WO2021256858A1 - Composition pour améliorer, prévenir ou traiter une stéatose hépatique non alcoolique et son procédé de préparation - Google Patents

Composition pour améliorer, prévenir ou traiter une stéatose hépatique non alcoolique et son procédé de préparation Download PDF

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WO2021256858A1
WO2021256858A1 PCT/KR2021/007565 KR2021007565W WO2021256858A1 WO 2021256858 A1 WO2021256858 A1 WO 2021256858A1 KR 2021007565 W KR2021007565 W KR 2021007565W WO 2021256858 A1 WO2021256858 A1 WO 2021256858A1
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fatty liver
liver disease
pharmaceutical composition
compound
nutmeg
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PCT/KR2021/007565
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English (en)
Korean (ko)
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장시영
서승현
김대환
조은애
전수빈
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주식회사 웰니스바이오
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    • 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)
    • 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
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • 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
    • A61K31/075—Ethers or acetals
    • A61K31/085—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon
    • A61K31/09—Ethers or acetals having an ether linkage to aromatic ring nuclear carbon having two or more such linkages
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6949—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes
    • A61K47/6951—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit inclusion complexes, e.g. clathrates, cavitates or fullerenes using cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • 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
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2250/00—Food ingredients
    • A23V2250/30—Other Organic compounds

Definitions

  • the present invention relates to a composition for improving, preventing or treating nonalcoholic fatty liver disease, comprising a nutmeg extract and/or a lignan compound derived therefrom, and a method for preparing the same.
  • the liver is a multifunctional organ that plays essential roles in body metabolism, biosynthesis, excretion, secretion and detoxification. Because this process requires energy, it makes the liver a highly aerobic, oxygen-dependent tissue. This process also confers vulnerability of the liver to anaerobes, increases susceptibility to harmful substances, and requires replacement of cells after tissue loss. Enhanced hepatocyte death and regeneration of damaged cells are indeed hallmarks of most liver diseases. The liver is able to regenerate from massive cell loss caused by cell proliferation. However, with many chronic liver diseases and chronic exposure to hepatic toxins such as alcohol, regeneration cannot keep pace with hepatocellular death.
  • Non-Patent Document 1 Fibrosis produced by hepatic stellate cells is gradually replaced and replaced by functional hepatocytes, which impairs liver function, resulting in liver dysfunction.
  • Liver disease ranges from mild reversible fatty liver disease to progressive chronic liver disease, which can lead to the development of life-threatening conditions such as cirrhosis of the liver, liver failure and liver cancer.
  • Chronic liver disease is the leading cause of mortality and morbidity worldwide. Most chronic liver diseases progress from mild inflammation to more severe inflammation, leading to fibrosis or cirrhosis. This may cause liver failure and portal hypertension, and increase the risk of liver cancer (Non-Patent Document 2).
  • Hepatitis B virus is the most common cause of liver disease in Koreans, followed by hepatitis C virus (HCV), alcoholic fatty liver disease, and non-alcoholic steatohepatitis (NASH). It consists of etc.
  • Non-alcoholic liver disease refers to a case where the cause of fatty liver is not caused by alcohol. to be.
  • non-alcoholic steatohepatitis refers to extensive liver damage, ranging from simple steatosis to fatty liver disease, cholestasis, hyperactive fibrosis and cirrhosis of the liver.
  • the pathological features are similar to those of alcohol-induced liver injury, but only occur in patients who do not abuse alcohol.
  • Nonalcoholic steatohepatitis should be distinguished from steatosis with or without hepatitis due to secondary causes, since these conditions clearly have different pathogens and consequences.
  • Secondary causes of fatty liver disease include nutrition (eg protein-calorie malnutrition, starvation, total parenteral nutrition, rapid weight loss, gastrointestinal surgery due to obesity), drugs (eg glucocorticoids, synthetic estrogens, aspirin, calcium-channels) Blockers, tetracyclines, valproic acid, cocaine, antiviral agents, pyaluridine, interferon- ⁇ , methotrexate, zidovudine), metabolizers or genetic agents (such as lipodystrophy, betalipoprotein disorder, Weber-Christian disease, galactosemia, glycogen storage disorders, gestational acute fatty liver) and other causes such as diabetes, obesity or hyperlipidemia (Non-Patent Documents 3 and 4).
  • Nonalcoholic steatohepatitis a liver disease that has been receiving the most attention recently, is increasing significantly along with the increase in the obese population due to westernized eating habits, lack of exercise, and changes in lifestyle. It is a disease that urgently needs to be addressed.
  • Nonalcoholic steatohepatitis is caused by not properly managing nonalcoholic fatty liver, in which more than 5% of fat (mainly triglycerides) has accumulated in the liver due to excessive calorie intake, lack of activity and lack of exercise, and a family history of liver disease. It causes circulation disorders in the lymphatic system, leading to a decrease in liver function.
  • liver cirrhosis liver cirrhosis
  • Nutmeg Myristica fragrans HOUTT.
  • Nutmeg is the seed of the nutmeg family (Myristicaceae), the acryl and seed peeled, soaked in lime, and then dried.
  • Nutmeg is an evergreen tree and is a dioecious plant grown in Sumatra and Java. The fruit is a spherical flesh with a seed surrounded by a red aril in the middle.
  • Nutmeg has been used extensively as a flavoring agent for foods such as sauces since ancient times, and in oriental medicine, it has been used as a flavoring agent for diarrhea, abdominal distension, vomiting, and loss of appetite.
  • nutmeg is known to exhibit growth inhibitory activity of Helicobacter pylori (Non-Patent Document 5) and activation of a liver detoxification mechanism (Non-Patent Document 6).
  • Lignans-based compounds having a mother nucleus such as alkyl aryl ether, dibenzylbutane, and tetrahydrofuran of nutmeg are PPAR ⁇ (Peroxisome) involved in hepatocyte differentiation, growth and metabolism.
  • PPAR ⁇ Peroxisome
  • NRF2 expressed by the NFE 2 L 2 (Nuclear factor erythroid-drived2-like2) gene, which has a mechanism to regulate proliferator activated receptor alpha and protects against oxidative damage induced by inflammation or toxic substances.
  • Non-Patent Document 7 is known to have a pharmacological mechanism, but the therapeutic effect related to nonalcoholic steatohepatitis has not been reported.
  • prior patents related to nutmeg extract include a composition for preventing or treating vascular disease containing nutmeg extract or a lignan-based compound isolated therefrom (Korea Patent No. 10-1338901), a lignan-based compound or nutmeg containing the same Novel use of extract or nutmeg agaric bark extract (Korean Patent No. 10-1088071), functional food containing nutmeg extract for the improvement and prevention of obesity symptoms (Korean Patent No. 10-1079916), nutmeg agar skin extract compound is effective Pharmaceutical composition for preventing and treating diabetes or PPAR-gamma (PPAR- ⁇ ) mediated disease comprising as a component (Korea Patent No.
  • composition for preventing or treating endometriosis comprising nutmeg extract (Korea) Patent No. 10-1787458) and the like, but none of them are related to non-alcoholic fatty liver disease or non-alcoholic steatohepatitis.
  • Patent Document 1 Republic of Korea Patent No. 10-1338901
  • Patent Document 2 2. Republic of Korea Patent No. 10-1088071
  • Patent Document 3 3. Republic of Korea Patent No. 10-1079916
  • Patent Document 4 4. Republic of Korea Patent No. 10-0959557
  • Patent Document 5 Republic of Korea Patent No. 10-1787458
  • Non-Patent Document 1 Malhi H. et al., Apoptosis and necrosis in the liver: a tale of two deaths? Hepatology, 43(2 Suppl 1); S31-44, 2006
  • Non-Patent Document 2 (Non-Patent Document 2)2. Iredale JP: Cirrhosis: new research provides a basis for rational and targeted treatments. BMJ, 327 (7407); 143-147, 2003
  • Non-Patent Document 3 Anguilo P., 2002, N. Engl. J. Med., 346: 1221-1231
  • Non-Patent Document 4 MacSween R.N.M. et al., 2002, Pathology of the Liver. Fourth Edition. Churchill Livingstone, Elsevier Science
  • Non-Patent Document 5 Bhamarapravati S. et al., Extracts of spice and food plants from Thai traditional medicine inhibit the growth of the human carcinogen Helicobacter pylori. In Vivo., 17(6); 541-4, 2003
  • Non-Patent Document 6 Non-Patent Document 6
  • Singh A. et al. Modulatory effect of Areca nut on the action of mace (Myristica fragrans, Houtt) on the hepatic detoxification system in mice.
  • Non-Patent Document 7 Xiao-Nan Yang et al., PPAR ⁇ Mediates the Hepatoprotective Effects of Nutmeg. J. Proteome Res. 17; 1887-1897, 2018
  • the present invention is to provide a composition effective for the prevention, treatment and improvement of non-alcoholic fatty liver disease and a method for preparing the same, specifically, a non-alcoholic nutmeg ( Myristica fragrans ) extract and a lignan-based compound derived therefrom as an active ingredient.
  • An object of the present invention is to provide a composition for preventing and treating alcoholic fatty liver disease and a method for preparing the same.
  • the present invention provides a pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease comprising a nutmeg extract and a lignan compound derived therefrom as an active ingredient, and preventing or improving non-alcoholic fatty liver disease comprising the same It provides a food composition for use, and a method for preparing the same.
  • composition according to the present invention is effective in preventing or treating non-alcoholic fatty liver disease, and thus can be usefully applied as a therapeutic agent for non-alcoholic fatty liver disease or as a health food for improving liver disease symptoms.
  • the present invention provides a pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, comprising as an active ingredient a nutmeg extract or a compound of Formula 1 or a pharmaceutically acceptable salt thereof.
  • the compound of Formula 1 is (1S,2R)-2-[2,6-dimethoxy-4-(prop-2-en-1-yl)phenoxy]-1-(4-hydroxy-3- Methoxyphenyl)propyl acetate ((1S,2R)-2-[2,6-dimethoxy-4-(prop-2-en-1-yl)phenoxy]-1-(4-hydroxy-3-methoxyphenyl)propyl acetate) (molecular formula: C 23 H 28 O 7 , molecular weight: 416.46).
  • nutmeg refers to dried seeds of nutmeg (Myristica fragrans).
  • extract means any substance that is wholly or partially liquid at about 20° C. to 50° C. and is hydrophobic but soluble in at least one organic solvent.
  • nutmeg extract means that obtained by grinding dried seeds of nutmeg, extracting it using supercritical or polar and non-polar solvents, and separating the extract.
  • active ingredient refers to a component that can exhibit a desired activity alone or can exhibit activity together with a carrier that has no activity by itself.
  • non-alcoholic fatty liver disease refers to a radiological examination or biopsy without significant alcohol intake, taking a drug that causes fatty liver, or liver disease due to other accompanying causes. It is a disease that shows intrahepatic fat deposition in the liver, and it is a diagnostic name that encompasses nonalcoholic fatty liver, nonalcoholic steatohepatitis, and nonalcoholic fatty liver-associated cirrhosis.
  • nonalcoholic fatty liver refers to a case in which fat deposition is observed in the liver, but there is no evidence of hepatocyte damage (balloon deformation) and fibrosis.
  • nonalcoholic steatohepatitis refers to a case in which inflammatory findings accompanied by hepatocyte damage (balloon deformation) while showing fat deposition in the liver, and sometimes accompanied by fibrosis.
  • nonalcoholic fatty liver-associated cirrhosis refers to cirrhosis occurring in patients with nonalcoholic fatty liver or steatohepatitis histologically, or histologically proven nonalcoholic fatty liver or steatohepatitis.
  • prevention means inhibiting or delaying the onset of a disease, disorder or disease. Prevention may be considered complete if the onset of the disease, disorder or condition is suppressed or delayed for a predetermined period of time.
  • treatment refers to a specific disease, disorder and/or symptom according to the disease or condition, partially or completely alleviating, ameliorating, alleviating, inhibiting or delaying, reducing the severity, or reducing the severity of one or more symptoms or characteristics. means to reduce the incidence.
  • the nonalcoholic fatty liver disease to which the composition according to the present invention is applied may be nonalcoholic fatty liver, nonalcoholic steatohepatitis, or nonalcoholic fatty liver-associated cirrhosis.
  • the nutmeg extract of the present invention may contain the compound of Formula 1 or a pharmaceutically acceptable salt thereof.
  • the compound of Formula 1 or a pharmaceutically acceptable salt thereof of the present invention may be isolated from a nutmeg extract.
  • Nutmeg extract according to the present invention comprises the steps of: 1) preparing an extract by adding an extraction solvent to nutmeg; 2) filtering the extract of step 1); and 3) drying the filtered filtrate of step 2) under reduced pressure.
  • the nutmeg extract of the present invention may be extracted with one or more solvents selected from the group consisting of water, methanol, ethanol, n-butanol, acetone, ethyl acetate, hexane and chloroform.
  • the solvent may be ethanol, more specifically 50 to 100% ethanol, more specifically 80 to 100% ethanol.
  • the extraction solvent may be added in an amount of preferably 1 to 50 mL, more preferably 1 to 30 mL, and most preferably 1 to 20 mL per 1 g of the weight of nutmeg used for extraction.
  • the extraction temperature, extraction time, and number of extractions may be appropriately selected.
  • the extraction temperature may be preferably 30 to 120 °C, more preferably 50 to 100 °C, and most preferably 70 to 90 °C.
  • the extraction time may be preferably 1 to 10 hours, more preferably 1 to 8 hours, and most preferably 1 to 5 hours.
  • the number of extractions may be preferably 1 to 5 times.
  • the extraction method may be preferably shaking extraction, Soxhlet extraction, reflux extraction, or supercritical fluid extraction, and most preferably reflux extraction or supercritical fluid extraction. Therefore, according to one embodiment of the present invention, the nutmeg extract of the present invention may be a supercritical fluid extract or a reflux extract. When the extract is used, it is preferable in terms of extraction efficiency of the extract, the yield of the compound of Formula 1, and the therapeutic effect of non-alcoholic fatty liver disease and the hepatocellular protective effect.
  • the term "supercritical fluid extraction” can be divided into a method of passing liquefied carbon dioxide through a reactor, and a method of passing a cosolvent auxiliary.
  • supercritical fluid extraction of the present invention is performed by passing liquefied carbon dioxide and an aqueous ethanol solution as a cosolvent through nutmeg powder in a reactor heated to a certain temperature to obtain an extract, and then the extract It may be a method of concentration by evaporation and freeze-drying of the concentrate.
  • the supercritical fluid extract may be extracted by a supercritical extraction method using carbon dioxide made in a supercritical state under a temperature of 35 to 100 °C and a pressure of 100 to 500 bar. More specifically, it may be at a temperature of 35 to 70° C., under a pressure of 200 to 450 bar, and even more specifically at a temperature of 40 to 60° C., under a pressure of 250 to 400 bar.
  • the extraction efficiency of the extract, the yield of the compound of Formula 1, and the therapeutic effect of nonalcoholic fatty liver disease and the hepatocellular protection effect are preferable.
  • the supercritical fluid extraction method may be to use a mixed fluid in which a cosolvent is additionally mixed with carbon dioxide made in a supercritical state.
  • the cosolvent may be at least one selected from the group consisting of water, methanol, ethanol, n-butanol, acetone, ethyl acetate, hexane and chloroform.
  • the cosolvent may be ethanol, and more specifically, ethanol at a concentration of 20 to 100%, more specifically, ethanol at a concentration of 50 to 100%, and even more specifically, ethanol at a concentration of 60 to 80%.
  • the solvent of the above conditions it is preferable in terms of extraction efficiency of the extract, the yield of the compound of Formula 1, and the therapeutic effect of non-alcoholic fatty liver disease and the hepatocellular protection effect.
  • the extraction time of the supercritical fluid extraction may be preferably 1 to 12 hours, more preferably 1 to 8 hours, and most preferably 1 to 4 hours.
  • reflux extraction may be a method of extracting under reflux by adding a solvent to nutmeg powder.
  • the reflux extraction solvent may be at least one selected from the group consisting of water, methanol, ethanol, n-butanol, acetone, ethyl acetate, hexane and chloroform.
  • the reflux extraction solvent may be ethanol, more specifically 50 to 100% ethanol, and even more specifically 80 to 100% ethanol.
  • the vacuum concentration may preferably be performed using a vacuum vacuum concentrator or a vacuum rotary evaporator.
  • the drying may be preferably reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying, and most preferably freeze drying.
  • composition according to the present invention comprises the compound (1S,2R)-2-[2,6-dimethoxy-4-(prop-2-en-1-yl)phenoxy]-1-(4-hydroxy- 3-methoxyphenyl)propyl acetate ((1S,2R)-2-[2,6-dimethoxy-4-(prop-2-en-1-yl)phenoxy]-1-(4-hydroxy-3-methoxyphenyl )propyl acetate) (molecular formula: C 23 H 28 O 7 , molecular weight: 416.46) may include, of course, a pharmaceutically acceptable salt thereof.
  • the "pharmaceutically acceptable salt” according to the present invention may be in the form of a therapeutically active, non-toxic base or acid addition salt capable of forming a compound represented by formula (1).
  • the non-toxic base is sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate chloride, bromide, iodide , fluoride, acetate, propionate, decanoate, caprylate, acrylate, formate, isobutyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, suberate, Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, methoxy Benzoate, phthalate, terephthalate, benzenesulfonate, toluenesulfon
  • the acid addition salt may be an acid addition salt formed with a pharmaceutically acceptable free acid, but is not limited thereto.
  • a pharmaceutically acceptable free acid e.g., hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, Inorganic acids such as nitrous acid or phosphorous acid; organic acids such as lactic acid, maleic acid, gluconic acid, methanesulfonic acid, 4-toluenesulfonic acid, tartaric acid, and fumaric acid.
  • the solvent of the solvate is not particularly limited, but may preferably be a hydrate or an alcoholate.
  • the compounds according to the invention may exist in different polymorphic forms. Although not explicitly indicated in the above formula, such forms are intended to be included within the scope of the present invention.
  • the composition of the present invention may contain 0.0001 to 90% by weight of the nutmeg extract or the compound of Formula 1 or a pharmaceutically acceptable salt thereof, based on the total weight of the composition, and specifically 0.1 to 50 It may be included in weight% or 0.1 to 30% by weight.
  • the pharmaceutical composition of the present invention is prepared in unit dose form or multi-dose by formulating using a pharmaceutically acceptable carrier according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. It can be prepared by introducing into a container.
  • the composition of the present invention may further include a pharmaceutically acceptable carrier.
  • carrier refers to a compound that facilitates the addition of a compound into a cell or tissue
  • pharmaceutically acceptable is physiologically acceptable and, when administered to a human, usually gastrointestinal It refers to a composition that does not cause an allergic reaction such as disorder or dizziness or a reaction similar thereto.
  • the pharmaceutically acceptable carriers are those commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl. pyrrolidone, cellulose, water, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.
  • the pharmaceutical composition of the present invention may further include additives such as fillers, anti-aggregants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives in addition to the above components.
  • additives such as fillers, anti-aggregants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives in addition to the above components.
  • the content of the additive included in the pharmaceutical composition is not particularly limited and may be appropriately adjusted within the content range used for conventional formulation.
  • the pharmaceutical composition of the present invention can be prepared according to a conventional method for each purpose of use, and oral formulations such as solutions, suspensions, powders, granules, tablets, capsules, pills, extracts, emulsions, syrups, aerosols, etc., and sterile injection solutions It can be formulated and used in various forms, such as injections, and can be administered through various routes including oral administration, intravenous, intraperitoneal, subcutaneous, rectal, topical administration, and the like.
  • the composition of the present invention may be formulated in the form of a liquid, powder, granule, tablet, capsule, pill, troche, or extract.
  • liquid preparation refers to a drug that is taken in the form of a drug dissolved in water or an organic solvent.
  • the liquid preparation has the advantage of more effective drug absorption from the intestinal tract into the systemic circulation compared to the suspension or solid preparation, and the liquid preparation may also contain additional solutes in addition to the pharmaceutical, providing color, odor, sweetness or stability. Additives may also be included.
  • the term "suspending agent” refers to any agent capable of providing the desired solubility and/or dispersibility of the alginate containing composition, i.e. providing an aqueous formulation that is substantially clear and free of sedimentation and lumps. it means.
  • binder refers to a finely divided drug, chemical, or a dry mixture of both.
  • the term "granule” refers to a pharmaceutical or a mixture of pharmaceuticals in granular form, which usually passes through a sieve of 4.76 to 20 mm.
  • Granules are generally produced by wetting the powder or powder mixture and passing the mass through a sieve or granulator of an appropriate mesh size depending on the size of the granules required. Since granules are in the form of particles, like powders, the degree of contact of the drug on the tongue is large, and when a drug having a bitter taste is used in the form of a granule, it may cause inconvenience to patients, especially children or the elderly.
  • tablette refers to a powdered pharmaceutical product made easy to take by compressing it into a small disk shape. Tablets may include uncoated tablets, film coated tablets, dragee tablets, multi-layer tablets, cored tablets, inner core tablets, orally disintegrating tablets, chewable tablets, effervescent tablets, dispersed tablets, dissolved tablets, and the like.
  • capsule refers to a pharmaceutical product made by filling a capsule in the form of a liquid, suspension, water, powder, granular, mini-tablet or pellet, or encapsulating it with a capsule base.
  • pill is meant to encompass small round solid dosage forms comprising composite particles mixed with a binder and other excipients.
  • extract refers to leaching of medicinal ingredients in plant or animal herbal medicines using an appropriate leaching agent, evaporating the solvent to concentrate it to a prescribed concentration, and adding excipients in cases where the content of the main ingredient is specified. It means a semi-solid or solid preparation made by adjusting.
  • the term "syrup" means a concentrated homemade sugar or sugar substitute.
  • the syrup is a drug having an unpleasant taste, for example, a bitter taste in a liquid form to make it easy to take, and is particularly suitable for children to take.
  • the syrup agent may include, in addition to purified water and nutmeg extract, sadang or a substitute for sadang used to give sweetness and viscosity, an antibacterial preservative, a flavoring agent, a flavoring agent, or a coloring agent, but is not limited thereto.
  • sweeteners examples include sucrose, mannitol, sorbitol, xylitol, aspartame, stevioside, fructose, lactose, sucralose, saccharin or menthol, but are not limited thereto.
  • Preferred dosage of the pharmaceutical composition of the present invention is the patient's condition and weight, age, sex, health condition, dietary constitution specificity, the nature of the preparation, the degree of disease, the administration time of the composition, administration method, administration period or interval, excretion
  • the range may vary depending on the rate and drug form, and may be appropriately selected by those skilled in the art. For example, it may be in the range of about 0.1 to 10,000 mg/kg, in the range of about 1 to 8,000 mg/kg, in the range of about 5 to 6,000 mg/kg, or in the range of about 10 to 4,000 mg/kg, preferably about It may be in the range of 50 to 2,000 mg/kg, but is not limited thereto, and may be administered in divided doses from once to several times a day.
  • the term “effective dosage of a pharmaceutical composition” refers to an amount of the composition of an active ingredient sufficient to treat a specific condition. It may vary depending on the formulation method, administration method, administration time and/or route of administration of the pharmaceutical composition, and the type and extent of the response to be achieved by administration of the pharmaceutical composition, the type, age, weight, and It may vary depending on a number of factors and similar factors well known in the pharmaceutical field, including general health conditions, symptoms or severity of disease, sex, diet, excretion, components of drugs or other compositions used simultaneously or concurrently with the subject. , a person of ordinary skill in the art can easily determine and prescribe an effective dosage for the desired treatment.
  • Administration of the pharmaceutical composition of the present invention may be administered once a day, may be administered divided into several times.
  • the pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. In consideration of all of the above factors, it can be administered in an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by a person skilled in the art to which the present invention pertains.
  • composition of the present invention may be formulated as a preparation containing an inclusion compound inclusion of beta-cyclodextrin ( ⁇ -cyclodextrin).
  • the beta-cyclodextrin is 2,6-dimethyl-beta-cyclodextrin (2,6-dimethyl- ⁇ -cyclodextrin), 2-hydroxyethyl-beta-cyclodextrin (2- hydroxyethyl- ⁇ -cyclodextrin) and 2-hydroxypropyl-beta-cyclodextrin (2-hydroxypropyl- ⁇ -cyclodextrin) may be at least one selected from the group consisting of.
  • the inclusion compound may be a beta-cyclodextrin inner cavity encapsulated in a nutmeg extract, a compound of Formula 1, or a pharmaceutically acceptable salt thereof.
  • oral administration means a substance prepared for digestion of the active substance, that is, administration to the gastrointestinal tract for absorption.
  • Non-limiting examples of the formulation for oral administration include tablets, troches, lozenges, aqueous suspensions, oily suspensions, prepared powders, granules, emulsions, hard capsules, soft capsules, syrups or elixirs, etc.
  • a binder such as lactose, saccharose, sorbitol, mannitol, starch, amylopectin, cellulose or gelatin, an excipient such as dicalcium phosphate, and a disintegrant such as corn starch or sweet potato starch , magnesium stearate, calcium stearate, sodium stearyl fumarate, etc. may be used, and sweeteners, fragrances, syrups, etc. may be used.
  • a liquid carrier such as fatty oil may be additionally used.
  • the term “excipient” refers to any material other than a therapeutic agent, and is used as a carrier or medium for delivery of a therapeutic agent or added to a pharmaceutical composition. Thereby improving handling and storage characteristics or permitting and facilitating unit dosage formation of the composition.
  • the present invention also provides a food composition for preventing or improving non-alcoholic fatty liver disease, comprising as an active ingredient a nutmeg extract or a compound of Formula 1 or a pharmaceutically acceptable salt thereof.
  • the nutmeg extract, the compound of Formula 1, or a pharmaceutically acceptable salt thereof included in the food composition of the present invention is the same as described for the pharmaceutical composition.
  • the nutmeg extract of the present invention may be added as it is, or it may be used together with other foods or food ingredients, and may be appropriately used according to a conventional method. .
  • the food composition of the present invention may also be a health functional food.
  • the term "functional food” means a food manufactured and processed using raw materials or ingredients useful for the human body, and in the present invention, it has a beneficial effect in improving liver disease.
  • the term “function” may refer to an effect useful for health purposes by regulating nutrients or physiological action with respect to the structure and function of the human body.
  • Nutmeg extract which is an active ingredient of the health functional food of the present invention, may be added as it is or used with other foods or food ingredients, and may be appropriately used according to a conventional method.
  • the food composition according to the present invention may further include a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier There is no particular limitation on the type of the food. Examples of the food include various foods, beverages, gum, tea, candy, vitamin complexes, health functional foods, powders, granules, tablets, capsules, jellies or beverages, and may include all health foods in a conventional sense. have.
  • the amount of the extract or the compound of Formula 1 or a pharmaceutically acceptable salt thereof in food or beverage may be added in an amount of 0.01 to 30% by weight of the total food weight, and the beverage composition is 0.01 to 90% by weight based on 100 mL , Preferably it can be added in a proportion of 0.01 to 50% by weight, but is not limited thereto.
  • the food of the present invention is not particularly limited in other ingredients except for containing the nutmeg extract, the compound of Formula 1 or a pharmaceutically acceptable salt thereof as an essential ingredient in the indicated ratio, and various flavoring agents such as conventional beverages Or it may contain natural carbohydrates as additives, but is not limited thereto.
  • the natural carbohydrate includes saccharides such as glucose, fructose, maltose, sucrose, dextrin, and cyclodextrin, and may include sugar alcohols such as xylitol, sorbitol, and erythritol.
  • the flavoring agent may include, but is not limited to, natural flavoring agents (taumatin, stevia extract, for example, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.).
  • the food of the present invention is a variety of nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents, pectic acid and its salts, alginic acid, citric acid, sodium citrate and salts thereof, organic acids , a protective colloidal thickener, a pH adjuster, a stabilizer, a preservative, glycerin, alcohol, a carbonation agent used in carbonated beverages, and the like, but is not limited thereto.
  • These additives are generally selected in the range of 0.001 to 90 parts by weight per 1 part by weight of the mixture of nutmeg extract as the active ingredient, but is not limited thereto.
  • the food composition according to the present invention may be formulated as a preparation comprising an inclusion compound inclusion with beta-cyclodextrin.
  • the beta-cyclodextrin-inclusion compound included in the food composition according to the present invention is as described above.
  • the present invention also provides a method for preparing a pharmaceutical composition for preventing and treating non-alcoholic fatty liver disease, comprising the step of extracting nutmeg.
  • the preparation method of the present invention may include extracting nutmeg, and contacting the obtained nutmeg with supercritical carbon dioxide to obtain an extract.
  • the step of contacting the supercritical carbon dioxide may be a step of putting nutmeg in a reactor, and injecting carbon dioxide at a reaction temperature of 30 to 100° C. and a pressure of 70 to 500 bar.
  • the manufacturing method of the present invention comprises the steps of selecting dried nutmeg; Washing the selected nutmeg and drying at 40 to 80° C. for 3 to 7 hours; Supercritical extraction treatment for 1 to 12 hours at a reaction temperature of 30 to 100° C. and a pressure of 70 to 500 bar; And it may be obtained through the step of concentration and freeze-drying.
  • the present invention also provides a method for preventing non-alcoholic fatty liver disease, comprising the steps of S1) extracting nutmeg, and S2) separating the compound of Formula 1 or a pharmaceutically acceptable salt thereof from the nutmeg extract obtained above. And it provides a method for preparing a pharmaceutical composition for treatment.
  • the present invention also provides a method for preparing a food composition for preventing and improving non-alcoholic fatty liver disease, comprising the step of extracting nutmeg.
  • the nutmeg extraction method applied in the present invention is the same as described above.
  • the present invention also provides a method for preventing non-alcoholic fatty liver disease, comprising the steps of S1) extracting nutmeg, and S2) separating the compound of Formula 1 or a pharmaceutically acceptable salt thereof from the nutmeg extract obtained above. And it provides a method for producing a food composition for improvement.
  • the composition according to the present invention does not contain toxic substances and has no cytotoxicity, and as a result of experiments through a methionine choline deficiency-induce fatty liver disease (MCD) model, hepatoprotective action and It shows prevention and treatment effects of nonalcoholic steatohepatitis, and can be usefully applied as an improvement, prevention and treatment for nonalcoholic fatty liver disease.
  • MCD methionine choline deficiency-induce fatty liver disease
  • FIG. 3 to 5 are 2D NMR data (FIG. 3 is COSY, FIG. 4 is HMBC, FIG. 5 is HSQC) of Compound 1 of the present invention.
  • FIG 9 and 10 are graphs showing the hepatocellular protective effect by H 2 O 2 of Compound 1 and nutmeg extract (NME) of the present invention (Mean ⁇ SD *p ⁇ 0.05).
  • FIG. 11 is a diagram showing the expression level of the AMPK signaling mechanism related protein of Compound 1 of the present invention.
  • FIG 12 and 13 are graphs of GOT and GPT activity inhibition by CCl 4 of Compound 1 and nutmeg extract (NME) of the present invention (Mean ⁇ SD *p ⁇ 0.05).
  • 16 and 17 are results of microscopic analysis of histopathological tests for ⁇ -SMA and TGF- ⁇ .
  • the present invention for achieving the above object is a pharmaceutical composition for the prevention or treatment of nonalcoholic fatty liver disease comprising a nutmeg extract or a compound of Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient, nonalcoholic fatty liver It is characterized in that it provides a food composition for preventing or ameliorating a disease and a method for preparing the same.
  • the extract obtained under the conditions of 250 bar and 60% aqueous ethanol solution was separated by high-performance liquid chromatography using a preparative column to separate Compound 1 by ESI-MS, ESI-MS/
  • the structure was confirmed using spectroscopic analysis methods such as MS, 1H, and 13C-NMR.
  • a high-resolution nuclear magnetic resonance spectrometer (600 MHz, AVANCE 600, Bruker, Germany) from Bruker was used for NMR analysis for the structural identification of the separated material, through which 1H, 13C-NMR, 2D NMR data (COSY, HSQC, HMBC, etc.) were used. ) was obtained and analyzed (Table 2, FIGS. 1 to 5).
  • HPLC for analysis for mass spectrometry of compound 1 was performed using Ultimate3000 (Thermo Scientific, USA) A: 0.1% Formic acid in water, B: 0.1% Formic acid in acetonitrile (0-1 min: 90% A, 1 -25 min: 100% B, 25-30 min: 90% B) HPLC [Waters BEH C18 (2.1 ⁇ 100 mm, 1.7 um particle size, flow rate: 0.4 mL/min, measurement wavelength: 280 nm, column temperature) : 45°C)], and the mass spectrophotometer was analyzed using Triple TOF 5600+ (AB Sciex, USA). As a result of the analysis, Compound 1 was a colorless oil and had a molecular weight of 416.46 (439 [M+Na] + ) ( FIGS. 6-7 ).
  • HepG2 human hepatocyte-derived cell line, ATCC, Manassas, VA, USA
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS fetal bovine serum
  • penicillin penicillin
  • streptomycin streptomycin
  • a 96-well plate 1 ⁇ 10 5 cells/well of HepG2 cells were dispensed using DMEM medium and cultured in a 5% CO 2 incubator for 24 hours. Then, the medium was removed and 0.1 M phosphate buffer (pH 7.0) was used. Cells were washed twice.
  • NME of Example 1 and Compound 1 of Example 4 were diluted in FBS-free DMEM medium at concentrations of 10, 20 and 30 ⁇ g/mL, respectively, and the sample was treated, followed by incubation at 37° C., 5% CO 2 condition for 24 hours did After removing the supernatant, 100 ⁇ L of MTT solution dissolved at a concentration of 5 mg/mL in 0.1 M phosphate buffer (pH 7.0) was added, followed by reaction in a CO 2 incubator for 2 hours. Thereafter, all the supernatant was removed and 100 ⁇ L of DMSO was added and reacted in the dark for 30 minutes, and the resulting formazan was measured at 540 nm in a Microplate reader (BKMPR-1096A, China). The cell viability (%) of the control group not treated with the sample was taken as 100%, and the following formula was used.
  • HepG2 cells were aliquoted in a 24-well plate at 3 ⁇ 10 5 cells/well, cultured for one day, starvated for 16 hours in a culture medium not containing FBS, treated with compound 1 at 100 ⁇ g/mL, and cultured for up to 6 hours.
  • HepG2 cells from which the culture medium was removed were washed twice with cold PBS, then placed on ice, and lysis buffer (40 mM Tris-HCl pH 7.5, 10 mM EDTA pH 8.0, 120 per 6-well plate well) After adding 200 ⁇ l of (mM NaCl, 1 mM PMSF, 1 mM NaF, proteinase inhibitor tablet) and scraping using a scraper, sonication at 4°C for 30 minutes, centrifugation at 11,000 g for 10 min. The concentration of the enzyme solution was measured by putting it in a tube, and the concentration of all enzymes was adjusted to 60 ⁇ g and stored at -70° C. until used in the experiment.
  • lysis buffer 40 mM Tris-HCl pH 7.5, 10 mM EDTA pH 8.0, 120 per 6-well plate well
  • the protein in the gel is transferred to the PVDF membrane using transfer buffer (Tris base, glycine, SDS, methyl alcohol), and the primary antibody AMPK ab (Sc-25792, Santa Cruz Biotechnology) and p-AMPK ab (# 07-626, Millipore Corporation) was diluted with 5% BSA at a ratio of 1:1,000, and the primary antibody was poured into a flat container enough to submerge the membrane. 5 minutes each for 1 hour.
  • transfer buffer Tris base, glycine, SDS, methyl alcohol
  • AMPK ab Sc-25792, Santa Cruz Biotechnology
  • p-AMPK ab # 07-626, Millipore Corporation
  • the washed membrane was attached to the secondary antibody, anti-rabbit (#7074, Cell membrane Signaling Technology), diluted at a ratio of 1:2,000 at room temperature for 1 hour, washed with PBS buffer for 5 minutes each for 1 hour, and ECL (Amersham After developing the membrane treated with Pharmacia Biotech, NJ, USA), the density of the protein band obtained was quantified using Image J software (NIH, Bethesda, MD, USA).
  • GPT glutamic pyruvic transaminase
  • GOT glutamic oxaloacetic acid
  • mice Male SD (Sprague-Dawley) rats weighing about 200 g were used, and 6 mice were assigned to each group as a normal control group, a carbon tetrachloride administration control group, the nutmeg extract of Example 1, and the Formula 1 administration group of Example 4 .
  • intubation was performed in the jugular vein of each experimental animal using a silastic tube (Silastic tubing, Dow Corning Co., USA) and a polyethylene tube (PE-50, Medichem, USA) for blood collection.
  • Carbon tetrachloride was suspended in olive oil at 50 mg/mL and administered intraperitoneally (50 mg/kg) to the carbon tetrachloride administration control group, NME and compound 1 administration group, respectively, and after 30 minutes, 200 mg/kg for the NME administration group, 100 mg/kg for the compound 1 administration group was suspended in 50% propylene glycol aqueous solution and orally administered, and the normal control group was administered with 50% propylene glycol aqueous solution. 24 hours after administration, blood was collected from each group through an intubated tube.
  • the activity against GOT was inhibited by NME and Compound 1 to 75.8 ⁇ 11.6 and 69.9 ⁇ 15.2 U/mL, respectively, and the activity against GPT was 40.5 ⁇ 10.2 and 35.2 ⁇ respectively.
  • the activity was significantly inhibited compared to the control group administered with CCl 4 at 8.2 U/mL, and it was confirmed that the cytoprotective effect against liver toxicity was excellent.
  • Example 9 Evaluation of the effect of nutmeg extract (NME) and compound 1 in a methionine choline deficiency (MCD) diet-induced fatty liver model
  • This experiment was started after adapting to the laboratory environment while feeding 5 week old C57BL/6NHsd male mice weighing about 20 g with a normal diet for 1 week. During the experiment, the laboratory environment was maintained with a 12-hour light-dark cycle (lights on at 8:00 am to off at 8:00 pm), temperature at 23 ⁇ 3°C, and humidity at 55 ⁇ 15%.
  • nutmeg extract and Compound 1 were weighed, dissolved in 10% DMSO of the total amount, and 0.5% carboxymethylcellulose (CMC) corresponding to 90% of the total liquid was added to prepare.
  • CMC carboxymethylcellulose
  • MCD diet was supplied for 5 days and normal food was supplied for 2 days thereafter.
  • MCD diet and normal diet were freely ingested alternately.
  • the administration dose was calculated as 10 mL/kg of each nutmeg extract and compound 1 based on the body weight measured on the latest weighing day, and the animal was fixed by the skin fixation method on the cervical region, and once / using a sonde for oral administration. Days and 4 weeks were administered.
  • ALT alanine transaminase, alanine transaminase
  • AST aspartate transaminase, aspartate transaminase
  • TG triglyceride
  • TCHO total cholesterol
  • the ALT and AST levels of all fatty liver-inducing groups were significantly higher than those of the normal control group (G1) (p ⁇ 0.001), G3 and The ALT level of G6 was significantly lower than that of G2 (p ⁇ 0.01 and p ⁇ 0.05), and the AST level of G3, G5 and G6 was significantly lower than that of G2 (p ⁇ 0.001).
  • ALT and AST are blood biochemical markers related to liver function, and administration of nutmeg extract and Compound 1 induced a decrease in liver function-related levels in the MCD diet-induced steatohepatitis model.
  • the weight was measured, and the left lobe of the excised liver was fixed in 10% neutral buffered formalin solution to perform histopathology. was performed.
  • the liver weight levels of G3, G4, G5 and G6 were statistically significantly lower than that of G1 (p ⁇ 0.001 or p ⁇ 0.01), and the liver weight levels of G3 and G6 were lower than those of G2. was significantly lower than that (p ⁇ 0.01).
  • liver relative weight levels of all fatty liver-inducing groups were significantly higher than those of G1 (p ⁇ 0.001), and the levels of liver relative weights of G3, G5 and G6 were statistically significantly lower than those of G2 (p ⁇ 0.001). 0.01).
  • the fixed tissue undergoes general tissue processing such as trimming, dehydration, paraffin embedding, and sectioning to prepare a sample for histopathological examination, Picrosirius red staining) and immunohistochemical staining (TGF- ⁇ , ⁇ -SMA) were performed, and histopathological changes were observed using an optical microscope (Olympus BX53, Japan).
  • Histopathological microscopy was evaluated according to the following microscopic standards (Liang et al, 2014), and analysis of Picrosirius red staining and immunohistochemical staining was performed using an Image analyzer (Zen 2.3 blue edition, Carl Zeiss, Germany), The staining area compared to the total area for each individual was compared between groups ( FIGS. 13-14 ).
  • macrophobic steatosis levels in all fatty liver-inducing groups were significantly higher than those in G1 (p ⁇ 0.001), and macrophobic steatosis levels in G3, G4, G5 and G6. was significantly lower than that of G2 (p ⁇ 0.01 or p ⁇ 0.05).
  • the level of microphobic steatosis in G2, G4, G5 and G6 was significantly higher than that of G1 (p ⁇ 0.001, p ⁇ 0.01 or p ⁇ 0.05), and the level of microphobic steatosis in G3 was significantly lower than that of G2 (p ⁇ 0.001, p ⁇ 0.01 or p ⁇ 0.05). ⁇ 0.01).
  • Inflammation levels of G2 and G4 were significantly higher than those of G1 (p ⁇ 0.01 and p ⁇ 0.05), and those of G3 were significantly lower than those of G2 (p ⁇ 0.05).
  • the macrophobic steatosis levels of all nutmeg extract-administered groups (G4-G6) were significantly lower than those of the negative control group (G2, negative control), and there was no significant difference, but microphobic steatosis and inflammation levels were also It showed a lower trend compared to the negative control group (G2).
  • the staining area level of G2 was significantly higher than that of G1 (p ⁇ 0.01), and the level of staining area of G6 was significantly lower than that of G2 (p ⁇ 0.05). It was observed that there was a dose correlation with the administration of nutmeg extract and compound 1, and the fibrosis area decreased.
  • TGF- ⁇ and ⁇ -SMA expression area level was significantly higher than that of G1 (p ⁇ 0.001).
  • TGF- ⁇ expression area levels of all nutmeg extract and compound 1 administration groups were significantly lower than those of G2 (p ⁇ 0.001 or p ⁇ 0.01), and ⁇ - of all nutmeg extract administration groups (G4-G6) SMA expression area level was significantly lower than that of G2 (p ⁇ 0.001 or p ⁇ 0.01).
  • the expression area level of ⁇ -SMA, a factor related to inflammation and fibrosis, showed a dose-related change trend and decreased with the administration of nutmeg extract and Compound 1, and the TGF- ⁇ expression area level was decreased in the nutmeg extract and Compound 1 administration groups ( G3-G6) showed a significant difference compared to the negative control group (G2) at all doses and decreased.
  • NEFA Non-esterified fatty acid
  • TCHO levels in liver tissues of G2, G4, G5 and G6 were significantly higher than those of G1 (p ⁇ 0.001 or p ⁇ 0.05), and TCHO levels in liver tissues of G5 were significantly higher than those of G2 (p ⁇ 0.01). , G3 and G6 TCHO levels in liver tissues were significantly lower than those of G2 (p ⁇ 0.001).
  • Free fatty acid (FFA) levels in serum and liver tissues of all fatty liver induction groups were significantly higher than those of G1 (p ⁇ 0.001), and FFA levels in serum of G3, G4 and G6 and FFA levels in the liver tissues of G5 and G6 were significantly lower than those of G2 (p ⁇ 0.001, p ⁇ 0.01 or p ⁇ 0.05).
  • TG and TCHO levels in liver tissue were significantly decreased in the nutmeg extract (NME) 400 mg/kg group (G6) compared to the negative control group (G2), and the nutmeg extract 200 mg/kg group (G5) and nutmeg extract FFA levels in the liver tissue of the 400 mg/kg administration group (G6), the serum FFA levels of the nutmeg extract 100 mg/kg administration group (G4) and the nutmeg extract 400 mg/kg administration group (G6) were also significantly reduced.
  • the test substance When the test substance was repeatedly administered to the C57BL/6 mouse model of nonalcoholic steatohepatitis (NASH) induced by methionine and choline deficiency (MCD) diet under these test conditions for 4 weeks, the nutmeg extract administration group showed a negative control group in the blood biochemical test results. In contrast, a decrease in liver function-related values was observed, and a significant decrease in the absolute and relative weight levels of the liver was observed. In addition, the histopathological examination results showed a dose-related change trend, and reduction of the level of macrophobic steatosis and reduction of the expression level of factors related to inflammation and fibrosis were observed. Significant reductions in cholesterol, FFA, and FFA levels in serum were observed.
  • NASH nonalcoholic steatohepatitis
  • MCD methionine and choline deficiency
  • the potential for development as a therapeutic agent for liver disease using the nutmeg extract and Compound 1 according to the present invention is expected to be high.
  • a formulation example of the pharmaceutical composition will be described, which is intended to describe only in detail, not to limit the present invention.
  • the dry nutmeg extract was mixed with lactose, calcium carboxymethylcellulose, light anhydrous silicic acid, polyoxyl stearate 40 and magnesium stearate in a speed mixer for 30 minutes. This mixture was filled into gelatin hard capsules in a capsule filling machine.
  • the dried nutmeg extract was added to lactose, sodium bicarbonate, and corn starch and mixed, and a binder solution made by adding corn starch to purified water was added thereto, and the mixture was kneaded in a mixing mixer for 30 minutes. After granulation by passing the mixture through a granulator, it was put into a dryer, dried for 5 hours, and then granulated in a granulator. Magnesium stearate, a lubricant, was added to the sized product, mixed, and then tableted to a weight of 288 mg per tablet.
  • Compound 1 inclusion complex of Formulation Example 2 was added to a mixture of xylitol, maltodextrin, and citric acid, and mixed in a soup mixer for 30 minutes. This mixture was passed through a granulator and granulated, then magnesium stearate and yogurt flavor were added, mixed, and then compressed with a tableting machine to a weight of 1 g per sugar.

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Abstract

La présente invention concerne une composition pharmaceutique destinée à la prévention ou au traitement d'une stéatose hépatique non alcoolique, une composition alimentaire, et son procédé de préparation, comprenant un extrait de noix de muscade (Myristica fragrans) et/ou un composé à base de lignane dérivé de celui-ci comme principe actif. La composition selon la présente invention ne contient pas de substances toxiques, ne possède aucune cytotoxicité, assure une excellente protection hépatocellulaire et présente des effets préventifs et thérapeutiques pour une stéatohépatite non alcoolique, et peut donc être utilement appliquée en tant qu'agent préventif et thérapeutique pour une stéatose hépatique non alcoolique.
PCT/KR2021/007565 2020-06-16 2021-06-16 Composition pour améliorer, prévenir ou traiter une stéatose hépatique non alcoolique et son procédé de préparation WO2021256858A1 (fr)

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