTCN2016078039-appb-000014
表4-2：试验化合物之体重增加量分析结果。
Figure PCTCN2016078039-appb-000015
Figure PCTCN2016078039-appb-000016
Figure PCTCN2016078039-appb-000017
结果显示，诱导脂肪肝动物腹部脂肪重量增加，在投予单一试验化合物时，甘露醇、薄荷醇、及三氯蔗糖可显著降低动物腹部脂肪重量。
此外，试验组投予不同试验化合物后，动物无任何异常症状，试验期间无造成任何动物的死亡，试验后牺牲动物剖检，亦无观察到因试验化合物所造成之病变或临床征状的发生，故该等试验化合物安全无虞。
2.2.2试验化合物具降低肝脏脂质的作用
图1显示诱导脂肪肝小鼠，在肝门管区(包括胆管、门静脉、肝动脉)附近肝脏细胞布满许多大泡状脂肪油滴，肝细胞出现气球化，成功诱导产生脂肪肝动物模式。
动物实验的结果显示，多种试验化合物在为期4周或8周的给药期间后，显示降低动物肝脏脂质的作用。结果显示于表5-1及表5-2。
表5-1：试验化合物可降低动物之肝脏脂质(4周的给药期间)
Figure PCTCN2016078039-appb-000018
Figure PCTCN2016078039-appb-000019
Figure PCTCN2016078039-appb-000020
表5-2：试验化合物可降低动物之肝脏脂质(8周的给药期间)
Figure PCTCN2016078039-appb-000021
结果显示，诱导脂肪肝小鼠肝脏中的三酸甘油酯及总胆固醇上升。在投予单一试验化合物时，橙皮、葛根素、圣草酚、根皮苷、甘露醇、薄荷醇、及三氯蔗糖可显著降低肝脏中的三酸甘油酯；尤其，圣草酚处理4周时，达优异效果，可降低约67％肝脏三酸甘油酯含量(p<0.005)。此外，橙皮、圣草酚、根皮苷、甘露醇、薄荷醇、三氯蔗糖、糖精可显著降低肝脏中的总胆固醇；尤其，糖精处理4周时，达优异效果，可降低约56％肝脏总胆固醇含量(p<0.005)。
在投予二种试验化合物之组合时，糖精与甘露醇的组合、薄荷醇与甘露醇的组合、三氯蔗糖与甘露醇的组合、圣草酚与甘露醇的组合、或圣草酚与三氯蔗糖的组合，可有效降低肝脏中的三酸甘油酯；尤其，薄荷醇与甘露醇的组合处理4周时，达优异效果，可降低约77％肝脏三酸甘油酯含量(p<0.005)；圣草酚与三氯蔗糖的组合处理8周时，达优异效果，可降低约78％肝脏三酸甘油酯含量(p<0.005)。此外，三氯蔗糖与甘露醇的组合、圣草酚与甘露醇的组合、或圣草酚与三氯蔗糖的组合，可显著降低肝脏中的总胆固醇；其中，圣草酚与三氯蔗糖的组合处理8周时，达优异效果，可降低约77％肝脏总胆固醇含量(p<0.005)。
在投予三种试验化合物之组合时，薄荷醇、甘露醇、圣草酚的组合、三氯蔗糖、甘露醇、圣草酚的组合，可有效降低肝脏中的三酸甘油酯；尤其，三氯蔗糖、甘露醇、圣草酚的组合处理8周时，达优异效果，可降低约79％肝脏三酸甘油酯含量(p<0.005)。此外，三氯蔗糖、甘露醇、圣草酚的组合，可显著降低肝脏中的总胆固醇。
2.2.3试验化合物具降低肝损伤的作用
2.2.3.1降低肝组织之脂肪肝及肝损伤的作用
动物实验的结果显示，多种试验化合物在为期4周的试验期间显示可降低动物之脂肪肝及肝组织损伤情形。图1显示，脂肪肝动物肝组织损伤，包括在肝门管区(包括胆管、门静脉、肝动脉)附近肝脏细胞布满许多大泡状脂肪油滴，肝细胞出现气球化。相较之下，经水飞蓟素、薄荷醇、圣草酚、或甘露醇治疗4周后，肝脏组织切片在肝脏细胞内大泡状脂肪油滴大量减少，其中，经水飞蓟素治疗的小鼠仍观察到部分小破状油滴，但经薄荷醇、圣草酚、或甘露醇治疗的小鼠的肝脏组织型态比较接近空白组，表示脂肪肝病变比较轻微。此外，NAS评分结果显示于表6。
表6：试验化合物可降低动物之肝损伤情形
Figure PCTCN2016078039-appb-000022
Figure PCTCN2016078039-appb-000023
NAS(Nonalcoholic Fatty Liver Disease Activity Score)分数指的是非酒精性脂肪肝病活性分数[Hepatology.2005Jun；41(6):1313-21.]，综合评估脂肪变性(steatosis)、肝小叶发炎(lobular inflammation)及肝脏细胞气球化(hepatocyte ballooning)程度，评分表如表7，分数越高表示肝损伤越严重。
Figure PCTCN2016078039-appb-000024
Figure PCTCN2016078039-appb-000025
结果显示，诱导脂肪肝小鼠肝组织损伤(NAS分数上升)。在投予单一试验化合物时，圣草酚与甘露醇可显著降低肝损伤。值得注意的是，在投予二种试验化合物之组合时，薄荷醇与甘露醇的组合，达优异效果，几乎未显现肝损伤，其NAS分数与空白组相同。
2.2.3.2降低肝功能损伤的作用
动物实验的结果显示，多种试验化合物在为期4周或8周的给药期间显示可降低动物之肝功能损伤情形。结果显示于表8-1及表8-2。
表8-1：试验化合物可降低动物之肝功能损伤情形(4周的给药期间)
Figure PCTCN2016078039-appb-000026
Figure PCTCN2016078039-appb-000027
Figure PCTCN2016078039-appb-000028
表8-2：试验化合物可降低动物之肝功能损伤情形(8周的给药期间)
Figure PCTCN2016078039-appb-000029
ALT和AST是最常用来反映肝脏生化功能损伤的酶指针。这些酶正常情况下存在于肝细胞内，当肝细胞受到破坏时便会泄露出来，血清中的ALT和AST数值上升通常可反映肝发炎及肝功能有损伤情形。
结果显示，诱导脂肪肝动物肝功能受损(ALT和AST数值上升)。在投予单一试验化合物时，橙皮、葛根素、圣草酚、根皮苷、甘露醇、薄荷醇、三氯蔗糖、 及糖精均可显著降低ALT和AST数值。尤其，甘露醇处理4周时，达优异效果，可降低约64％ALT数值(p<0.005)及约60％AST数值(p<0.005)。
在投予二种试验化合物之组合时，薄荷醇与甘露醇的组合或圣草酚与三氯蔗糖的组合可显著降低ALT数值；薄荷醇与甘露醇的组合、三氯蔗糖与甘露醇的组合、或糖精与甘露醇的组合，可显著降低AST数值。尤其，薄荷醇与甘露醇的组合处理4周时，达优异效果，可降低约76％ALT数值(p<0.005)及约62％AST数值(p<0.005)。
在投予三种试验化合物之组合时，三氯蔗糖、甘露醇、圣草酚的组合可显著降低ALT数值(p<0.005)。
2.2.4试验化合物具提高肝脏抗氧化能力
动物实验的结果显示，多种试验化合物在为期4周的试验期间显示可提升动物肝脏抗氧化能力。结果显示于表9-1及表9-2。
表9-1：试验化合物可提升动物肝脏抗氧化能力(Gpx及GSH)
Figure PCTCN2016078039-appb-000030
Figure PCTCN2016078039-appb-000031
表9-2：试验化合物可提升动物肝脏抗氧化能力(Grd及SOD)
Figure PCTCN2016078039-appb-000032
Figure PCTCN2016078039-appb-000033
Gpx、GSH、Grd及SOD是常见的肝脏抗氧化系统成员，可降低肝脏的氧化压力，使肝脏免于受到氧化压力导致的伤害。Gpx、GSH、Grd及SOD的数值提高代表肝脏维持较佳的抗氧化活性。
结果显示，诱导脂肪肝小鼠动物抗氧化活性降低。在投予单一试验化合物时，橙皮、葛根素、圣草酚、根皮苷、甘露醇、三氯蔗糖均可显著提高脏抗氧化活性。尤其，甘露醇处理4周时，达优异效果，大幅提高Gpx、GSH、Grd及SOD的水平(p<0.005)。
综上，本发明所提供之化合物可降低肝脂肪含量、降低肝损伤及提高肝脏抗氧化活性。该等化合物属于低分子天然植物酚类化合物，系广泛存在于蔬果、谷物、根茎、花卉、茶叶与红葡萄酒等，经动物试验确认安全无虞，具有发展成为降肝脂、改善相关病症，如脂肪肝、急性与慢性酒精性脂肪肝、急性与慢性非酒精性脂肪肝(Non-alcoholic Fatty Liver Disease,NAFLD)、急性与慢性酒精性肝炎、急性与慢性非酒精性脂肪肝炎、非酒精性肝硬化、酒精性肝硬化(ICD-9-CM Diagnosis Code571.8,571.0,571.1,571.2,571.3,571.4,571.5,571.9)等病症之保健食品或药物之潜力。
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Claims (34)

  1. 一种化合物用于制备预防或治疗脂肪肝、保护肝功能、或改善脂肪肝引起的肝病变或其他相关病症之组合物的用途，其中该化合物系选自于以下所组成群组：硫酸月桂酸钠(sodium lauryl sulfate)、薄荷醇(menthol)、三氯蔗糖(sucralose)、甘露醇(mannitol)、山梨醇(sorbitol)、糖精(saccharin)、甘油(glycerin)、苯甲酸钠(sodium benzoate)、氧化铁红(oxide red)、预糊化淀粉(pregelatinized starch)、环己烷氨基磺酸钠(sodium cyclamate)、山梨酸(sorbic acid)、柠檬油(lemon oil)、柠檬酸(citric acid)、及丁基羟基茴香醚(butylated hydroxyanisole)、枸橘苷(poncirin)、异牡荆素(isovitexin)、圣草酚(eriodictyol)、麦角固醇(ergosterol)、月桂烯(β-myrcene)、金丝桃苷(hyperoside)、儿茶素((+)-catechin)、高良姜素(galangin)、桑叶素(morin)、金松双黄酮(sciadopitysin)、香风草甙(didymin)、棉纤维素(gossypin)、木犀草素-7-葡萄糖甙(luteolin-7-Glucoside)、双氢槲皮素((+)-taxifolin)、肉桂酸(trans-cinnamic acid)、香叶木素(diosmin)、蒙花甙(linarin)木糖醇(xylitol)、木犀草素(luteolin)、獐牙菜苦素(swertiamarin)、及其任何组合。
  2. 根据请求项1之用途，其中该化合物系选自于以下所组成群组：硫酸月桂酸钠(sodium lauryl sulfate)、薄荷醇(menthol)、三氯蔗糖(sucralose)、甘露醇(mannitol)、山梨醇(sorbitol)、糖精(saccharin)、甘油(glycerin)、苯甲酸钠(sodium benzoate)、氧化铁红(oxide red)、预糊化淀粉(pregelatinized starch)、环己烷氨基磺酸钠(sodium cyclamate)、山梨酸(sorbic acid)、柠檬油(lemon oil)、柠檬酸(citric acid)、及丁基羟基茴香醚(butylated hydroxyanisole)、及其任何组合。
  3. 根据请求项1之用途，其中该化合物系选自于以下所组成群组：枸橘苷(poncirin)、异牡荆素(isovitexin)、圣草酚(eriodictyol)、麦角固醇(ergosterol)、月桂烯(β-myrcene)、金丝桃苷(hyperoside)、儿茶素((+)-catechin)、高良姜素(galangin)、桑叶素(morin)、金松双黄酮(sciadopitysin)、香风草甙(didymin)、棉纤维素(gossypin)、木犀草素-7-葡萄糖甙(luteolin-7-Glucoside)、双氢槲皮素 ((+)-taxifolin)、肉桂酸(trans-cinnamic acid)、香叶木素(diosmin)、蒙花甙(linarin)、木糖醇(xylitol)、木犀草素(luteolin)、獐牙菜苦素(swertiamarin)、及其任何组合。
  4. 根据请求项1之用途，其中该化合物系选自于以下所组成群组：圣草酚(eriodictyol)、甘露醇(mannitol)、薄荷醇(menthol)、三氯蔗糖(sucralose)、糖精(saccharin)、及其任何组合。
  5. 根据请求项1之用途，其中该化合物系选自于以下所组成群组：(1)糖精与甘露醇的组合、(2)薄荷醇与甘露醇的组合、(3)三氯蔗糖与甘露醇的组合、(4)圣草酚与甘露醇的组合、(5)圣草酚与三氯蔗糖、(6)薄荷醇、甘露醇、圣草酚、或(7)三氯蔗糖、甘露醇、圣草酚的组合。
  6. 根据请求项1至5中任一项之用途，其中该化合物进一步包括葛根素(puerarin)、根皮苷(phloridzin)、甜橙黄酮(sinensetin)、(-)-表没食子儿茶素((-)-epigallocatechin)、山柰(kaempferol)、熊果酸(ursolic acid)、水飞蓟素、(silymarin)、(+)-柠檬油精((+)-limonene)、橙皮甙(hesperidin)、(-)-表儿茶素-3-没食子酸酯((-)-epicatechin-3-gallate)、水飞蓟宾(silybin)、芒柄花黄素(formononetin)、十四烷酸乙酯(myristic acid ethyl ester)、二十碳五烯酸(eicosapentaenoic acid,EPA)、汉黄芩素(wongonin)、聚维酮K-30(povidone K-30)、原儿茶酸(protocatechuic acid)、伞形酮(umbelliferone)、橙皮(hesperitin)、去甲二氢愈创木酸(nordihydroguaiaretic acid)、新橙皮苷(neohesperidin)、柚皮苷(naringin)、(-)-表儿茶素((-)-epicatechin)、甘草甜素(glycyrrhizin)、黄芩苷(baicalin)、斛皮素(quercitrin)、及/或黄芩甙元(baicalein)。
  7. 根据请求项6之用途，其中该组合物系用于降低个体之肝脂肪含量。
  8. 根据请求项6之用途，其中该组合物系用于降低个体之肝细胞之脂肪含量。
  9. 根据请求项6之用途，其中该组合物系用于降低个体之肝损害。
  10. 根据请求项9之用途，其中该肝损害包括肝组织损害或肝功能损害。
  11. 根据请求项6之用途，其中该组合物系用于提高肝脏抗氧化活性。
  12. 根据请求项6之用途，其中该肝病变或其他相关病症系选自于以下所组成的群组、急性与慢性酒精性脂肪肝、急性与慢性非酒精性脂肪肝、急性与慢性酒精性肝炎、急性与慢性非酒精性脂肪肝炎、非酒精性肝硬化、及酒精性肝硬化。
  13. 根据请求项1至5中任一项之用途，其中该组合物可施用于非酒精性脂肪肝疾病之患者或肥胖者。
  14. 根据请求1至5中任一项之用途，其中该组合物为药物、食品添加物或健康食品。
  15. 一种组合物，其包括选自于以下所组成之群组的任何两种或以上的化合物：硫酸月桂酸钠(sodium lauryl sulfate)、薄荷醇(menthol)、三氯蔗糖(sucralose)、甘露醇(mannitol)、山梨醇(sorbitol)、糖精(saccharin)、甘油(glycerin)、苯甲酸钠(sodium benzoate)、氧化铁红(oxide red)、预糊化淀粉(pregelatinized starch)、环己烷氨基磺酸钠(sodium cyclamate)、山梨酸(sorbic acid)、柠檬油(lemon oil)、柠檬酸(citric acid)、丁基羟基茴香醚(butylated hydroxyanisole)、枸橘苷(poncirin)、异牡荆素(isovitexin)、圣草酚(eriodictyol)、麦角固醇(ergosterol)、月桂烯(β-myrcene)、金丝桃苷(hyperoside)、儿茶素((+)-catechin)、高良姜素(galangin)、桑叶素(morin)、金松双黄酮(sciadopitysin)、香风草甙(didymin)、棉纤维素(gossypin)、木犀草素-7-葡萄糖甙(luteolin-7-Glucoside)、双氢槲皮素((+)-taxifolin)、肉桂酸(trans-cinnamic acid)、香叶木素(diosmin)、蒙花甙(linarin)、异红草素(homoorientin)、木糖醇(xylitol)、木犀草素(luteolin)、獐牙菜苦素(swertiamarin)、。
  16. 根据请求项15之组合物，其包括选自于以下所组成之群组的任何两种或以上的化合物：硫酸月桂酸钠(sodium lauryl sulfate)、薄荷醇(menthol)、三氯蔗糖(sucralose)、甘露醇(mannitol)、山梨醇(sorbitol)、糖精(saccharin)、甘油(glycerin)、苯甲酸钠(sodium benzoate)、氧化铁红(oxide red)、预糊化淀粉(pregelatinized starch)、环己烷氨基磺酸钠(sodium cyclamate)、山梨酸(sorbic acid)、柠檬油(lemon oil)、柠檬酸(citric acid)、及丁基羟基茴香醚(butylated hydroxyanisole)。
  17. 根据请求项15之组合物，其包括选自于以下所组成之群组的任何两种或以上的化合物：枸橘苷(poncirin)、异牡荆素(isovitexin)、圣草酚(eriodictyol)、麦角固醇(ergosterol)、月桂烯(β-myrcene)、金丝桃苷(hyperoside)、儿茶素((+)-catechin)、高良姜素(galangin)、桑叶素(morin)、金松双黄酮(sciadopitysin)、香风草甙(didymin)、棉纤维素(gossypin)、木犀草素-7-葡萄糖甙(luteolin-7-Glucoside)、双氢槲皮素((+)-taxifolin)、肉桂酸(trans-cinnamic acid)、香叶木素(diosmin)、蒙花甙(linarin)、木糖醇(xylitol)、木犀草素(luteolin)、及獐牙菜苦素(swertiamarin)。
  18. 根据请求项15之组合物，其包括选自于以下所组成之群组的任何两种或以上的化合物：、圣草酚(eriodictyol)、甘露醇(mannitol)、薄荷醇(menthol)、三氯蔗糖(sucralose)、及糖精(saccharin)。
  19. 根据请求项15之组合物，其包括选自于以下所组成之群组的组合：(1)糖精与甘露醇的组合、(2)薄荷醇与甘露醇的组合、(3)三氯蔗糖与甘露醇的组合、(4)圣草酚与甘露醇的组合、(5)圣草酚与三氯蔗糖、(6)薄荷醇、甘露醇、圣草酚、或(7)三氯蔗糖、甘露醇、圣草酚的组合。
  20. 根据请求项15至19中任一项之组合物，其进一步包括葛根素(puerarin)、根皮苷(phloridzin)、甜橙黄酮(sinensetin)、(-)-表没食子儿茶素((-)-epigallocatechin)、山柰(kaempferol)、熊果酸(ursolic acid)、水飞蓟素、(silymarin)、(+)-柠檬油精((+)-limonene)、橙皮甙(hesperidin)、(-)-表儿茶素-3-没食子酸酯((-)-epicatechin-3-gallate)、水飞蓟宾(silybin)、芒柄花黄素(formononetin)、十四烷酸乙酯(myristic acid ethyl ester)、二十碳五烯酸(eicosapentaenoic acid,EPA)、汉黄芩素(wongonin)、聚维酮K-30(povidone K-30)、原儿茶酸(protocatechuic acid)、伞形酮(umbelliferone)、橙皮(hesperitin)、去甲二氢愈创木酸(nordihydroguaiaretic acid)、新橙皮苷(neohesperidin)、柚皮苷(naringin)、(-)-表儿茶素((-)-epicatechin)、甘草甜素(glycyrrhizin)、黄芩苷(baicalin)、斛皮素(quercitrin)、及/或黄芩甙元(baicalein)。
  21. 根据请求项20之组合物，其中该化合物各自或组合系以可降低个体之肝脂肪含量或改善相关病症之有效量存在于该组合物中。
  22. 根据请求项20之组合物，其中该化合物各自或组合系以可降低个体之肝细胞之脂肪含量之有效量存在于该组合物中。
  23. 根据请求项20之组合物，其中该化合物各自或组合系以可降低个体之肝损害之有效量存在于该组合物中。
  24. 根据请求项23之组合物，其中该肝损害包括肝组织损害或肝功能损害。
  25. 根据请求项20之组合物，其中该化合物各自或组合系以可提高肝脏抗氧化活性之有效量存在于该组合物中。
  26. 根据请求项20之组合物，其中该化合物各自或组合系以可治疗或预防脂肪肝、急性与慢性酒精性脂肪肝、急性与慢性非酒精性脂肪肝、急性与慢性酒精性肝炎、急性与慢性非酒精性脂肪肝炎、非酒精性肝硬化、及酒精性肝硬化之有效量存在于该组合物中。
  27. 根据请求20之组合物，其中该组合物为药物、食品添加物或健康食品。
  28. 一种在有需要的个体预防或治疗脂肪肝、保护肝功能、或改善脂肪肝引起的肝病变或其他相关病症之方法，其包括将有效量的化合物投用于该个体，其中该化合物系选自于以下所组成群组：硫酸月桂酸钠(sodium lauryl sulfate)、薄荷醇(menthol)、三氯蔗糖(sucralose)、甘露醇(mannitol)、山梨醇(sorbitol)、糖精(saccharin)、甘油(glycerin)、苯甲酸钠(sodium benzoate)、氧化铁红(oxide red)、预糊化淀粉(pregelatinized starch)、环己烷氨基磺酸钠(sodium cyclamate)、山梨酸(sorbic acid)、柠檬油(lemon oil)、柠檬酸(citric acid)、及丁基羟基茴香醚(butylated hydroxyanisole)、枸橘苷(poncirin)、异牡荆素(isovitexin)、圣草酚(eriodictyol)、麦角固醇(ergosterol)、月桂烯(β-myrcene)、金丝桃苷(hyperoside)、儿茶素((+)-catechin)、高良姜素(galangin)、桑叶素(morin)、金松双黄酮(sciadopitysin)、香风草甙(didymin)、棉纤维素(gossypin)、木犀草素-7-葡萄糖甙(luteolin-7-Glucoside)、双氢槲皮素((+)-taxifolin)、肉桂酸(trans-cinnamic acid)、香叶木素(diosmin)、蒙花甙(linarin)、异红草素(homoorientin)、木糖醇(xylitol)、犀草素(luteolin)、獐牙菜苦素(swertiamarin)、及其任何组合。
  29. 一种化合物用于制备降低肝脂肪含量及改善相关病症之组合物的用途，其中该化合物系选自于以下所组成群组：硫酸月桂酸钠(sodium lauryl sulfate)、薄荷醇(menthol)、三氯蔗糖(sucralose)、甘露醇(mannitol)、山梨醇(sorbitol)、糖精(saccharin)、甘油(glycerin)、苯甲酸钠(sodium benzoate)、氧化铁红(oxide red)、预糊化淀粉(pregelatinized starch)、环己烷氨基磺酸钠(sodium cyclamate)、山梨酸(sorbic acid)、柠檬油(lemon oil)、柠檬酸(citric acid)、及丁基羟基茴香醚(butylated hydroxyanisole)、枸橘苷(poncirin)、异牡荆素(isovitexin)、圣草酚(eriodictyol)、麦角固醇(ergosterol)、月桂烯(β-myrcene)、金丝桃苷(hyperoside)、儿茶素((+)-catechin)、高良姜素(galangin)、桑叶素(morin)、金松双黄酮(sciadopitysin)、香风草甙(didymin)、棉纤维素(gossypin)、木犀草素-7-葡萄糖甙(luteolin-7-Glucoside)、双氢槲皮素((+)-taxifolin)、肉桂酸(trans-cinnamic acid)、香叶木素(diosmin)、蒙花甙(linarin)木糖醇(xylitol)、木犀草素(luteolin)、獐牙菜苦素(swertiamarin)、及其任何组合。
  30. 根据请求项29之用途，其中该化合物系选自于以下所组成群组：硫酸月桂酸钠(sodium lauryl sulfate)、薄荷醇(menthol)、三氯蔗糖(sucralose)、甘露醇(mannitol)、山梨醇(sorbitol)、糖精(saccharin)、甘油(glycerin)、苯甲酸钠(sodium benzoate)、氧化铁红(oxide red)、预糊化淀粉(pregelatinized starch)、环己烷氨基磺酸钠(sodium cyclamate)、山梨酸(sorbic acid)、柠檬油(lemon oil)、柠檬酸(citric acid)、及丁基羟基茴香醚(butylated hydroxyanisole)、及其任何组合。
  31. 根据请求项29之用途，其中该化合物系选自于以下所组成群组：枸橘苷(poncirin)、异牡荆素(isovitexin)、圣草酚(eriodictyol)、麦角固醇(ergosterol)、月桂烯(β-myrcene)、金丝桃苷(hyperoside)、儿茶素((+)-catechin)、高良姜素(galangin)、桑叶素(morin)、金松双黄酮(sciadopitysin)、香风草甙(didymin)、棉纤维素(gossypin)、木犀草素-7-葡萄糖甙(luteolin-7-Glucoside)、双氢槲皮素((+)-taxifolin)、肉桂酸(trans-cinnamic acid)、香叶木素(diosmin)、蒙花甙(linarin)、木糖醇(xylitol)、木犀草素(luteolin)、獐牙菜苦素(swertiamarin)、及其任何组合。
  32. 根据请求项29之用途，其中该化合物系选自于以下所组成群组：圣草酚(eriodictyol)、甘露醇(mannitol)、薄荷醇(menthol)、三氯蔗糖(sucralose)、糖精(saccharin)、及其任何组合。
  33. 根据请求项29之用途，其中该化合物系选自于以下所组成群组：(1)糖精与甘露醇的组合、(2)薄荷醇与甘露醇的组合、(3)三氯蔗糖与甘露醇的组合、(4)圣草酚与甘露醇的组合、(5)圣草酚与三氯蔗糖、(6)薄荷醇、甘露醇、圣草酚、或(7)三氯蔗糖、甘露醇、圣草酚的组合。
  34. 根据请求项29至33中任一项之用途，其中该化合物进一步包括葛根素(puerarin)、根皮苷(phloridzin)、甜橙黄酮(sinensetin)、(-)-表没食子儿茶素((-)-epigallocatechin)、山柰(kaempferol)、熊果酸(ursolic acid)、水飞蓟素、(silymarin)、(+)-柠檬油精((+)-limonene)、橙皮甙(hesperidin)、(-)-表儿茶素-3-没食子酸酯((-)-epicatechin-3-gallate)、水飞蓟宾(silybin)、芒柄花黄素(formononetin)、十四烷酸乙酯(myristic acid ethyl ester)、二十碳五烯酸(eicosapentaenoic acid,EPA)、汉黄芩素(wongonin)、聚维酮K-30(povidone K-30)、原儿茶酸(protocatechuic acid)、伞形酮(umbelliferone)、橙皮(hesperitin)、去甲二氢愈创木酸(nordihydroguaiaretic acid)、新橙皮苷(neohesperidin)、柚皮苷(naringin)、(-)-表儿茶素((-)-epicatechin)、甘草甜素(glycyrrhizin)、黄芩苷(baicalin)、斛皮素(quercitrin)、及/或黄芩甙元(baicalein)。
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EP3991724A3 (en) * 2015-09-24 2022-07-27 Sinew Pharma Inc. Compounds effective in treating hepatotoxicity and fatty liver diseases and uses thereof
CN112471376A (zh) * 2020-11-02 2021-03-12 辽宁康汇医学临床研究有限公司 一种防治酒精性脂肪肝的固体饮料及其制备方法

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