WO2021256858A1 - Composition for improving, preventing or treating non-alcoholic fatty liver disease and preparation method therefor - Google Patents
Composition for improving, preventing or treating non-alcoholic fatty liver disease and preparation method therefor Download PDFInfo
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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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- WIPO (PCT)
- Prior art keywords
- fatty liver
- liver disease
- pharmaceutical composition
- compound
- nutmeg
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- MSRILKIQRXUYCT-UHFFFAOYSA-M valproate semisodium Chemical compound [Na+].CCCC(C(O)=O)CCC.CCCC(C([O-])=O)CCC MSRILKIQRXUYCT-UHFFFAOYSA-M 0.000 description 1
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Images
Classifications
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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)
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- 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
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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
- A61K31/075—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
The present invention relates to a pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease, a food composition, and a preparation method therefor, comprising a nutmeg (Myristica fragrans) extract and/or a lignan compound derived therefrom as an active ingredient. The composition of the present invention does not contain toxic substances, has no cytotoxicity, and exhibits excellent hepatocellular protection and preventive and therapeutic effects for non-alcoholic steatohepatitis, and thus can be usefully applied as a preventive and therapeutic agent for non-alcoholic fatty liver disease.
Description
본 발명은 육두구 추출물 및/또는 그로부터 유래한 리그난계 화합물을 포함하는 비알코올성 지방간 질환의 개선, 예방 또는 치료용 조성물 및 그 제조방법에 관한 것이다. 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.
간은 체내 신진 대사, 생합성, 배설, 분비 및 해독에 필수적인 역할을 하는 다기능 기관이다. 이러한 과정은 에너지를 필요로 하기 때문에 간을 호기성이 높은 산소 의존성 조직으로 만든다. 이 과정은 또한 무산소에 간의 취약성을 부여하고, 유해한 물질에 대한 감수성을 증가시키며, 조직 손실 후 세포의 교체가 요구된다. 강화된 간세포 사멸과 손상된 세포의 재생은 실제로 대부분의 간질환의 특징이다. 간은 세포 증식에 의한 막대한 세포 손실로부터 재생성 할 수 있다. 그러나, 많은 만성 간질환 및 알코올과 같은 간 독소에 만성적으로 노출되면 재생은 간세포 사멸과 보조를 맞출 수 없다. 간 성상 세포에 의해 만들어진 섬유화는 기능성 간세포가 점차적으로 대체되고 치환되어 간 기능을 손상시키고 결과적으로 간 기능 장애를 일으키게 된다 (비특허문헌 1). 간질환의 범주는 경증의 가역적인 지방간에서부터 점진적 만성 간질환까지 다양하며, 간 경화, 간 부전 및 간암과 같은 생명을 위협하는 상태의 발생을 초래할 수 있다. 만성 간질환은 전 세계적으로 사망률과 이환율의 주요 원인이다. 대부분의 만성 간질환은 가벼운 염증에서 더 심한 염증으로 진행되어 섬유증이나 간경변으로 이어진다. 이것은 간 기능 부전 및 문맥압 항진을 일으킬 수 있으며 간암의 위험이 증가하게 된다 (비특허문헌 2).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. Fibrosis produced by hepatic stellate cells is gradually replaced and replaced by functional hepatocytes, which impairs liver function, resulting in liver dysfunction (Non-Patent Document 1). 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).
한국인 간질환의 원인으로는 B형 간염 바이러스 (Hepatitis B virus; HBV)가 가장 많고, C형 간염 바이러스 (Hepatitis C virus; HCV), 알코올성 지방간질환, 비알코올성 지방간염 (Non-alcoholic steatohepatitis; NASH) 등으로 구성되어 있다. 비알코올성 지방간질환(non-alcoholic liver disease)은 그 중 지방간의 원인이 알코올에 기인되지 않은 경우를 말하며 간 내 단순 지방침착(simple steatosis)부터 지방간염, 간경변에 이르는 일련의 과정을 모두 포함하는 용어이다. 특히, 비-알코올성 지방간염은 광범위한 간 손상, 즉 간단한 지방증에서부터 지방간질환, 쓸개즙정체, 항진성 섬유증 및 간경화에 이르기까지 다양한 것을 지칭한다. 병리학적 특징은 알코올-유도성 간 손상의 병리학적 특징과 유사하지만, 이것은 알코올을 남용하지 않는 환자에서만 생긴다. 비알코올성 지방간염은 부차적인 원인에 기인한 간염에 의하거나 간염에 의하지 않는 지방증과는 구별되어야 하는데, 이것은 이들 상태가 분명히 상이한 병원체 및 결과를 갖기 때문이다. 지방간질환(지방증)의 부차적 원인은 영양(예컨대 단백질-칼로리 영양 이상, 기아, 총 비경구적 영양, 급속한 체중 감소, 비만으로 인한 위장관 수술), 약물(예컨대 글루코코르티코이드, 합성 에스트로겐, 아스피린, 칼슘-채널 차단제, 테트라사이클린, 발프로익산, 코카인, 항바이러스제, 피알유리딘, 인터페론-α, 메토트렉세이트, 지도부딘), 대사제 또는 유전제(예컨대 지방이상증, 베타지질단백장애, 웨버-크리스티안 질환, 갈락토오스혈증, 글리코겐 저장 장애, 임신성 급성 지방간) 및 당뇨병, 비만 또는 고지혈증과 같은 기타 원인이 있다(비특허문헌 3 및 4). Hepatitis B virus (HBV) 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. In particular, 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 (steatosis) 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-α, 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).
최근 가장 주목 받고 있는 간질환인 비알코올성 지방간염은 서구화된 식습관, 운동부족, 생활양식의 변화 등으로 비만 인구 증가와 더불어 현저히 증가하고 있고, 대사증후군, 당뇨병, 심혈관 질환과 밀접한 연관이 있어 국가적인 대책 마련이 시급한 질환이다. 비알코올성 지방간염은 과도한 칼로리 섭취와 활동량 및 운동부족, 간 질환의 가족력 등에 의해 간에 5% 이상의 지방 (주로 중성지방)이 쌓인 비알코올성 지방간을 제대로 관리하지 않아 생기는데, 간세포에 지방이 축적되면 혈액 및 림프계에 순환장애를 일으켜 간 기능이 저하된다. 특히 만성적인 염증 반응으로 간의 섬유화 과정이 지속되면 간 조직이 딱딱해지면서 기능을 잃어가는 간경변 (간경화)으로 진행하게 된다. 그러나, 이러한 만성 간질환의 발생빈도가 점점 높아짐에도 불구하고 이러한 부류의 대부분의 질환에 유효한 예방 또는 치료법이 존재하지 않고 있다. 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. In particular, if the fibrosis process of the liver continues due to a chronic inflammatory response, the liver tissue becomes hard and loses its function, leading to cirrhosis (liver cirrhosis). However, despite the increasing frequency of these chronic liver diseases, there is no effective prevention or treatment for most of these types of diseases.
육두구 (Myristica fragrans HOUTT.)는 육두구과 (Myristicaceae)의 씨를 가종피 및 씨 껍질을 벗기고 석회에 담근 다음 건조한 것이다. 육두구는 상록교목으로 수마트라나 자바에서 재배되는 자웅이주 식물이다. 과실은 난구형 육질로서 가운데에 홍색의 가종피에 둘러 싸여진 종자를 포함한다. 육두구는 예로부터 소스 등 식품의 향미료로서 광범위하게 이용되어 왔고, 한방에서는 설사, 복부팽만, 구토, 식욕감퇴 등의 방향성 건위제로 사용되었다. 이 외에도 육두구는 헬리코박터 파일로리 (Helicobacter pylori)의 성장 억제 활성 (비특허문헌 5), 간의 해독 작용 기전의 활성화 (비특허문헌 6) 등을 나타낸다고 알려져 있다. Nutmeg ( Myristica fragrans HOUTT.) 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. In addition, 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).
특히, 육두구의 알킬 아릴 에테르(Alkyl aryl ether), 다이벤질부탄(Dibenzylbutane), 테트라하이드로퓨란(Tetrahydrofuran) 등의 모핵을 가지는 리그난 (Lignans)계 화합물은 간세포 분화, 성장 및 대사에 관여하는 PPARα (Peroxisome proliferator activated receptor alpha)를 조절하는 기전을 가지며, 염증이나 독성물질에 의해 유발된 산화적 손상에 대한 보호작용을 하는 NFE2L2 (Nuclear factor erythroid-drived2-like2) gene에 의해 발현되는 NRF2를 활성화하는 (비특허문헌 7) 약리기전을 가지고 있다고 알려져 있으나, 비알코올성 지방간염과 관련된 치료 효과는 보고된 바가 없는 실정이다. In particular, Lignans-based compounds having a mother nucleus such as alkyl aryl ether, dibenzylbutane, and tetrahydrofuran of nutmeg are PPARα (Peroxisome) involved in hepatocyte differentiation, growth and metabolism. Activates 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.
한편, 육두구 추출물과 관련된 선행 특허로는, 육두구 추출물 또는 이로부터 분리된 리그난계 화합물을 함유하는 혈관 질환의 예방 또는 치료용 조성물 (대한민국특허 제 10-1338901호), 리그난계 화합물 또는 이를 함유하는 육두구 추출물 또는 육두구 가종피 추출물의 신규한 용도 (대한민국특허 제10-1088071호), 비만 증상의 개선 및 예방을 위한 육두구 추출물 함유 기능성식품 (대한민국특허 제10-1079916호), 육두구 가종피 추출 화합물을 유효성분으로 포함하는 당뇨병 또는 피피에이알-감마 (PPAR-γ) 매개 질환 예방 및 치료용 약학적 조성물 (대한민국특허 제10-0959557호), 육두구 추출물을 포함하는 자궁내막증의 예방 또는 치료용 조성물 (대한민국특허 제10-1787458호) 등이 있으나, 모두 비알코올성 지방간 질환 내지 비알코올성 지방간염과는 무관하다. On the other hand, 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-γ) mediated disease comprising as a component (Korea Patent No. 10-0959557), 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.
따라서, 현재까지 치료제가 개발되지 않은 비알코올성 지방간 질환, 특히 비알코올성 지방간염의 예방, 치료 및 개선에 효과적이면서도 간독성 및 인체 부작용이 적은 천연물 소재의 약재 개발이 요망된다.Therefore, there is a demand for the development of drugs made from natural materials that are effective for the prevention, treatment and improvement of non-alcoholic fatty liver disease, in particular, non-alcoholic steatohepatitis, for which no therapeutic agent has been developed yet, but with less hepatotoxicity and side effects to the human body.
[선행기술문헌][Prior art literature]
[특허문헌][Patent Literature]
(특허문헌 1) 1. 대한민국 등록특허 제10-1338901호(Patent Document 1) 1. Republic of Korea Patent No. 10-1338901
(특허문헌 2) 2. 대한민국 등록특허 제10-1088071호(Patent Document 2) 2. Republic of Korea Patent No. 10-1088071
(특허문헌 3) 3. 대한민국 등록특허 제10-1079916호(Patent Document 3) 3. Republic of Korea Patent No. 10-1079916
(특허문헌 4) 4. 대한민국 등록특허 제10-0959557호(Patent Document 4) 4. Republic of Korea Patent No. 10-0959557
(특허문헌 5) 5. 대한민국 등록특허 제10-1787458호(Patent Document 5) 5. Republic of Korea Patent No. 10-1787458
[비특허문헌][Non-patent literature]
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(비특허문헌 6)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. Food Chem Toxicol, 31(7); 517-21, 1993(Non-Patent Document 6)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. Food Chem Toxicol, 31(7); 517-21, 1993
(비특허문헌 7)7. Xiao-Nan Yang et al., PPARα Mediates the Hepatoprotective Effects of Nutmeg. J. Proteome Res. 17; 1887-1897, 2018(Non-Patent Document 7)7. Xiao-Nan Yang et al., PPARα Mediates the Hepatoprotective Effects of Nutmeg. J. Proteome Res. 17; 1887-1897, 2018
본 발명은 비알코올성 지방간 질환의 예방, 치료 및 개선에 효과적인 조성물 및 그 제조방법을 제공하기 위한 것으로, 구체적으로 천연물인 육두구 (Myristica fragrans) 추출물 및 그로부터 유래된 리그난계 화합물을 유효 성분으로 포함하는 비알코올성 지방간 질환의 예방 및 치료용 조성물 및 이의 제조 방법을 제공하기 위한 것이다.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.
상기 상술한 목적을 달성하기 위하여 본 발명은, 육두구 추출물 및 그로부터 유래된 리그난 화합물을 유효 성분으로 포함하는 비알코올성 지방간 질환의 예방 또는 치료용 약학적 조성물, 이를 포함하는 비알코올성 지방간 질환의 예방 또는 개선용 식품 조성물, 및 이의 제조 방법을 제공한다.In order to achieve the above object, 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.
본 발명에 따른 조성물은 비알코올성 지방간 질환의 예방 또는 치료에 효과가 있어, 비알코올성 지방간 질환 치료제 또는 간질환 증세의 개선용 건강식품으로 유용하게 적용될 수 있다.The 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.
본 발명은 육두구 추출물, 또는 하기 화학식 1의 화합물 또는 이의 약학적으로 허용되는 염을 유효 성분으로 포함하는 비알코올성 지방간 질환의 예방 또는 치료용 약학적 조성물을 제공한다.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.
[화학식 1][Formula 1]
상기 화학식 1의 화합물은 (1S,2R)-2-[2,6-디메톡시-4-(프로프-2-엔-1-일)페녹시]-1-(4-하이드록시-3-메톡시페닐)프로필 아세테이트 ((1S,2R)-2-[2,6-dimethoxy-4-(prop-2-en-1-yl)phenoxy]-1-(4-hydroxy-3-methoxyphenyl)propyl acetate) (분자식: C23H28O7, 분자량: 416.46)이다.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).
본 발명에서 용어 "육두구"는, 육두구(Myristica fragrans)의 건조된 씨를 의미한다. As used herein, the term "nutmeg" refers to dried seeds of nutmeg (Myristica fragrans).
본 발명에서 용어 "추출물"은, 약 20℃내지 50℃에서 전체적으로 또는 부분적으로 액체이며, 소수성이나 적어도 하나의 유기 용매에 가용성인 임의의 물질을 의미한다.As used herein, the term “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.
본 발명에서 용어 "육두구 추출물"은, 육두구의 건조된 씨를 마쇄하여 초임계 또는 극성 및 비극성 용매를 이용하여 추출하고, 추출물을 분리하여 수득된 것을 의미한다. In the present invention, the term "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.
본 발명에서 용어 "유효성분"은, 단독으로 목적하는 활성을 나타내거나 또는 그 자체는 활성이 없는 담체와 함께 활성을 나타낼 수 있는 성분을 의미한다. In the present invention, the term "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.
본 발명에서 용어 "비알코올성 지방간질환(non-alcoholic fatty liver diseases, NAFLD)"는 유의한 알코올 섭취, 지방간을 초래하는 약물의 복용, 동반된 다른 원인에 의한 간질환이 없으면서 영상의학 검사나 조직검사에서 간 내 지방침착의 소견을 보이는 질환으로, 비알코올성 지방간에서 비알코올성 지방간염, 비알코올성 지방간연관 간경변증을 포괄하는 진단명이다.In the present invention, the term "non-alcoholic fatty liver disease (NAFLD)" 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.
본 발명에서 용어 "비알코올성 지방간"은 간 내 지방침착을 보이지만 간세포 손상(풍선변형) 및 섬유화의 소견은 없는 경우를 의미한다.In the present invention, the term "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.
본 발명에서 용어 "비알코올성 지방간염"은 간 내 지방침착을 보이면서 간세포 손상(풍선변형)을 동반한 염증소견이 잇는 경우를 의미하며, 섬유화를 동반하기도 한다.In the present invention, the term "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.
본 발명에서 용어 "비알코올성 지방간연관 간경변증"은 조직학적으로 비알코올성 지방간이나 지방간염의 소견이 동반된 간경변증, 혹은 과거 조직학적으로 증명된 비알코올성 지방간, 지방간염 환자에서 발생한 간경변증을 의미한다.As used herein, the term "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.
본 발명에서 용어 "예방"은, 질병, 장애 또는 질환의 발병을 억제하거나 지연을 의미한다. 질병, 장애 또는 질환의 발병이 예정된 기간 동안 억제되거나 지연된 경우 예방은 완전한 것으로 간주될 수 있다.In the present invention, the term "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.
본 발명에서 용어 "치료"란, 특정 질병, 장애 및/또는 질환 또는 질환에 따른 증상을 부분적으로 또는 완전히 경감, 개선, 완화, 저해 또는 지연시키며, 중증도를 감소시키거나, 하나 이상의 증상 또는 특징의 발생을 감소시키는 것을 의미한다. As used herein, the term "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.
본 발명의 일 구현예에 따르면, 본 발명에 따른 조성물이 적용되는 비알코올성 지방간 질환은 비알코올성 지방간, 비알코올성 지방간염, 또는 비알코올성 지방간연관 간경변증일 수 있다.According to one embodiment of the present invention, 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.
본 발명의 일 구현예에 따르면, 본 발명의 육두구 추출물은 화학식 1의 화합물 또는 이의 약학적으로 허용되는 염을 함유할 수 있다.According to one embodiment of the present invention, the nutmeg extract of the present invention may contain the compound of Formula 1 or a pharmaceutically acceptable salt thereof.
본 발명의 일 구현예에 따르면, 본 발명의 상기 화학식 1의 화합물 또는 이의 약학적으로 허용되는 염은 육두구 추출물로부터 분리된 것일 수 있다.According to one embodiment of the present invention, the compound of Formula 1 or a pharmaceutically acceptable salt thereof of the present invention may be isolated from a nutmeg extract.
본 발명에 따른 육두구 추출물은, 1) 육두구에 추출용매를 가하여 추출물을 제조하는 단계; 2) 단계 1)의 추출물을 여과하는 단계; 및 3) 단계 2)의 여과된 여과물을 감압농축한 후 건조하는 단계를 포함하는 방법에 의해 제조될 수 있다.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.
본 발명의 일 구현예에 따르면, 본 발명의 육두구 추출물은 물, 메탄올, 에탄올, n-부탄올, 아세톤, 에틸아세테이트, 헥산 및 클로로포름으로 이루어진 군으로부터 선택되는 하나 이상의 용매로 추출될 수 있다. 구체적으로 용매는 에탄올일 수 있으며, 더 구체적으로 50 내지 100%의 에탄올, 보다 더 구체적으로 80 내지 100%의 에탄올일 수 있다. 상기 조건의 용매를 사용하는 경우 추출물의 추출 효율, 화학식 1의 화합물의 수율, 및 비알코올성 지방간 질환의 치료 효과 및 간 세포 보호 효과면에서 바람직하다.According to one embodiment of the present invention, 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. Specifically, the solvent may be ethanol, more specifically 50 to 100% ethanol, more specifically 80 to 100% ethanol. When the solvent of the above conditions 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 protection effect.
또한, 상기 추출용매는 추출에 사용되는 육두구의 중량 1 g당 바람직하게는 1 내지 50 mL, 더 바람직하게는 1 내지 30 mL, 가장 바람직하게는 1 내지 20 mL의 양으로 첨가될 수 있다.In addition, 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.
상기 추출방법에서 추출 온도, 추출 시간, 추출 횟수는 적절하게 선택할 수 있다. 상기 추출 온도는 바람직하게는 30 내지 120℃, 더 바람직하게는 50 내지 100℃, 가장 바람직하게는 70 내지 90℃일 수 있다. 상기 추출 시간은 바람직하게는 1 내지 10시간, 더 바람직하게는 1 내지 8시간, 가장 바람직하게는 1 내지 5시간일 수 있다. 상기 추출 횟수는 바람직하게는 1 내지 5회일 수 있다. 상기 조건에서 추출하는 경우 추출물의 추출 효율, 화학식 1의 화합물의 수율, 및 비알코올성 지방간 질환의 치료 효과 및 간 세포 보호 효과면에서 바람직하다.In the extraction method, 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. When extracted under 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.
또한, 상기 추출방법은 바람직하게는 진탕 추출, Soxhlet 추출, 또는 환류 추출, 초임계 유체 추출일 수 있고, 가장 바람직하게는 환류 추출 또는 초임계 유체 추출일 수 있다. 따라서, 본 발명의 일 구현예에 따르면, 본 발명의 육두구 추출물은 초임계 유체 추출물 또는 환류 추출물일 수 있다. 상기 추출물을 사용하는 경우 추출물의 추출 효율, 화학식 1의 화합물의 수율, 및 비알코올성 지방간 질환의 치료 효과 및 간 세포 보호 효과면에서 바람직하다.In addition, 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.
본 발명에서 용어 "초임계 추출"(Supercritical Fluid Extraction)은, 반응기에 액화 이산화탄소를 통과시키는 방법과, 공용매를 보조적으로 통과시키는 방법으로 나뉠 수 있다.In the present invention, the term "supercritical fluid extraction" (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.
본 발명의 일 구현예에 따르면, 본 발명의 초임계 추출(Supercritical Fluid Extraction)은 일정 온도로 가온된 반응기에 액화 이산화탄소와 공용매로서 에탄올 수용액을 육두구 분말에 통과시켜 추출물을 수득한 후 상기 추출물을 증발 농축하고 농축물을 동결 건조하는 방법일 수 있다.According to one embodiment of the present invention, 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.
본 발명의 일 구현예에 따르면, 상기 초임계 유체 추출물은 35 내지 100℃ 온도, 100 내지 500 bar의 압력 하에 초임계 상태로 만든 이산화탄소를 이용한 초임계 추출 방법으로 추출한 것일 수 있다. 보다 구체적으로, 35 내지 70℃ 온도, 200 내지 450 bar의 압력 하일 수 있고, 보다 더 구체적으로는 40 내지 60℃ 온도, 250 내지 400 bar의 압력 하일 수 있다. 상기 조건을 사용하는 경우 추출물의 추출 효율, 화학식 1의 화합물의 수율, 및 비알코올성 지방간 질환의 치료 효과 및 간 세포 보호 효과면에서 바람직하다.According to one embodiment of the present invention, 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. When the above conditions are used, 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.
본 발명의 일 구현예에 따르면, 상기 초임계 유체 추출 방법은 초임계 상태로 만든 이산화탄소에 추가적으로 공용매를 혼합한 혼합 유체를 이용하는 것일 수 있다.According to one embodiment of the present invention, 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.
본 발명의 일 구현예에 따르면, 상기 공용매는 물, 메탄올, 에탄올, n-부탄올, 아세톤, 에틸아세테이트, 헥산 및 클로로포름으로 이루어진 군으로부터 선택되는 하나 이상일 수 있다. 구체적으로, 공용매는 에탄올일 수 있으며, 더 구체적으로 농도 20 내지 100%의 에탄올, 더 구체적으로 50 내지 100%의 에탄올, 보다 더 구체적으로 60 내지 80%의 에탄올일 수 있다. 상기 조건의 용매를 사용하는 경우 추출물의 추출 효율, 화학식 1의 화합물의 수율, 및 비알코올성 지방간 질환의 치료 효과 및 간 세포 보호 효과면에서 바람직하다.According to one embodiment of the present invention, the cosolvent may be at least one selected from the group consisting of water, methanol, ethanol, n-butanol, acetone, ethyl acetate, hexane and chloroform. Specifically, 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%. When the solvent of the above conditions 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 protection effect.
또한, 상기 초임계 유체 추출의 추출 시간은 바람직하게는 1 내지 12시간, 더 바람직하게는 1 내지 8시간, 가장 바람직하게는 1 내지 4시간일 수 있다. 상기 조건에서 추출하는 경우 추출물의 추출 효율, 화학식 1의 화합물의 수율, 및 비알코올성 지방간 질환의 치료 효과 및 간 세포 보호 효과면에서 바람직하다.In addition, 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. When extracted under 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.
본 발명의 일 구현예에 따르면, 환류 추출은 육두구 분말에 용매를 넣고 환류 냉각 추출하는 방법일 수 있다. According to one embodiment of the present invention, reflux extraction may be a method of extracting under reflux by adding a solvent to nutmeg powder.
본 발명의 일 구현예에 따르면, 상기 환류 추출 용매는 물, 메탄올, 에탄올, n-부탄올, 아세톤, 에틸아세테이트, 헥산 및 클로로포름으로 이루어진 군으로부터 선택되는 하나 이상일 수 있다. 구체적으로, 환류 추출 용매는 에탄올일 수 있으며, 더 구체적으로 50 내지 100%의 에탄올, 보다 더 구체적으로 80 내지 100%의 에탄올일 수 있다. 상기 조건의 용매를 사용하는 경우 추출물의 추출 효율, 화학식 1의 화합물의 수율, 및 비알코올성 지방간 질환의 치료 효과 및 간 세포 보호 효과면에서 바람직하다.According to one embodiment of the present invention, 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. Specifically, the reflux extraction solvent may be ethanol, more specifically 50 to 100% ethanol, and even more specifically 80 to 100% ethanol. When the solvent of the above conditions 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 protection effect.
상기 감압농축은 바람직하게는 진공감압농축기 또는 진공회전증발기를 이용할 수 있다.The vacuum concentration may preferably be performed using a vacuum vacuum concentrator or a vacuum rotary evaporator.
상기 건조는 바람직하게는 감압건조, 진공건조, 비등건조, 분무건조 또는 동결건조일 수 있고, 가장 바람직하게는 동결건조일 수 있다.The drying may be preferably reduced pressure drying, vacuum drying, boiling drying, spray drying or freeze drying, and most preferably freeze drying.
본 발명에 따른 조성물은, 화합물 (1S,2R)-2-[2,6-디메톡시-4-(프로프-2-엔-1-일)페녹시]-1-(4-하이드록시-3-메톡시페닐)프로필 아세테이트 ((1S,2R)-2-[2,6-dimethoxy-4-(prop-2-en-1-yl)phenoxy]-1-(4-hydroxy-3-methoxyphenyl)propyl acetate) (분자식: C23H28O7, 분자량: 416.46)은 물론 그의 약학적으로 허용되는 염을 포함할 수 있다.The 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.
본 발명에 따른 "약제학적으로 허용되는 염"은 화학식 1로 표시되는 화합물을 형성할 수 있는 치료적으로 활성인, 비-독성 염기 또는 산 부가염의 형태일 수 있다.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).
이때, 상기 비-독성 염기는 설페이트, 피로설페이트, 바이설페이트, 설파이트, 바이설파이트, 니트레이트, 포스페이트, 모노하이드로겐 포스페이트, 디하이드로겐 포스페이트, 메타포스페이트, 피로포스페이트 클로라이드, 브로마이드, 아이오다이드, 플루오라이드, 아세테이트, 프로피오네이트, 데카노에이트, 카프릴레이트, 아크릴레이트, 포메이트, 이소부티레이트, 카프레이트, 헵타노에이트, 프로피올레이트, 옥살레이트, 말로네이트, 석시네이트, 수베레이트, 세바케이트, 푸마레이트, 말리에이트, 부틴-1,4-디오에이트, 헥산-1,6-디오에이트, 벤조에이트, 클로로벤조에이트, 메틸벤조에이트, 디니트로 벤조에이트, 하이드록시벤조에이트, 메톡시벤조에이트, 프탈레이트, 테레프탈레이트, 벤젠설포네이트, 톨루엔설포네이트, 클로로벤젠설포네이트, 크실렌설포네이트, 페닐아세테이트, 페닐프로피오네이트, 페닐부티레이트, 시트레이트, 락테이트, β-하이드록시부티레이트, 글리콜레이트, 말레이트, 타트레이트, 메탄설포네이트, 프로판설포네이트, 나프탈렌-1-설포네이트, 나프탈렌-2-설포네이트 또는 만델레이트를 포함할 수 있다.In this case, 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, toluenesulfonate, chlorobenzenesulfonate, xylenesulfonate, phenylacetate, phenylpropionate, phenylbutyrate, citrate, lactate, β-hydroxybutyrate, glycolate , malate, tartrate, methanesulfonate, propanesulfonate, naphthalene-1-sulfonate, naphthalene-2-sulfonate or mandelate.
또한 상기 산 부가 염은 약학적으로 허용 가능한 유리산(free acid)에 의해 형성된 산 부가염일 수 있으며, 이에 한정되지는 않으나 바람직하게는, 염산, 질산, 인산, 황산, 브롬화수소산, 요드화수소산, 아질산 또는 아인산과 같은 무기산류이거나 지방족 모노 또는 디카르복실레이트, 페닐-치환된 알카노에이트, 하이드록시 알카노에이트 또는 알칸디오에이트와 같은 방향족 산류이거나 지방족 또는 방향족 설폰산류이거나 아세트산, 안식향산, 구연산, 젖산, 말레인산, 글루콘산, 메탄설폰산, 4-톨루엔설폰산, 주석산, 푸마르산과 같은 유기산일 수 있다.In addition, the acid addition salt may be an acid addition salt formed with a pharmaceutically acceptable free acid, but is not limited thereto. Preferably, 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.
상기 용매화물의 용매는 특별히 제한되지는 않으나, 바람직하게는 수화물 또는 알콜화물 일 수 있다.The solvent of the solvate is not particularly limited, but may preferably be a hydrate or an alcoholate.
또한 본 발명에 관하여 화합물의 언급은 특정의 이성질체 형태가 특히 언급되지 않는다면 각각의 그의 가능한 이성질체 형태 및 이의 혼합물인 화합물을 포함하는 것으로 의도된다.Reference to a compound in the context of the present invention is also intended to include the compound in each of its possible isomeric forms and mixtures thereof, unless a specific isomeric form is specifically stated.
본 발명에 따른 화합물은 상이한 다형 형태로 존재할 수 있다. 비록 상기 식에서 명시적으로 표시되지는 않았지만, 그러한 형태는 본 발명의 범위 내에 포함되는 것으로 의도된다.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.
본 발명의 일 구현예에 따르면, 본 발명의 조성물은 육두구 추출물 또는 화학식 1의 화합물 또는 이의 약학적으로 허용되는 염을 조성물 전체 중량에 대하여 0.0001 내지 90 중량% 포함할 수 있고, 구체적으로 0.1 내지 50 중량% 또는 0.1 내지 30 중량%로 포함할 수 있다.According to one embodiment 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.
본 발명의 약학적 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약학적으로 허용되는 담체를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다.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.
본 발명의 일 구현예에 따르면, 본 발명의 조성물은 약학적으로 허용 가능한 담체를 더 포함할 수 있다.According to one embodiment of the present invention, the composition of the present invention may further include a pharmaceutically acceptable carrier.
본 발명에서 용어 "담체"는, 세포 또는 조직 내로의 화합물의 부가를 용이하게 하는 화합물을 의미하고, 용어 "약학적으로 허용되는"이란, 생리학적으로 허용되고 인간에게 투여될 때, 통상적으로 위장 장애, 현기증과 같은 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 조성물을 말한다. As used herein, the term "carrier" refers to a compound that facilitates the addition of a compound into a cell or tissue, and the term "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.
상기 약학적으로 허용되는 담체는 제제 시에 통상적으로 이용되는 것으로서, 락토오스, 덱스트로오스, 수크로오스, 소르비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미결정셀룰로스, 폴리비닐피롤리돈, 셀룰로오스, 물, 메틸셀룰로오스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함할 수 있다.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.
본 발명의 약학적 조성물은 상기 성분들 이외에 충전제, 항응집제, 윤활제, 습윤제, 향료, 유화제, 방부제 등의 첨가제를 추가로 포함할 수 있다. 본 발명에 있어서, 상기 약학적 조성물에 포함되는 첨가제의 함량은 특별히 한정되는 것은 아니며 통상의 제제화에 사용되는 함량 범위 내에서 적절하게 조절될 수 있다. 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. In the present invention, 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.
본 발명의 약학적 조성물은 각각의 사용 목적에 맞게 통상의 방법에 따라 액제, 현탁제, 산제, 과립제, 정제, 캡슐제, 환제, 엑스제, 에멀젼, 시럽제, 에어로졸 등의 경구 제형, 멸균 주사 용액의 주사제 등 다양한 형태로 제형화하여 사용할 수 있으며, 경구 투여하거나 정맥 내, 복강 내, 피하, 직장, 국소 투여 등을 포함한 다양한 경로를 통해 투여될 수 있다.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.
본 발명의 일 구현예에 따르면, 본 발명의 조성물은 액제, 산제, 과립제, 정제, 캅셀제, 환제, 트로키제, 또는 엑스제의 형태로 제형화될 수 있다.According to one embodiment of the present invention, the composition of the present invention may be formulated in the form of a liquid, powder, granule, tablet, capsule, pill, troche, or extract.
본 발명에서 용어 "액제"는, 의약품을 물 또는 유기 용매에 용해한 물약 상태로 먹는 약을 의미한다. 상기 액제는 현탁제 또는 고형제에 비하여 장관에서의 전신 순환계로의 약물 흡수가 보다 효과적인 이점을 지니며, 상기 액제는 의약품 외에도 부가적인 용질도 포함할 수 있으며, 색깔, 냄새, 감미 혹은 안정성을 주는 첨가제도 포함할 수 있다. As used herein, the term “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.
본 발명에서 용어 "현탁제"는, 알기네이트 함유 조성물의 요망되는 용해도 및/또는 분산도를 제공할 수 있는, 즉, 실질적으로 투명하며 침강 및 덩어리가 없는 수성 제제를 제공할 수 있는 어떠한 작용제를 의미한다. As used herein, 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.
본 발명에서 용어 "산제"는, 잘게 분할된 약물, 화학물질 또는 양자의 건조 상태의 혼합물을 의미한다. As used herein, the term "powder" refers to a finely divided drug, chemical, or a dry mixture of both.
본 발명에서 용어 "과립제"는, 의약용 또는 의약품의 혼합물을 입상으로 한 것으로, 보통 4.76 내지 20 ㎜의 체를 통과하는 범위의 것을 말한다. 과립은 일반적으로 분말, 또는 분말 혼합물을 적셔서 그 덩어리를 필요로 하는 과립의 크기에 따라 적당한 메시 사이즈의 체 또는 과립기를 통과시킴으로써 생성한다. 과립제 역시 산제와 마찬가지로, 입자 상태이기 때문에 약물이 혀에 닿는 정도가 커서, 쓴맛을 가지는 약물을 과립제 형태로 사용할 경우에는 환자 특히 어린이나 노약자에게는 불편함을 야기할 수 있다.In the present invention, 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.
본 발명에서 용어 "정제"는, 분말상의 의약품을 작은 원판 모양으로 압축하여 복용하기 쉽게 만든 것을 의미한다. 정제는 나정, 필름 코팅정, 당의정, 다층정, 유핵정, 내핵정, 구강붕해정, 츄어블정, 발포정, 분산정, 용해정 등이 포함될 수 있다. In the present invention, the term "tablet" 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.
본 발명에서 용어 "캡슐제'는, 의약품을 액상, 현탁상, 물상, 분말상, 과립상, 미니정제 또는 펠렛 등의 형태로 캡슐에 충전하거나 캡슐 기제로 피포 성형하여 만든 것을 의미한다. In the present invention, the term "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.
본 발명에서 용어 "환제"는, 결합제 및 기타 부형제와 혼합된 복합 입자를 포함하는 작고 둥근 고체 투여형을 포괄하는 의미이다. As used herein, the term "pill" is meant to encompass small round solid dosage forms comprising composite particles mixed with a binder and other excipients.
본 발명에서 용어 "엑스제"는, 식물성 또는 동물성 생약 중의 약효 성분을 적당한 침출제를 사용해서 침출하고 용매를 증발시켜 규정된 농도로까지 농축하고 주성분 함량의 규정이 있는 것에서는 부형제를 가해 함량을 조절해서 만든 반고형 또는 고형의 제제를 의미한다. In the present invention, the term "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.
본 발명에서 용어 "시럽제"는, 설탕 또는 설탕대용제의 농조한 수제를 의미한다. 본 발명에서 상기 시럽제는, 불쾌한 맛, 예컨대 쓴맛이 있는 의약품을 액제로 하여 복용하기 쉽게 한 것으로, 특히 어린이들이 복용하기에 적합한 제형이다. 본 발명에서 상기 시럽제는 정제수 및 육두구 추출물 외에 사당 또는 감미와 점성을 주기 위하여 쓰이는 사당의 대용 약품, 항균성 보존제, 착미제 향료, 또는 착색제 등을 포함할 수 있으나, 이에 한정되는 것은 아니다. 이러한 시럽제에 포함될 수 있는 감미제의 예로는 백당, 만니톨, 소르비톨, 자일리톨, 아스파탐, 스테비오사이드, 과당, 유당, 수크랄로오스, 사카린 또는 멘톨 등이 있으나, 이에 한정되는 것은 아니다.In the present invention, the term "syrup" means a concentrated homemade sugar or sugar substitute. In the present invention, 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. In the present invention, 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. Examples of sweeteners that may be included in the syrup include sucrose, mannitol, sorbitol, xylitol, aspartame, stevioside, fructose, lactose, sucralose, saccharin or menthol, but are not limited thereto.
본 발명의 약학적 조성물의 바람직한 투여량은 환자의 상태 및 체중, 연령, 성별, 건강상태, 식이 체질 특이성, 제제의 성질, 질병의 정도, 조성물의 투여시간, 투여방법, 투여기간 또는 간격, 배설율 및 약물 형태에 따라 그 범위가 다양할 수 있으며, 이 분야 통상의 기술자에 의해 적절하게 선택될 수 있다. 예컨대, 약 0.1 내지 10,000 mg/kg의 범위, 약 1 내지 8,000 mg/kg의 범위, 약 5 내지 6,000 mg/kg의 범위, 또는 약 10 내지 4,000 mg/kg의 범위일 수 있으며, 바람직하게는 약 50 내지 2,000 mg/kg의 범위일 수 있으나 이에 제한되지 않으며, 하루 일회 내지 수회에 나누어 투여될 수 있다. 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.
본 발명에서 용어 "약학적 조성물의 유효 투여량"은, 특정한 증상을 치료하기 위해 충분한 활성 성분의 조성물의 양을 의미한다. 약학적 조성물의 제제화 방법, 투여 방식, 투여 시간 및/또는 투여 경로 등에 의해 다양해질 수 있으며, 약학 조성물의 투여로 달성하고자 하는 반응의 종류와 정도, 투여 대상이 되는 개체의 종류, 연령, 체중, 일반적인 건강 상태, 질병의 증세나 정도, 성별, 식이, 배설, 해당 개체에 동시 또는 일시에 함께 사용되는 약물 기타 조성물의 성분 등을 비롯한 여러 인자 및 의약 분야에서 잘 알려진 유사 인자에 따라 다양해질 수 있으며, 당해 기술 분야에서 통상의 지식을 가진 자가 목적하는 치료에 효과적인 투여량을 용이하게 결정하고 처방할 수 있다. As used herein, 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.
본 발명의 약학적 조성물의 투여는 하루에 1회 투여될 수 있고, 수회에 나누어 투여될 수도 있다. 본 발명의 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있다. 상기 요소를 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양으로 투여할 수 있으며, 이는 본 발명이 속하는 기술 분야의 통상의 기술자에 의해 용이하게 결정될 수 있다.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.
본 발명의 일 구현예에 따르면, 본 발명의 조성물은 베타-사이클로덱스트린(β-cyclodextrin)으로 포접된 포접 화합물을 포함하는 제제로 제형화될 수 있다.According to one embodiment of the present invention, the composition of the present invention may be formulated as a preparation containing an inclusion compound inclusion of beta-cyclodextrin (β-cyclodextrin).
본 발명의 일 구현예에 따르면, 상기 베타-사이클로덱스트린은 2,6-디메틸-베타-사이클로덱스트린(2,6-dimethyl-β-cyclodextrin), 2-히드록시에틸-베타-사이클로덱스트린(2-hydroxyethyl-β-cyclodextrin) 및 2-히드록시프로필-베타-사이클로덱스트린(2-hydroxypropyl-β-cyclodextrin) 중에서 선택된 어느 하나 이상일 수 있다.According to one embodiment of the present invention, the beta-cyclodextrin is 2,6-dimethyl-beta-cyclodextrin (2,6-dimethyl-β-cyclodextrin), 2-hydroxyethyl-beta-cyclodextrin (2- hydroxyethyl-β-cyclodextrin) and 2-hydroxypropyl-beta-cyclodextrin (2-hydroxypropyl-β-cyclodextrin) may be at least one selected from the group consisting of.
본 발명의 일 구현예에 따르면, 상기 포접 화합물은 베타-사이클로덱스트린 내부 공동에 육두구 추출물, 화학식 1의 화합물 또는 이의 약학적으로 허용되는 염이 봉입된 것일 수 있다.According to one embodiment of the present invention, 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.
본 발명에서 용어 "경구 투여"는, 활성물질이 소화되도록 제조된 물질, 즉 흡수를 위한 위장기관으로 투여되는 것을 의미한다. 상기 경구 투여용 제제의 비제한적인 예로는, 정제, 트로키제 (troches), 로젠지 (lozenge), 수용성 현탁액, 유성 현탁액, 조제 분말, 과립, 에멀젼, 하드 캡슐, 소프트 캡슐, 시럽 또는 엘릭실제 등을 들 수 있다. 본 발명의 약학적 조성물을 경구 투여용으로 제제화하기 위하여, 락토오스, 사카로오스, 소르비톨, 만니톨, 전분, 아밀로펙틴, 셀룰로오스 또는 젤라틴 등과 같은 결합제, 디칼슘 포스페이트 등과 같은 부형제, 옥수수 전분 또는 고구마 전분 등과 같은 붕해제, 스테아르산 마그네슘, 스테아르산 칼슘, 스테아릴 푸마르산 나트륨 등을 사용할 수 있으며, 감미제, 방향제, 시럽제 등도 사용할 수 있다. 나아가 캡슐제의 경우에는 상기 언급한 물질 외에도 지방유와 같은 액체 담체 등을 추가로 사용할 수 있다.In the present invention, the term “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. can be heard In order to formulate the pharmaceutical composition of the present invention for oral administration, 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. Furthermore, in the case of capsules, in addition to the above-mentioned substances, a liquid carrier such as fatty oil may be additionally used.
본 발명에서 용어 "부형제"는, 치료제가 아닌, 어느 물질을 의미하며, 치료제의 전달을 위한 담체 또는 매체로 이용되거나 또는 약학적 조성물에 추가되는 것을 의미한다. 이에 의해, 취급 및 저장 특성을 개선하거나 또는 조성물의 단위 투여량 형성을 허용 및 촉진시키게 된다.As used herein, 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.
본 발명은 또한, 육두구 추출물, 또는 하기 화학식 1의 화합물 또는 이의 약학적으로 허용되는 염을 유효 성분으로 포함하는 비알코올성 지방간 질환의 예방 또는 개선용 식품 조성물을 제공한다.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.
[화학식 1][Formula 1]
본 발명의 식품 조성물에 포함되는 육두구 추출물, 화학식 1의 화합물 또는 이의 약학적으로 허용되는 염에 대해서는 상기 약학적 조성물에서 설명된 바와 동일하다.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.
본 발명에 따른 식품 조성물은 본 발명의 육두구 추출물, 화학식 1의 화합물 또는 이의 약학적으로 허용되는 염을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. In the food composition according to the present invention, the nutmeg extract of the present invention, the compound of Formula 1, or a pharmaceutically acceptable salt thereof 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. .
본 발명의 식품 조성물은 또한 건강기능식품일 수 있다.The food composition of the present invention may also be a health functional food.
본 발명에서 용어 "기능식품" 이란, 인체에 유용한 기능성을 가진 원료나 성분을 사용하여 제조 및 가공한 식품을 의미하며, 본 발명에 있어서는 간질환 개선에 그 유익한 효과가 있다.In the present invention, 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.
본 발명에서 용어 "기능" 이란, 인체의 구조 및 기능에 대하여 영양소를 조절하거나 생리학적인 작용으로 보건 용도에 유용한 효과를 의미할 수 있다. 본 발명인 건강기능식품의 유효성분인 육두구 추출물을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. In the present invention, 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.
본 발명에 따른 식품 조성물은 약학적으로 허용 가능한 담체를 더 포함할 수 있다. 상기 식품의 종류에는 특별한 제한은 없다. 상기 식품의 예로는 각종 식품류, 음료, 껌, 차, 캔디, 비타민 복합제, 건강 기능성 식품류, 분말, 과립, 정제, 캡슐, 젤리 또는 음료 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함할 수 있다. The food composition according to the present invention may further include 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.
간질환 예방 및 치료를 목적으로 식품 또는 음료에 첨가될 수 있다. 이 때, 식품 또는 음료 중의 상기 추출물 또는 화학식 1의 화합물 또는 이의 약학적으로 허용되는 염의 양은 전체 식품 중량의 0.01 내지 30 중량%로 가할 수 있으며, 음료 조성물은 100 mL를 기준으로 0.01 내지 90 중량%, 바람직하게는 0.01 내지 50 중량%의 비율로 가할 수 있고, 이에 한정되는 것은 아니다. It may be added to food or beverage for the purpose of preventing and treating liver disease. At this time, 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.
본 발명의 식품은 지시된 비율로 필수 성분으로서 상기 육두구 추출물, 화학식 1의 화합물 또는 이의 약학적으로 허용되는 염을 함유하는 것 외에는 다른 성분에는 특별한 제한이 없으며, 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 첨가제로서 함유할 수 있고, 이에 한정되는 것은 아니다. 상기 천연 탄수화물은 포도당, 과당, 말토오스, 슈크로오스, 덱스트린, 사이클로덱스트린과 같은 사카라이드류가 있으며, 자일리톨, 소르비톨, 에리트리톨 등의 당알코올이 있을 수 있다. 상기 향미제에는 천연 향미제 (타우마틴, 스테비아 추출물, 예를 들어, 레바우디오시드 A, 글리시리진 등) 및 합성 향미제 (사카린, 아스파탐 등)를 사용할 수 있으나, 이에 한정되는 것은 아니다. 또한 본 발명의 식품은 추가로 여러 가지 영양제, 비타민, 광물 (전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제, 펙트산 및 그의 염, 알긴산, 구연산, 구연산 나트륨 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있으나, 이에 한정되는 것은 아니다. 이러한 첨가제는 유효성분인 육두구 추출물의 혼합물 1 중량부 당 0.001 내지 90 중량부의 범위에서 선택되는 것이 일반적이지만, 이에 한정되는 것은 아니다. 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.). In addition, 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.
본 발명에 따른 식품 조성물은 베타-사이클로덱스트린으로 포접된 포접 화합물을 포함하는 제제로 제형화될 수 있다.The food composition according to the present invention may be formulated as a preparation comprising an inclusion compound inclusion with beta-cyclodextrin.
본 발명에 따른 식품 조성물에 포함되는 베타-사이클로덱스트린으로 포접된 포접 화합물은 상기 상술한 바와 같다.The beta-cyclodextrin-inclusion compound included in the food composition according to the present invention is as described above.
본 발명의 조성물, 제제, 식품 및 건강기능식품에서 언급된 사항은 서로 모순되지 않는 한 동일하게 적용된다.Matters mentioned in the composition, formulation, food, and health functional food of the present invention are equally applicable as long as they do not contradict each other.
본 발명은 또한, 육두구를 추출하는 단계를 포함하는 비알코올성 지방간 질환의 예방 및 치료용 약학적 조성물의 제조방법을 제공한다.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.
본 발명의 일 구현예에 있어서, 본 발명의 제조방법은, 육두구를 추출하는 단계, 및 상기 수득된 육두구를 초임계 이산화탄소와 접촉시켜 추출물을 수득하는 단계를 포함할 수 있다.In one embodiment of the present invention, the preparation method of the present invention may include extracting nutmeg, and contacting the obtained nutmeg with supercritical carbon dioxide to obtain an extract.
본 발명의 일 구현예에 있어서, 상기 초임계 이산화탄소를 접촉시키는 단계는 육두구를 반응기에 넣고 반응 온도를 30 내지 100℃, 압력을 70 내지 500 bar로 하여 이산화탄소를 주입하는 단계일 수 있다.In one embodiment of the present invention, 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.
구체적으로, 본 발명의 제조방법은, 건조된 육두구를 선별하는 단계; 선별된 육두구를 세척한 후 40 내지 80℃ 에서 3 내지 7시간 동안 건조하는 단계; 반응 온도를 30 내지 100℃, 압력을 70 내지 500 bar로 하여 1 내지 12시간 동안 초임계 추출 처리하는 단계; 및 농축하여 동결 건조하는 단계를 통해 수득된 것일 수 있다. Specifically, 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.
본 발명은 또한, S1) 육두구를 추출하는 단계, 및 S2) 상기에서 수득한 육두구 추출물로부터 하기 화학식 1의 화합물 또는 또는 이의 약학적으로 허용되는 염을 분리하는 단계를 포함하는 비알코올성 지방간 질환의 예방 및 치료용 약학적 조성물의 제조방법을 제공한다.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.
[화학식 1][Formula 1]
본 발명은 또한, 육두구를 추출하는 단계를 포함하는 비알코올성 지방간 질환의 예방 및 개선용 식품 조성물의 제조방법을 제공한다.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.
본 발명에 있어 적용되는 육두구 추출 방법은 상기에서 상술한 바와 같다.The nutmeg extraction method applied in the present invention is the same as described above.
본 발명은 또한, S1) 육두구를 추출하는 단계, 및 S2) 상기에서 수득한 육두구 추출물로부터 하기 화학식 1의 화합물 또는 또는 이의 약학적으로 허용되는 염을 분리하는 단계를 포함하는 비알코올성 지방간 질환의 예방 및 개선용 식품 조성물의 제조방법을 제공한다.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.
[화학식 1][Formula 1]
본 발명에 따른 조성물은 독성물질을 포함하지 않고 세포 독성이 없으며, 메티오닌 콜린 결핍식이로 유도된 비알코올성 지방간 질환(Methionine choline deficiency-induce fatty liver disease; MCD) 모델을 통한 실험 결과, 간세포 보호작용 및 비알코올성 지방간염의 예방 및 치료 효과를 보여, 비알코올성 지방간 질환의 개선, 예방 및 치료제로 유용하게 적용될 수 있다.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.
도 1은 본 발명의 화합물 1의 1H-NMR 자료이다. 1 is 1 H-NMR data of Compound 1 of the present invention.
도 2는 본 발명의 화합물 1의 13C-NMR 자료이다. 2 is 13 C-NMR data of Compound 1 of the present invention.
도 3 내지 5는 본 발명의 화합물 1의 2D NMR data (도 3은 COSY, 도 4는 HMBC, 도 5는 HSQC) 자료이다.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.
도 6 및 7은 본 발명의 화합물 1의 질량분석자료이다.6 and 7 are mass spectrometry data of Compound 1 of the present invention.
도 8은 본 발명의 화합물 1(Compound 1)과 육두구 추출물(NME)의 HepG2 세포에 대한 독성시험 그래프이다 (Mean±S.D. *p<0.05).8 is a graph showing the toxicity test for HepG2 cells of Compound 1 and nutmeg extract (NME) of the present invention (Mean±S.D. *p<0.05).
도 9 및 10은 본 발명의 화합물 1(Compound 1)과 육두구 추출물(NME)의 H2O2에 의한 간세포 보호 효과를 나타낸 그래프이다 (Mean±S.D. *p<0.05). 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).
도 11은 본 발명의 화합물 1(Compound 1)의 AMPK 신호전달 기전 관련 단백질의 발현 수준을 나타낸 도이다.11 is a diagram showing the expression level of the AMPK signaling mechanism related protein of Compound 1 of the present invention.
도 12 및 13는 본 발명의 화합물 1(Compound 1)과 육두구 추출물(NME)의 CCl4에 의한 GOT 및 GPT 활성 억제에 대한 그래프이다 (Mean±S.D. *p<0.05). 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).
도 14 및 15는 Hematoxylin & Eosin (H&E), Picrosirius red staining 염색에 따른 Histopathological 시험을 현미경으로 분석한 결과이다.14 and 15 are results of microscopic analysis of Histopathological tests according to Hematoxylin & Eosin (H&E) and Picrosirius red staining.
도 16 및 17은 α-SMA, TGF-β에 대한 Histopathological 시험을 현미경으로 분석한 결과이다.16 and 17 are results of microscopic analysis of histopathological tests for α-SMA and TGF-β.
상기 상술한 목적을 달성하기 위한 본 발명은 육두구 추출물, 또는 하기 화학식 1의 화합물 또는 이의 약학적으로 허용되는 염을 유효 성분으로 포함하는 비알코올성 지방간 질환의 예방 또는 치료용 약학적 조성물, 비알코올성 지방간 질환의 예방 또는 개선용 식품 조성물 및 이의 제조방법을 제공하는 것을 특징으로 한다.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.
[화학식 1][Formula 1]
이하, 실시 예를 통해서 본 발명을 보다 상세히 설명하기로 한다. 하지만, 이들은 본 발명을 보다 상세하게 설명하기 위한 것일 뿐 본 발명의 권리범위가 이에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail through examples. However, these are only for describing the present invention in more detail, and the scope of the present invention is not limited thereto.
<실시예 1> 초임계 추출기를 이용한 육두구 추출물의 분리
<Example 1> Separation of nutmeg extract using supercritical extractor
인도네시아산 건조된 육두구를 구입하여 시료로 하고 깨끗이 수세한 후 건조한 다음, 육두구 1 kg을 마쇄한 후 초임계 추출기 (Supercritical fluid extractor, 5 L SCF SYSTEM, 포스엔텍, 한국)의 추출조 (용량: 5 L)에 넣고, 액화 이산화탄소 공급펌프를 통하여 이산화탄소를 70 cc/min의 속도로 공급하여 추출조 온도 60℃, 분리조 온도 40℃ 및 압력 250~400 bar 조건에서 초임계 상태로 만들고, 고속액체크로마토그래피 펌프 (Supercritical fluid chromatograph, 포스엔텍, 한국)를 이용하여 공용매 (co-solvent)로 에탄올 및 농도별 에탄올 수용액을 15 mL/min의 속도로 공급하면서 분리조를 통하여 2 시간 동안 추출물을 회수하였다. 상기 추출액을 회전감압농축기 (EYELA A-3S, 일본)로 농축하고 동결건조기 (BK-10N50, 중국)로 동결 건조하여 표 1과 같이 수득하였다.Purchase dried nutmeg from Indonesia, wash it clean, dry it, grind 1 kg of nutmeg, and then use the supercritical fluid extractor (5 L SCF SYSTEM, POS Entec, Korea) extraction tank (capacity: 5) L), and supplying carbon dioxide at a rate of 70 cc/min through a liquefied carbon dioxide supply pump to make it supercritical at the extraction tank temperature of 60 °C, the separation tank temperature of 40 °C, and the pressure of 250 to 400 bar, high-speed liquid chromatography The extract was recovered for 2 hours through a separation tank while supplying ethanol and an aqueous ethanol solution according to concentration as a co-solvent at a rate of 15 mL/min using a graph pump (Supercritical fluid chromatograph, POS Entec, Korea). . The extract was concentrated with a rotary vacuum concentrator (EYELA A-3S, Japan) and freeze-dried with a freeze dryer (BK-10N50, China) to obtain as shown in Table 1.
<실시예 2> 환류추출을 이용한 육두구 추출물의 분리
<Example 2> Separation of nutmeg extract using reflux extraction
분쇄한 육두구 분말 100 g에 에탄올 및 각 농도별 에탄올 수용액 1 L를 넣고 80℃ 에서 2 시간 동안 환류 냉각 추출한 후 여지 (Whatman No. 4, 미국)로 여과한 다음 여과 추출물을 회전감압농축기 (EYELA A-3S, 일본)로 감압 농축하고 동결건조기 (BK-10N50, 중국)로 동결 건조하여 표 1과 같이 수득하였다.To 100 g of ground nutmeg powder, add ethanol and 1 L of an aqueous ethanol solution for each concentration, extract under reflux at 80° C. for 2 hours, and then filter through a filter paper (Whatman No. 4, USA), and then extract the filtered extract with a rotary vacuum concentrator (EYELA A -3S, Japan), and freeze-drying with a freeze dryer (BK-10N50, China) to obtain as shown in Table 1.
<실시예 3> 화합물 1의 고속액체크로마토그래프법 (HPLC)에 따른 분석 <Example 3> Analysis according to high performance liquid chromatography (HPLC) of compound 1
실시예 1~2의 추출물을 동일한 농도로 제조하여 화합물 1을 다음과 같은 조건에서 분석한 화합물 1의 표준품은 Aurora fine chemicals (No. A67.867.655)에서 구입하였다. 각 조건의 육두구 추출물을 Waters 2996 (Waters, 미국)를 사용하여 아세토니트릴 : 0.1% 아세트산 용액 (50 : 50 v/v %) 조건의 HPLC [Agilent Eclipse XDB-C18 (4.6×250mm, 입자 크기 5 μm), 유속: 0.6 mL/분, 측정파장: 280 nm]를 이용하여 분석하였다. 육두구 추출물의 화합물 1의 함량은 표 1과 같이 초임계 추출방법을 이용한 추출압력 250 bar, 에탄올 60%의 공용매 조건에서 가장 높은 12.7%의 함량을 나타내었다.A standard of Compound 1, prepared by preparing the extracts of Examples 1 and 2 at the same concentration and analyzing Compound 1 under the following conditions, was purchased from Aurora fine chemicals (No. A67.867.655). Nutmeg extract under each condition was subjected to HPLC [Agilent Eclipse XDB-C18 (4.6×250 mm, particle size 5 μm) in acetonitrile: 0.1% acetic acid solution (50: 50 v/v %) conditions using Waters 2996 (Waters, USA). ), flow rate: 0.6 mL/min, measurement wavelength: 280 nm]. As shown in Table 1, the content of Compound 1 in the nutmeg extract showed the highest content of 12.7% under the extraction pressure of 250 bar and the co-solvent condition of 60% ethanol using the supercritical extraction method.
<실시예 4> 육두구로부터 추출된 화합물 1의 확인<Example 4> Identification of compound 1 extracted from nutmeg
실시예 1의 추출 조건에 따라 수득한 추출물 중 250 bar, 60% 에탄올 수용액의 조건에서 수득한 추출물을 분취용 컬럼을 이용한 고속액체크로마토그래피법으로 화합물 1을 분리하여 ESI-MS, ESI-MS/MS, 1H, 13C-NMR 등의 분광학적 분석방법을 이용하여 구조를 확인하였다.Among the extracts obtained according to the extraction conditions of Example 1, 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.
<실시예 4-1> 분취용 컬럼을 이용한 고속액체크로마토그래피법으로 화합물 1의 분리 <Example 4-1> Separation of compound 1 by high-performance liquid chromatography using a preparative column
실시예 1의 추출 조건에 따라 수득한 추출물 중 250 bar, 60% 에탄올 수용액의 조건에서 수득한 추출물 50 g을 이동상에 용해하고 여과한 후 Waters DeltaprepLC300 (Waters, 미국)을 사용하여 1 회 주입량 10 mL로 하고 아세토니트릴 : 0.1% 아세트산 용액 (50 : 50 v/v%) 조건의 HPLC [Luna® 5 μm C18(2) 100Å, LC Column (21.2×250 mm, 입자크기: 5 μm), 유속: 20mL/분, 측정파장: 280 nm]를 이용하여 분리하였다. 분취액을 진공동결건조기 (ChemStar 1402N, Welch®, IL, USA)로 동결 건조하여 무색의 오일상인 화합물 1을 1.2 g 수득하였다.Among the extracts obtained according to the extraction conditions of Example 1, 50 g of the extract obtained under the conditions of 250 bar and 60% aqueous ethanol solution was dissolved in a mobile phase, filtered, and then filtered using Waters DeltaprepLC300 (Waters, USA) for a single injection amount of 10 mL HPLC with acetonitrile: 0.1% acetic acid solution (50: 50 v/v%) conditions [Luna ® 5 μm C18(2) 100Å, LC Column (21.2×250 mm, particle size: 5 μm), flow rate: 20mL /min, measuring wavelength: 280 nm]. An aliquot was freeze-dried using a vacuum freeze dryer (ChemStar 1402N, Welch ® , IL, USA) to obtain 1.2 g of Compound 1 as a colorless oil.
<실시예 4-2> 화합물 1의 1H, 13C-NMR, ESI-MS, ESI-MS/MS 분석<Example 4-2> 1H, 13C-NMR, ESI-MS, ESI-MS/MS analysis of compound 1
<실시예 4-2-1> 화합물 1의 1H, 13C-NMR 분석<Example 4-2-1> 1H, 13C-NMR analysis of compound 1
분리 물질의 구조 규명을 위한 NMR 분석에는 Bruker사의 고해상도 핵자기공명분광기 (600 MHz, AVANCE 600, Bruker, Germany)를 사용하였고, 이를 통해 1H, 13C-NMR, 2D NMR data (COSY, HSQC, HMBC 등)를 확보하여 분석하였다 (표 2, 도 1~5).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).
<실시예 4-2-2> 화합물 1의 LC/MS 분석<Example 4-2-2> LC/MS analysis of compound 1
화합물 1의 질량분석을 위한 분석을 위한 HPLC는 Ultimate3000 (Thermo Scientific, USA)를 사용하여 A: 0.1% Formic acid in water, B: 0.1% Formic acid in acetonitrile (0-1분: 90% A, 1-25분: 100% B, 25-30분: 90% B) 조건의 HPLC [Waters BEH C18 (2.1×100 mm, 1.7 um 입자크기, 유속: 0.4 mL/min, 측정파장: 280 nm, 컬럼온도: 45℃)]을 이용하였고, Mass spectrophotometer는 Triple TOF 5600+(AB Sciex, USA)를 이용하여 분석하였다. 분석결과, 화합물 1은 무색의 오일(oil) 상으로 분자량은 416.46 (439[M+Na]+)으로 측정되었다 (도 6~7).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 ).
<실시예 5> HepG2 세포주를 이용한 세포독성실험<Example 5> Cytotoxicity test using HepG2 cell line
<실시예 5-1> HepG2 세포주 배양<Example 5-1> HepG2 cell line culture
인간 간세포주인 HepG2 (human hepatocyte-derived cell line, ATCC, Manassas, VA, 미국)를 둘베코수정이글배지 (Dulbecco's Modified Eagle's Medium, DMEM)에 10% 소태아혈청 (fetal bovine serum, FBS), 100 U/mL 페니실린 (penicillin) 및 100 μg/mL 스트렙토마이신 (streptomycin)을 첨가하여 37℃, 5% CO2를 유지하는 조건으로 배양기 (Forma Series II, Thermo Electron Corp., MA, 미국)에서 배양하였다.HepG2 (human hepatocyte-derived cell line, ATCC, Manassas, VA, USA), a human hepatocyte cell line, was added to Dulbecco's Modified Eagle's Medium (DMEM) with 10% fetal bovine serum (FBS), 100 U /mL penicillin (penicillin) and 100 μg/mL streptomycin (streptomycin) was added and cultured in an incubator (Forma Series II, Thermo Electron Corp., MA, USA) under conditions of maintaining 37°C and 5% CO 2 .
<실시예 5-2> MTT 분석을 이용한 HepG2 세포독성실험<Example 5-2> HepG2 cytotoxicity test using MTT assay
실시예 1의 추출 조건에 따라 수득한 추출물 중 250 bar, 60% 에탄올 수용액의 조건에서 수득한 육두구 추출물 (NME)과 실시예 4에서 수득한 화합물 1에 대한 세포독성 여부를 확인하기 위하여, MTT (3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) 분석을 이용하여 측정하였다. Among the extracts obtained according to the extraction conditions of Example 1, 250 bar, in order to determine whether the nutmeg extract (NME) obtained under the conditions of a 60% aqueous ethanol solution and the cytotoxicity to the compound 1 obtained in Example 4, MTT ( 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide) analysis was used.
96-well plate에 DMEM 배지를 이용하여 1×105 cells/well의 HepG2 세포를 분주하고 24 시간 동안 5% CO2 배양기에서 배양한 후 배지를 제거하고 0.1 M 인산염완충액 (pH 7.0)을 사용하여 cell을 2 회 세척하였다. 실시예 1의 NME와 실시예 4의 화합물 1을 각각 10, 20 및 30 μg/mL의 농도로 FBS-free DMEM 배지에 희석하여 시료를 처리한 후 37℃, 5% CO2 조건 하에서 24시간 배양하였다. 상등액을 제거한 후 0.1 M 인산염완충액 (pH 7.0)에 5 mg/mL의 농도로 용해시킨 MTT solution 100 μL를 첨가하여 CO2 배양기에서 2 시간 반응시켰다. 이후, 상등액을 모두 제거하고 DMSO 100 μL씩을 첨가하여 30 분간 암실에서 반응시켰으며, 생성된 포르마잔(formazan)을 Microplate reader (BKMPR-1096A, 중국)에서 540 nm로 측정하였다. 시료를 처리하지 않은 대조군의 세포생존율을 100%로 하여 세포생존율 (%)은 다음과 같은 식을 사용하였다. In 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 μg/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 μL 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 μL 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.
세포생존율 (%)=(시료처리군의 흡광도/대조군의 흡광도)×100Cell viability (%) = (absorbance in the sample treatment group / absorbance in the control group) × 100
그 결과, NME 및 화합물 1의 투여군에서 90% 이상의 세포생존율을 나타내어 세포 독성을 나타내지 않음을 확인하였다 (도 8).As a result, it was confirmed that the NME and compound 1 administration group exhibited a cell viability of 90% or more, indicating no cytotoxicity (FIG. 8).
실시예 5-2에 따른 NME와 실시예 4의 화합물 1의 과산화수소 (H2O2)에 의한 간세포 보호 효과를 확인하기 위하여, 간 기능의 지표 효소인 GPT (glutamic pyruvic transaminase) 및 GOT (glutamic oxaloacetic transaminase)의 활성을 측정하였다. In order to confirm the hepatocellular protective effect of NME according to Example 5-2 and hydrogen peroxide (H 2 O 2 ) of Compound 1 of Example 4, GPT (glutamic pyruvic transaminase) and GOT (glutamic oxaloacetic acid), which are indicator enzymes of liver function transaminase) activity was measured.
24-well plate에 DMEM 배지를 이용하여 5×105 cells/well의 HepG2 세포를 분주하고 24 시간 동안 5% CO2 배양기에서 배양하였다. 배양된 세포에 과산화수소 200 μM, NME 및 화합물 1을 50 μg/mL의 농도로 가하고, 24 시간 동안 반응시킨 후 3,000xg에서 5 분 동안 원심분리 후 상등액을 회수하여 GOT 측정용 키트 (ASAN GOT-Lq Reagents, 아산제약, 한국)를 사용하여 Microplate reader (BKMPR-1096A, 중국)로 340 nm에서 측정하였다. 5×10 5 cells/well of HepG2 cells were dispensed in a 24-well plate using DMEM medium and cultured in a 5% CO 2 incubator for 24 hours. 200 μM of hydrogen peroxide, NME, and Compound 1 were added to the cultured cells at a concentration of 50 μg/mL, reacted for 24 hours, centrifuged at 3,000xg for 5 minutes, and the supernatant was recovered and a kit for GOT measurement (ASAN GOT-Lq) Reagents, Asan Pharmaceutical, Korea) were used and measured at 340 nm with a Microplate reader (BKMPR-1096A, China).
도 9 및 10에서 나타낸 바와 같이, HepG2 세포에 과산화수소 200 μM를 처리하였을 때 H2O2 투여 대조군에 비하여, NME와 화합물 1에 의하여 GOT에 대한 활성이 각각 54.2±6.6, 49.3±8.4 U/L로 억제되고, GPT에 대한 활성이 각각 39.5±11.2, 35.6±5.1 U/L로 억제되어 유의한 간세포 보호 효과를 확인하였다.As shown in FIGS. 9 and 10 , when 200 μM of hydrogen peroxide was treated in HepG2 cells, the activity on GOT by NME and Compound 1 was 54.2±6.6 and 49.3±8.4 U/L, respectively, compared to the H 2 O 2 administration control group. was inhibited, and the activity against GPT was inhibited to 39.5±11.2 and 35.6±5.1 U/L, respectively, confirming a significant hepatocellular protective effect.
<실시예 7> 화합물 1의 AMPK 활성화 측정<Example 7> Measurement of AMPK activation of compound 1
HepG2 세포를 24-well plate에 3×105 cells/well 분취한 후 하룻동안 배양하고 FBS가 포함되어 있지 않은 배양액으로 16 시간 starvation 시킨 후 화합물 1을 100 μg/mL로 처리한 후 6 시간까지 배양하였다. 배양액이 제거된 HepG2 세포를 차가운 PBS로 2 회 세척한 다음 얼음 위에 플레이트를 올려놓고 6-well plate well 당 라이시스 버퍼(lysis buffer) (40 mM Tris-HCl pH 7.5, 10 mM EDTA pH 8.0, 120 mM NaCl, 1 mM PMSF, 1 mM NaF, proteinase inhibitor tablet)를 200 ㎕ 넣고 스크랩퍼를 이용하여 긁어서 모은 세포를 4℃ 조건에서 30 분간 sonication 후 11,000 g에서 10 min 원심분리를 한 다음 상등액을 다른 Ep-tube에 담아 효소액의 농도를 측정하고 모든 효소의 농도를 60 ㎍으로 맞춰서 실험에 이용하기 전까지 -70℃에 보관하였다. Western blot 실험을 위해 효소 샘플을 얼음에 꽂아 놓고 녹이고 4× 샘플 버퍼(250 mM Tris-HCl pH 6.8, 40% 글리세롤, 8% SDS, 4% β-메르캅토에탄올, 0.2% 브로모페놀 블루)를 샘플에 함께 섞은 후 끓는 물에서 5 분간 끓여 준 후 60 ㎍의 단백질을 만들어 놓은 SDS-폴리아크릴아마이드 겔(1.5 M Tris-HCl pH 8.0, DDW, 30% 아크릴아마이드, 10% SDS, TEMED, 10% APS)에 로딩한 후 10% SDS PAGE 버퍼 (Tris 베이스, 글라이신, SDS)를 이용하여 120 V에서 단백질을 분리하였다. 전기영동이 끝나면 gel의 단백질을 Transfer 버퍼 (Tris base, 글라이신, SDS, 메틸 알코올)를 이용하여 PVDF 멤브레인으로 옮기고 1차 항체인 AMPK ab (Sc-25792, Santa Cruz Biotechnology)와 p-AMPK ab (#07-626, Millipore Corporation)를 5% BSA로 1 : 1,000의 비율로 희석하고, Primary antibody를 납작한 통에 멤브레인이 잠길 정도로 부어준 후 냉장 상태에서 overnight shaking 후 Primary antibody를 overnight해 준 후 PBS 버퍼를 이용하여 5 분씩 1 시간 동안 세척하였다. 세척한 멤브레인은 1 : 2,000의 비율로 희석한 2차 항체인 anti-rabbit (#7074, Cell membrane Signaling Technology)에 1 시간 동안 상온에서 부착시킨 후 PBS 버퍼로 5 분씩 1 시간 동안 세척하고 ECL (Amersham Pharmacia Biotech, NJ, USA)을 처리한 멤브레인을 현상한 후 얻어진 단백질 밴드의 밀도는 Image J software (NIH, Bethesda, MD, USA)를 사용하여 정량하였다. 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 μg/mL, and cultured for up to 6 hours. did 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 μl of (mM NaCl, 1 mM PMSF, 1 mM NaF, proteinase inhibitor tablet) and scraping using a scraper, sonication at 4℃ 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 μg and stored at -70° C. until used in the experiment. For Western blot experiments, the enzyme samples were placed on ice, thawed, and 4x sample buffer (250 mM Tris-HCl pH 6.8, 40% glycerol, 8% SDS, 4% β-mercaptoethanol, 0.2% bromophenol blue) was added. SDS-polyacrylamide gel (1.5 M Tris-HCl pH 8.0, DDW, 30% acrylamide, 10% SDS, TEMED, 10%) prepared with 60 µg protein after mixing with the sample and boiling in boiling water for 5 minutes APS), the protein was separated at 120 V using 10% SDS PAGE buffer (Tris base, glycine, SDS). After electrophoresis, 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. 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).
실험결과 도 11과 같이, 화합물 1은 3 시간 이후 유의하게 AMPK의 인산화를 증가시켰으며, AMPK의 하위 단백질인 ACC의 인산화는 6 시간에 가장 높게 증가하였다.Experimental results As shown in FIG. 11 , Compound 1 significantly increased phosphorylation of AMPK after 3 hours, and phosphorylation of ACC, a subprotein of AMPK, was highest at 6 hours.
<실시예 8> 사염화탄소 ( )에 의한 간독성에 대한 보호 효과 <Example 8> Carbon tetrachloride ( ) protective effect against hepatotoxicity by
실시예 5-2에 따른 NME와 실시예 4의 화합물 1의 사염화탄소 (CCl4)에 의한 간독성에 대한 보호 효과를 확인하기 위하여, 간 기능의 지표 효소인 GPT (glutamic pyruvic transaminase) 및 GOT (glutamic oxaloacetic transaminase)의 활성을 측정하였다. In order to confirm the protective effect against hepatotoxicity by NME according to Example 5-2 and carbon tetrachloride (CCl 4 ) of Compound 1 of Example 4, GPT (glutamic pyruvic transaminase) and GOT (glutamic oxaloacetic acid), which are indicator enzymes of liver function transaminase) activity was measured.
실험 동물은 체중 200 g 내외의 웅성 SD (Sprague-Dawley)계 흰쥐를 이용하였고, 정상대조군, 사염화탄소 투여 대조군, 실시예 1의 육두구 추출물 및 실시예 4의 화학식 1 투여군으로 하여 각 군당 6 마리씩 배정하였다. 실험 하루 전에 채혈을 위해 각 실험 동물의 경정맥에 실라스틱 튜브 (Silastic tubing, Dow Corning Co., 미국)와 폴리에틸렌 튜브 (PE-50, Medichem, 미국)를 이용하여 삽관을 실시하였다. 사염화탄소를 올리브유에 50 mg/mL로 현탁하여 사염화탄소 투여 대조군, NME 및 화합물 1 투여군에 각각 복강 투여 (50 mg/kg)하고, 30 분 후 NME 투여군 200 mg/kg, 화합물 1 투여군은 100 mg/kg을 50% 프로필렌글리콜 수용액에 현탁하여 경구 투여하였고, 정상대조군은 50% 프로필렌글리콜 수용액을 투여하였다. 투여 24 시간 후 각각의 군에서 삽관된 튜브를 통하여 혈액을 채취하였다. 채혈된 혈액을 원심분리기 (Union 32R, 한일, 한국)로 3,000Хg에서 5분 동안 원심 분리한 다음 혈장을 분리하여 GPT 및 GOT 측정용 키트 (ASAN GPT-Lq Reagents, ASAN GOT-Lq Reagents, 아산제약, 한국)를 사용하여 Microplate reader (SmartReader 96, ACCURIS Instruments, 미국)로 340 nm에서 측정하였다.As the experimental animals, 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 . One day before the experiment, 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. After centrifuging the collected blood at 3,000Хg for 5 minutes with a centrifuge (Union 32R, Hanil, Korea), plasma is separated and a kit for GPT and GOT measurement (ASAN GPT-Lq Reagents, ASAN GOT-Lq Reagents, Asan Pharmaceutical) , Korea) using a Microplate reader (SmartReader 96, ACCURIS Instruments, USA) was measured at 340 nm.
실험결과, 도 12 및 13에서 나타낸 바와 같이, NME와 화합물 1에 의하여 GOT에 대한 활성이 각각 75.8±11.6, 69.9±15.2 U/mL로 억제되었으며, GPT에 대한 활성은 각각 40.5±10.2, 35.2±8.2 U/mL로 CCl4 투여 대조군에 비하여 유의하게 활성이 억제되어, 간 독성에 대한 세포 보호 효과가 우수함을 확인하였다.As a result of the experiment, as shown in FIGS. 12 and 13 , 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.
<통계분석><Statistical Analysis>
대조군과 각 시료로부터 얻은 실험 결과들의 유의성을 검정하기 위하여 SAS, version 9.0 (SAS Institute Inc., Cary, NC, USA)를 사용하여 분산분석 (ANOVA)을 실시한 후, p<0.05 수준에서 Duncan's multiple range test를 실시하였다. 모든 실험결과는 평균±standard deviation로 표시하였다.In order to test the significance of the experimental results obtained from the control group and each sample, analysis of variance (ANOVA) was performed using SAS, version 9.0 (SAS Institute Inc., Cary, NC, USA), and then Duncan's multiple range at p<0.05 level. The test was carried out. All experimental results were expressed as mean±standard deviation.
<실시예 9> 메티오닌 콜린 결핍 (MCD) 식이로 유도된 지방간 모델에서의 육두구 추출물(NME)과 화합물 1의 효과 평가<Example 9> Evaluation of the effect of nutmeg extract (NME) and compound 1 in a methionine choline deficiency (MCD) diet-induced fatty liver model
20g 내외의 생후 5 주령 C57BL/6NHsd 수컷 생쥐를 1 주 동안 정상 식이를 공급하면서 실험실 환경에 적응시킨 후 본 실험을 시작하였다. 실험기간 동안 명암주기 12 시간 간격(오전 8시 점등 ~ 오후 8시 소등), 온도 23±3℃, 습도 55±15%로 실험실 환경을 유지하였다. 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%.
<실시예 9-1> 시험군의 구성 및 투여량 설정<Example 9-1> Test group composition and dosage setting
적량의 육두구 추출물 및 화합물 1을 칭량하고, 전체 액량의 10% DMSO에 녹인 뒤, 전체 액량의 90%에 해당하는 0.5% 카르복시메틸셀룰로오스 (CMC)를 첨가하여 조제하였다. 사료는 MCD 식이를 5 일간 공급하고 이후 2 일간은 정상사료를 공급하는 방식으로 12주 (8주 유발+4주 치료) 동안 MCD 식이 및 정상사료를 번갈아가며 자유롭게 섭취하도록 하였다. 투여 용량은 최근 체중측정일에 측정한 체중을 기준으로 각각의 육두구 추출물 및 화합물 1을 10 mL/kg로 산출하였고, 동물을 경배부 피부 고정법으로 고정하고, 경구투여용 존데를 이용하여 1 회/일, 4 주간 투여하였다.An appropriate amount of 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. For the feed, MCD diet was supplied for 5 days and normal food was supplied for 2 days thereafter. For 12 weeks (8 weeks of induction + 4 weeks of treatment), 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.
<실시예 9-2> 혈액생화학적 검사<Example 9-2> Blood biochemical test
각 시험군 별 육두구 추출물 및 화합물 1의 투여 전 및 투여 후 4 주째 (부검일)에 채혈하여 분리한 혈청으로 혈액생화학분석기 (7180 Hitachi, Japan)를 이용하여 ALT (알라닌 트랜스아미네이즈, alanine transaminase), AST (아스파르테이트 트랜스아미네이즈, aspartate transaminase), TG (트리글리세라이드, Triglyceride), TCHO (총 콜레스테롤, Total cholesterol) 항목을 검사하였다.ALT (alanine transaminase, alanine transaminase) using a blood biochemistry analyzer (7180 Hitachi, Japan) with blood collected and separated from the serum before and 4 weeks after administration of nutmeg extract and compound 1 for each test group (the day of autopsy) , AST (aspartate transaminase, aspartate transaminase), TG (triglyceride), and TCHO (total cholesterol) were tested.
부검 전 모든 생존 동물은 하룻밤 동안 절식 (물은 제공)하였다. 부검일에 동물을 에테르로 흡입마취하고, 마취가 확인되면 개복하여 후대정맥에서 주사기를 이용하여 채혈을 실시한 뒤, 복대동맥 및 후대정맥을 절단하여 방혈/치사시켰다. 혈액은 clot activator가 들어 있는 vacutainer tube에 주입하고 약 15 분간 실온에 방치하여 응고시킨 후 12,000 rpm으로 3 분간 원심분리하여 혈청을 분리하였다. 혈청은 분석 전까지 -70℃ 이하로 설정되어 있는 Deep freezer에 보관하였으며, 혈액생화학적 검사에 사용하였다. All surviving animals were fasted (water provided) overnight before necropsy. On the day of autopsy, animals were anesthetized with ether by inhalation, and when anesthesia was confirmed, the animals were laparotized and blood was collected from the posterior vena cava using a syringe, and then the abdominal aorta and posterior vena cava were cut and exsanguinated/killed. Blood was injected into a vacutainer tube containing a clot activator, left at room temperature for about 15 minutes to solidify, and then centrifuged at 12,000 rpm for 3 minutes to separate serum. Serum was stored in a deep freezer set at -70°C or lower until analysis, and used for blood biochemical tests.
실험결과 표 4 및 표 5에서와 같이, 각 시험군별 육두구 추출물 및 화합물 1의 투여 개시 전 모든 지방간 유발군 (G2-G6)의 ALT 및 AST 수준은 정상대조군(G1)에 비하여 유의하게 높았으며 (p<0.001), G5의 TCHO 수준은 G1에 비하여 유의하게 낮았다 (p<0.01).Experimental Results As shown in Tables 4 and 5, the ALT and AST levels of all fatty liver-inducing groups (G2-G6) before the start of administration of nutmeg extract and Compound 1 for each test group were significantly higher than those of the normal control group (G1) ( p<0.001), the TCHO level of G5 was significantly lower than that of G1 (p<0.01).
각 시험군 별 육두구 추출물 및 화합물 1의 투여개시 후 4 주째에 모든 지방간 유발군 (G2-G6)의 ALT, AST 수준은 정상대조군(G1)에 비하여 유의하게 높았으며 (p<0.001), G3 및 G6의 ALT 수준은 G2에 비하여 유의하게 낮았고 (p<0.01 및 p<0.05), G3, G5 및 G6의 AST 수준은 G2 대비 유의하게 낮았다 (p<0.001). At 4 weeks after the start of administration of nutmeg extract and Compound 1 for each test group, the ALT and AST levels of all fatty liver-inducing groups (G2-G6) 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).
각 시험군 별 육두구 추출물 및 화합물 1의 투여개시 후 4 주째에 모든 지방간 유발군 (G2-G6)의 TCHO 및 TG 수준은 정상대조군(G1)에 비하여 유의하게 낮았다 (p<0.001). ALT 및 AST는 간 기능과 관련된 혈액생화학적 지표로써, 육두구 추출물 및 화합물 1의 투여가 MCD 식이에 의하여 유도된 지방간염 모델에서 간 기능 관련 수치의 감소를 유도하였다.At 4 weeks after the start of administration of nutmeg extract and Compound 1 for each test group, the TCHO and TG levels of all fatty liver-inducing groups (G2-G6) were significantly lower than those of the normal control group (G1) (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.
<실시예 9-3> 간 중량 측정<Example 9-3> Liver weight measurement
부검 시 간을 적출하여 중량을 측정하고, 적출한 간의 좌엽은 10% 중성완충포르말린용액에 고정하여 조직병리를 수행하였으며, 우엽은 급속 냉동 후 -70 ℃이하로 설정되어 있는 Deep freezer에 보관하여 ELISA를 수행하였다. At the time of autopsy, 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.
실험결과, 표 6에서와 같이, G3, G4, G5 및 G6의 간 중량 수준은 G1에 비하여 통계학적으로 유의하게 낮았고 (p<0.001 또는 p<0.01), G3 및 G6의 간 중량 수준은 G2에 비하여 유의하게 낮았다 (p<0.01). As a result of the experiment, as shown in Table 6, 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).
모든 지방간 유발군 (G2-G6)의 간 상대중량 수준은 G1 대비 유의하게 높았으며 (p<0.001), G3, G5 및 G6의 간 상대중량 수준은 G2에 비하여 통계학적으로 유의하게 낮았다 (p<0.01).The liver relative weight levels of all fatty liver-inducing groups (G2-G6) 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).
상기 결과로 미루어 보아, 본 발명의 화합물 1 및 육두구 추출물 투여가 지방간 유도에 따른 간 비대를 억제한 것으로 사료된다.Judging from the above results, it is considered that the administration of Compound 1 and nutmeg extract of the present invention suppressed hepatomegaly caused by fatty liver induction.
<실시예 9-4> 조직병리학적 검사<Example 9-4> Histopathological examination
고정된 조직은 삭정, 탈수, 파라핀 포매, 박절 등 일반적인 조직처리 과정을 거쳐 조직병리학적 검사를 위한 검체를 제작한 뒤, 헤마톡실린 & 에오신 (Hematoxylin & Eosin, H&E), 피크로시리우스 적색 착색 (Picrosirius red staining) 염색 및 면역조직화학염색 (TGF-β, α-SMA)을 실시하고, 광학 현미경 (Olympus BX53, Japan)을 이용하여 조직병리학적 변화를 관찰하였다. 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-β, α-SMA) were performed, and histopathological changes were observed using an optical microscope (Olympus BX53, Japan).
조직병리학적 검경은 하기 검경 기준에 따라 평가하였으며 (Liang et al, 2014), Picrosirius red 염색 및 면역조직화학염색의 분석은 Image analyzer (Zen 2.3 blue edition, Carl Zeiss, Germany)를 이용하여 실시하였으며, 각 개체 별 전체 면적 대비 염색면적을 군간 비교하였다 (도 13-14).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 ).
실험결과 표 8에서와 같이, 모든 지방간 유발군 (G2-G6)의 거대공포성 지방증 수준은 G1에 비하여 유의하게 높았으며 (p<0.001), G3, G4, G5 및 G6의 거대공포성 지방증 수준은 G2에 비하여 유의하게 낮았다 (p<0.01 또는 p<0.05). As shown in Table 8, macrophobic steatosis levels in all fatty liver-inducing groups (G2-G6) 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).
G2, G4, G5 및 G6의 미세공포성 지방증 수준은 G1에 비하여 유의하게 높았고 (p<0.001, p<0.01 또는 p<0.05), G3의 미세공포성 지방증 수준은 G2에 비하여 유의하게 낮았다 (p<0.01). 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).
G2 및 G4의 염증 수준은 G1에 비하여 유의하게 높았고 (p<0.01 및 p<0.05), G3의 염증 수준은 G2에 비하여 유의하게 낮았다 (p<0.05). 모든 육두구 추출물 투여군 (G4-G6)의 거대공포성 지방증 수준은 음성대조군 (G2, negative control)에 비하여 유의하게 낮은 병변 수준을 나타내었으며, 유의한 차이는 아니었으나, 미세공포성 지방증 및 염증 수준 또한 음성대조군 (G2) 대비 낮은 경향을 나타내었다.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).
Picrosirius red 염색을 이용한 섬유화 면적 분석 결과, G2의 염색 면적 수준은 G1에 비하여 유의하게 높았으며 (p<0.01), G6의 염색 면적 수준은 G2에 비하여 유의하게 낮았다 (p<0.05). 육두구 추출물 및 화합물 1의 투여와 용량상관성을 보이며 섬유화 면적의 감소 경향을 나타내었고, 육두구 추출물 400 mg/kg 투여군 (G6)의 섬유화 면적 수준은 음성대조군 (G2) 대비 유의하게 감소한 것으로 관찰되었다. As a result of the analysis of the fibrosis area using Picrosirius red staining, 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.
면역조직화학염색을 이용한 TGF-β 및 α-SMA 발현 면적 분석 결과, 모든 지방간 유도군 (G2-G6)의 TGF-β 및 α-SMA 발현 면적 수준은 G1에 비하여 유의하게 높았고 (p<0.001), 모든 육두구 추출물 및 화합물 1 투여군 (G3-G6)의 TGF-β 발현 면적 수준은 G2에 비하여 유의하게 낮았으며 (p<0.001 또는 p<0.01), 모든 육두구 추출물 투여군 (G4-G6)의 α-SMA 발현 면적 수준은 G2에 비하여 유의하게 낮았다 (p<0.001 또는 p<0.01). 염증 및 섬유화 관련 인자인 α-SMA의 발현 면적 수준이 육두구 추출물 및 화합물 1의 투여와 용량상관성 있는 변화경향을 나타내며 감소하는 양상을 나타내었고, TGF-β 발현 면적 수준은 육두구 추출물 및 화합물 1 투여군 (G3-G6) 전 용량에서 음성대조군 (G2) 대비 유의한 차이를 나타내며 감소하였다.As a result of analysis of TGF-β and α-SMA expression area using immunohistochemical staining, the expression area level of TGF-β and α-SMA in all fatty liver-inducing groups (G2-G6) was significantly higher than that of G1 (p<0.001). , TGF-β expression area levels of all nutmeg extract and compound 1 administration groups (G3-G6) were significantly lower than those of G2 (p<0.001 or p<0.01), and α- 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 α-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-β 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.
<실시예 9-5> ELISA 분석<Example 9-5> ELISA analysis
부검일에 채혈한 혈청을 이용하여 NEFA (Non-esterified fatty acid)를 측정하였으며, 적출한 간 조직을 이용하여 간 조직 내 TG, TCHO 및 NEFA를 측정하였다. NEFA (Non-esterified fatty acid) was measured using the serum collected on the day of autopsy, and TG, TCHO and NEFA in the liver tissue were measured using the extracted liver tissue.
ELISA 분석 결과 표 9에서와 같이, G2, G4 및 G5의 간 조직 내 TG 수준은 G1에 비하여 유의하게 높았고 (p<0.001), G3 및 G6의 간 조직 내 TG 수준은 G2에 비하여 유의하게 낮았다 (p<0.001). As a result of ELISA analysis, as shown in Table 9, the TG levels in the liver tissues of G2, G4, and G5 were significantly higher than those of G1 (p<0.001), and the TG levels in the liver tissues of G3 and G6 were significantly lower than those of G2 ( p<0.001).
G2, G4, G5 및 G6의 간 조직 내 TCHO 수준은 G1에 비하여 유의하게 높았고 (p<0.001 또는 p<0.05), G5의 간 조직 내 TCHO 수준은 G2에 비하여 유의하게 높았으며 (p<0.01), G3 및 G6의 간 조직 내 TCHO 수준은 G2에 비하여 유의하게 낮았다 (p<0.001).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).
모든 지방간 유도군 (G2-G6)의 혈청 및 간 조직 내 자유 지방산 (free fatty acid, FFA) 수준은 G1에 비하여 유의하게 높았으며 (p<0.001), G3, G4 및 G6의 혈청 내 FFA 수준 및 G5 및 G6의 간 조직 내 FFA 수준은 G2에 비하여 유의하게 낮았다 (p<0.001, p<0.01 또는 p<0.05).Free fatty acid (FFA) levels in serum and liver tissues of all fatty liver induction groups (G2-G6) 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).
ELISA 분석 결과, 간 조직 내 TG 및 TCHO 수준이 육두구 추출물(NME) 400 mg/kg 투여군 (G6)에서 음성대조군 (G2) 대비 유의하게 감소하였으며, 육두구 추출물 200 mg/kg 투여군 (G5) 및 육두구 추출물 400 mg/kg 투여군 (G6)의 간 조직 내 FFA 수준, 육두구 추출물 100 mg/kg 투여군 (G4) 및 육두구 추출물 400 mg/kg 투여군 (G6)의 혈청 내 FFA 수준 또한 유의하게 감소하는 것으로 나타났다. As a result of ELISA analysis, 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.
상기 조직병리학적 검사 및 ELISA 분석 결과, 육두구 추출물 및 화합물 1의 투여가 지방간 유도에 따른 지방 침착 및 염증 등 지방간 병변 수준의 감소와 염증 및 섬유화와 관련된 인자의 발현을 유의하게 억제하는 것으로 나타났다.As a result of the histopathological examination and ELISA analysis, it was found that administration of nutmeg extract and Compound 1 significantly suppressed the reduction of fatty liver lesion levels such as fat deposition and inflammation, and the expression of factors related to inflammation and fibrosis following fatty liver induction.
본 시험조건 하에서 메티오닌 및 콜린 결핍(MCD) 식이로 유발된 비알코올성 지방간염 (NASH) C57BL/6 mouse 모델에 시험물질을 4 주 반복 투여하였을 때, 육두구 추출물 투여군은 혈액생화학적 검사 결과에서 음성대조군 대비 간 기능 관련 수치의 감소가 관찰되었고, 간 절대중량 및 상대중량 수준의 유의한 감소 양상이 관찰되었다. 또한, 조직병리학적 검사 결과에서 용량상관성 있는 변화 경향을 나타내며 거대공포성 지방증 수준의 경감 및 염증 및 섬유화 관련 인자의 발현 수준 감소가 관찰되었고, 육두구 추출물 고용량 투여군에서는 상기 결과와 더불어 간 조직 내 TG, cholesterol, FFA, 혈청 내 FFA 수준의 유의한 감소가 관찰되었다. 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.
따라서, 메티오닌 및 콜린 결핍(MCD) 식이로 유발된 비알코올성 지방간염 (NASH) C57BL/6 mouse 모델에서 본 발명의 4 주간 반복 투여는 비알코올성 지방간염의 개선에 효과적임을 확인할 수 있었다.Therefore, it was confirmed that repeated administration of the present invention for 4 weeks in the C57BL/6 mouse model of nonalcoholic steatohepatitis (NASH) induced by methionine and choline deficiency (MCD) diet was effective in improving nonalcoholic steatohepatitis.
<통계학적 분석><Statistical Analysis>
본 실험의 결과에 대하여 자료의 정규성을 가정하고, 모수적인 다중비교 (parametric multiple comparison procedures) 또는 비모수적인 다중비교 (non-parametric multiple comparison procedures)를 이용하여 분석하였다. Normality of the data was assumed for the results of this experiment, and analysis was performed using parametric multiple comparison procedures or non-parametric multiple comparison procedures.
모수적 일원분산분석 (One-way ANOVA) 결과가 유의하였을 경우, Dunnett's multiple comparison test를 이용하여 사후검정을 실시하고, 비모수적 Kruskal-Wallis'H-test 분석 결과가 유의하였을 경우, Mann-Whitney U test를 이용하여 사후검정을 실시하였다. 통계학적 분석은 Prism 7.04 (GraphPad Software Inc., San Diego, CA, USA)을 이용하여 실시하였으며, p값이 0.05 미만일 경우, 통계학적으로 유의한 것으로 판정하였다.If the parametric one-way ANOVA result was significant, a post hoc test was performed using Dunnett's multiple comparison test, and if the nonparametric Kruskal-Wallis'H-test analysis result was significant, Mann-Whitney U A post-hoc test was performed using the test. Statistical analysis was performed using Prism 7.04 (GraphPad Software Inc., San Diego, CA, USA), and when the p-value was less than 0.05, it was determined to be statistically significant.
이러한 결과들에 따라서 본원발명에 따른 육두구 추출물 및 화합물 1을 이용한 간질환 치료제로서의 개발 가능성은 높을 것으로 보인다. 하기에 상기 약학조성물의 제제예를 설명하나, 이는 본 발명을 한정하고자 함이 아닌 단지 구체적으로 설명하고자 함이다.According to these results, 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. Hereinafter, a formulation example of the pharmaceutical composition will be described, which is intended to describe only in detail, not to limit the present invention.
제제예Formulation example
<제제예 1> 육두구 추출물과 2-히드록시프로필 베타-사이클로덱스트린 포접물의 제조<Formulation Example 1> Preparation of nutmeg extract and 2-hydroxypropyl beta-cyclodextrin inclusion product
ㆍ육두구 추출물 10 gㆍ10 g nutmeg extract
ㆍ2-히드록시프로필 베타-사이클로덱스트린 20 gㆍ20 g of 2-hydroxypropyl beta-cyclodextrin
30 v/v % 에탄올 수용액 100 mL에 2-히드록시프로필 베타-사이클로덱스트린 20 g을 가하여 녹인 후, 육두구 추출물을 가하고 균질기 (Homogenizer, ULTRA TURRAX®IKA®T18basic)를 이용하여 1 분당 6,000 rpm으로 30 분간 교반한 다음, 동결건조기 (BK-10N50, 중국)로 동결 건조하였다.After dissolving 20 g of 2-hydroxypropyl beta-cyclodextrin in 100 mL of 30 v/v % ethanol aqueous solution, add nutmeg extract and use a homogenizer (Homogenizer, ULTRA TURRAX ® IKA ® T18basic) at 6,000 rpm per minute After stirring for 30 minutes, it was freeze-dried with a freeze dryer (BK-10N50, China).
<제제예 2> 화합물 1과 2-히드록시프로필 베타-사이클로덱스트린 포접물의 제조<Formulation Example 2> Preparation of compound 1 and 2-hydroxypropyl beta-cyclodextrin inclusion product
ㆍ본 발명의 화학식 1의 화합물(화합물 1) 100 mgㆍ100 mg of compound of formula 1 of the present invention (compound 1)
ㆍ2-히드록시프로필 베타-사이클로덱스트린 300 mgㆍ2-hydroxypropyl beta-cyclodextrin 300 mg
30 v/v % 에탄올 수용액 50 mL에 2-히드록시프로필 베타-사이클로덱스트린 300 mg을 가하여 녹인 후 화합물 1을 100 mg을 가하고 균질기 (Homogenizer, ULTRA TURRAX®IKA®T18basic)를 이용하여 1 분당 6,000 rpm으로 30 분간 교반한 다음, 동결건조기 (BK-10N50, 중국)로 동결 건조하였다.After adding and dissolving 300 mg of 2-hydroxypropyl beta-cyclodextrin in 50 mL of 30 v/v % aqueous ethanol solution, 100 mg of compound 1 was added and 6,000 per minute using a homogenizer (Homogenizer, ULTRA TURRAX ® IKA ® T18basic) After stirring at rpm for 30 minutes, freeze-drying was performed using a freeze dryer (BK-10N50, China).
<제제예 3> 캅셀의 제조<Formulation Example 3> Preparation of capsules
ㆍ육두구 추출물 100 mgㆍNutmeg extract 100 mg
ㆍ유당 80 mg ㆍLactose 80 mg
ㆍ카르복시메틸셀룰로오스칼슘 4 mg ㆍCarboxymethylcellulose calcium 4 mg
ㆍ경질무수규산 4 mgㆍLight anhydrous silicic acid 4 mg
ㆍ스테아린산폴리옥실40 2 mg ㆍPolyoxyl stearate 40 2 mg
ㆍ스테아린산마그네슘 1 mg ㆍMagnesium Stearate 1 mg
육두구 건조추출물을 유당, 카르복시메틸셀룰로오스칼슘, 경질무수규산, 스테아린산폴리옥실40 및 스테아린산마그네슘을 스피드믹서에서 30 분간 혼합하였다. 이 혼합물을 캅셀충진기에서 젤라틴 경질캅셀에 충진하였다.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.
<제제예 4> 정제의 제조<Formulation Example 4> Preparation of tablets
ㆍ육두구 추출물 100 mgㆍNutmeg extract 100 mg
ㆍ유당 160 mg ㆍLactose 160 mg
ㆍ옥수수전분 22 mgㆍCorn Starch 22 mg
ㆍ탄산수소나트륨 5 mgㆍSodium bicarbonate 5 mg
ㆍ스테아린산마그네슘 1 mg ㆍMagnesium Stearate 1 mg
ㆍ정제수 적량ㆍAppropriate amount of purified water
육두구 건조추출물을 유당, 탄산수소나트륨, 옥수수전분에 가하여 혼합하고 여기에 옥수수전분을 정제수에 가하여 호화시킨 결합액을 가하여 30분 동안 혼합믹서에서 연합하였다. 이 연합물을 제립기를 통과시켜 제립 후 건조기에 넣어 5시간 동안 건조 후 정립기에서 정립하였다. 정립물에 활택제인 스테아린산마그네슘을 가하고 혼합한 후 정당 288 mg의 중량으로 타정하였다.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.
<제제예 5> 액제의 제조<Formulation Example 5> Preparation of liquid formulation
ㆍ제제예 1의 포접물 300 mgㆍ300 mg of clathrate of Formulation Example 1
ㆍ자일리톨 30 gㆍXylitol 30 g
ㆍ체리향 20 mg ㆍCherry flavor 20 mg
ㆍ정제수 적량ㆍAppropriate amount of purified water
제제예 1의 육두구 추출물과 2-히드록시프로필 베타-사이클로데스트린의 포접물 300 mg을 정제수에 가하여 녹이고, 자일리톨과 체리향을 가하여 호모믹서로 20분간 교반한 후 전량을 100 mL로 하여 액제를 제조하였다.300 mg of the inclusion product of the nutmeg extract of Formulation Example 1 and 2-hydroxypropyl beta-cyclodestrin was added to purified water to dissolve, added xylitol and cherry flavor, stirred for 20 minutes with a homomixer, and the total amount was made 100 mL to prepare a solution prepared.
<제제예 6> 츄어블정의 제조<Formulation Example 6> Preparation of chewable tablets
ㆍ제제예 2의 포접물 60 mgㆍ60 mg of clathrate of Formulation Example 2
ㆍ자일리톨 620 mgㆍXylitol 620 mg
ㆍ말토덱스트린 260 mgㆍMaltodextrin 260 mg
ㆍ구연산 50 mg ㆍCitric acid 50 mg
ㆍ요구르트향 10 mg ㆍYogurt flavor 10 mg
ㆍ스테아린산마그네슘 1 mg ㆍMagnesium Stearate 1 mg
제제예 2의 화합물 1 포접물을 자일리톨, 말토덱스트린, 구연산 혼합물에 가하여 30분 동안 스프드믹서에서 혼합하였다. 이 혼합물을 제립기를 통과시켜 제립 후 활택제인 스테아린산마그네슘과 요구르트향을 가하고 혼합한 후 타정기로 정당 1 g의 중량으로 타정하였다. 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.
이상에서, 출원인은 본 발명의 바람직한 실시 예들을 설명하였지만, 이와 같은 실시예들은 본 발명의 기술적 사상을 구현하는 일 실시예일 뿐이며 본 발명의 기술적 사상을 구현하는 한 어떠한 변경예 또는 수정예도 본 발명의 범위에 속하는 것으로 해석되어야 한다.In the above, the applicant has described preferred embodiments of the present invention, but these embodiments are only one embodiment that implements the technical idea of the present invention, and any changes or modifications as long as the technical idea of the present invention is implemented. should be construed as within the scope.
Claims (20)
- 육두구 추출물, 또는 하기 화학식 1의 화합물 또는 이의 약학적으로 허용되는 염을 유효 성분으로 포함하는 비알코올성 지방간 질환의 예방 또는 치료용 약학적 조성물.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.[화학식1][Formula 1]
- 제1항에 있어서, 상기 비알코올성 지방간 질환은 비알코올성 지방간, 비알코올성 지방간염, 또는 비알코올성 지방간연관 간경변증인 것을 특징으로 하는 비알코올성 지방간 질환의 예방 또는 치료용 약학적 조성물.The pharmaceutical composition for preventing or treating nonalcoholic fatty liver disease according to claim 1, wherein the nonalcoholic fatty liver disease is nonalcoholic fatty liver disease, nonalcoholic steatohepatitis, or nonalcoholic fatty liver-associated cirrhosis.
- 제1항에 있어서, 상기 육두구 추출물은 화학식 1의 화합물 또는 이의 약학적으로 허용되는 염을 함유하는 것을 특징으로 하는 비알코올성 지방간 질환의 예방 또는 치료용 약학적 조성물.The pharmaceutical composition for preventing or treating nonalcoholic fatty liver disease according to claim 1, wherein the nutmeg extract contains the compound of Formula 1 or a pharmaceutically acceptable salt thereof.
- 제1항에 있어서, 상기 화학식 1의 화합물 또는 이의 약학적으로 허용되는 염은 육두구 추출물로부터 분리된 것을 특징으로 하는 비알코올성 지방간 질환의 예방 또는 치료용 약학적 조성물.[Claim 2] The pharmaceutical composition for preventing or treating nonalcoholic fatty liver disease according to claim 1, wherein the compound of Formula 1 or a pharmaceutically acceptable salt thereof is isolated from a nutmeg extract.
- 제1항에 있어서, 상기 육두구 추출물은 물, 메탄올, 에탄올, n-부탄올, 아세톤, 에틸아세테이트, 헥산 및 클로로포름으로 이루어진 군으로부터 선택되는 하나 이상의 용매로 추출되는 것을 특징으로 하는 비알코올성 지방간 질환의 예방 또는 치료용 약학적 조성물.The method of claim 1, wherein the nutmeg extract is extracted with one or more solvents selected from the group consisting of water, methanol, ethanol, n-butanol, acetone, ethyl acetate, hexane and chloroform. Prevention of nonalcoholic fatty liver disease or a therapeutic pharmaceutical composition.
- 제1항에 있어서, 상기 육두구 추출물은 초임계 유체 추출물 또는 환류 추출물인 것을 특징으로 하는 비알코올성 지방간 질환의 예방 또는 치료용 약학적 조성물.The pharmaceutical composition for preventing or treating nonalcoholic fatty liver disease according to claim 1, wherein the nutmeg extract is a supercritical fluid extract or a reflux extract.
- 제6항에 있어서, 상기 초임계 유체 추출물은 35 내지 100℃ 온도, 100 내지 500 bar의 압력 하에 초임계 상태로 만든 이산화탄소를 이용한 초임계 추출 방법으로 추출한 것을 특징으로 하는 비알코올성 지방간 질환의 예방 또는 치료용 약학적 조성물.The method of claim 6, wherein the supercritical fluid extract is 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. A therapeutic pharmaceutical composition.
- 제7항에 있어서, 상기 초임계 유체 추출 방법은 초임계 상태로 만든 이산화탄소에 추가적으로 공용매를 혼합한 혼합 유체를 이용하는 것을 특징으로 하는 비알코올성 지방간 질환의 예방 또는 치료용 약학적 조성물.The pharmaceutical composition for preventing or treating nonalcoholic fatty liver disease according to claim 7, wherein the supercritical fluid extraction method uses a mixed fluid obtained by mixing a cosolvent in addition to carbon dioxide made in a supercritical state.
- 제8항에 있어서, 상기 공용매는 물, 메탄올, 에탄올, n-부탄올, 아세톤, 에틸아세테이트, 헥산 및 클로로포름으로 이루어진 군으로부터 선택되는 하나 이상인 것을 특징으로 하는 비알코올성 지방간 질환의 예방 또는 치료용 약학적 조성물.The pharmaceutical for preventing or treating non-alcoholic fatty liver disease according to claim 8, wherein the cosolvent is at least one selected from the group consisting of water, methanol, ethanol, n-butanol, acetone, ethyl acetate, hexane and chloroform. composition.
- 제1항에 있어서, 상기 약학적 조성물은 육두구 추출물, 또는 화학식 1의 화합물 또는 이의 약학적으로 허용되는 염을 조성물 전체 중량에 대하여 0.0001 내지 90 중량% 포함하는 것을 특징으로 하는 비알코올성 지방간 질환의 예방 또는 치료용 약학적 조성물.[Claim 2] The prevention of nonalcoholic fatty liver disease according to claim 1, wherein the pharmaceutical composition comprises 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. or a therapeutic pharmaceutical composition.
- 제1항에 있어서, 상기 약학적 조성물은 약학적으로 허용 가능한 담체를 더 포함하는 것을 특징으로 하는 비알코올성 지방간 질환의 예방 또는 치료용 약학적 조성물.[Claim 2] The pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease according to claim 1, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.
- 제1항에 있어서, 상기 약학적 조성물은 액제, 산제, 과립제, 정제, 캅셀제, 환제, 트로키제, 또는 엑스제의 형태로 제형화된 것을 특징으로 하는 비알코올성 지방간 질환의 예방 또는 치료용 약학적 조성물.According to claim 1, wherein the pharmaceutical composition is a pharmaceutical composition for the prevention or treatment of non-alcoholic fatty liver disease, characterized in that it is formulated in the form of a liquid, powder, granule, tablet, capsule, pill, troche, or extract. composition.
- 제1항에 있어서, 상기 약학적 조성물은 베타-사이클로덱스트린으로 포접된 포접 화합물을 포함하는 제제로 제형화된 것을 특징으로 하는 비알코올성 지방간 질환의 예방 또는 치료용 약학적 조성물.The pharmaceutical composition for preventing or treating nonalcoholic fatty liver disease according to claim 1, wherein the pharmaceutical composition is formulated as a formulation comprising an inclusion compound inclusion with beta-cyclodextrin.
- 제13항에 있어서, 상기 베타-사이클로덱스트린은 2,6-디메틸-베타-사이클로덱스트린, 2-히드록시에틸-베타-사이클로덱스트린 및 2-히드록시프로필-베타-사이클로덱스트린 중에서 선택된 어느 하나 이상인 것을 특징으로 하는 비알코올성 지방간 질환의 예방 또는 치료용 약학적 조성물.14. The method of claim 13, wherein the beta-cyclodextrin is at least one selected from 2,6-dimethyl-beta-cyclodextrin, 2-hydroxyethyl-beta-cyclodextrin, and 2-hydroxypropyl-beta-cyclodextrin. A pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, characterized in that.
- 제13항에 있어서, 상기 포접 화합물은 베타-사이클로덱스트린 내부 공동에 육두구 추출물, 또는 화학식 1의 화합물 또는 이의 약학적으로 허용되는 염이 봉입된 것을 특징으로 하는 비알코올성 지방간 질환의 예방 또는 치료용 약학적 조성물.[Claim 14] The pharmaceutical for preventing or treating non-alcoholic fatty liver disease according to claim 13, wherein the inclusion compound is beta-cyclodextrin in which a nutmeg extract or a compound of Formula 1 or a pharmaceutically acceptable salt thereof is encapsulated in the internal cavity of the beta-cyclodextrin. enemy composition.
- 육두구 추출물, 또는 하기 화학식 1의 화합물 또는 이의 약학적으로 허용되는 염을 유효 성분으로 포함하는 비알코올성 지방간 질환의 예방 또는 개선용 식품 조성물.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.[화학식1][Formula 1]
- 육두구를 추출하는 단계를 포함하는 비알코올성 지방간 질환의 예방 및 치료용 약학적 조성물의 제조방법.A method for preparing a pharmaceutical composition for the prevention and treatment of non-alcoholic fatty liver disease, comprising the step of extracting nutmeg.
- S1) 육두구를 추출하는 단계, 및S1) extracting nutmeg, andS2) 상기에서 수득한 육두구 추출물로부터 하기 화학식 1의 화합물 또는 이의 약학적으로 허용되는 염을 분리하는 단계를 포함하는 비알코올성 지방간 질환의 예방 또는 치료용 약학적 조성물의 제조방법.S2) A method of preparing a pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease, comprising the step of isolating the compound of Formula 1 or a pharmaceutically acceptable salt thereof from the nutmeg extract obtained above.[화학식1][Formula 1]
- 육두구를 추출하는 단계를 포함하는 비알코올성 지방간 질환의 예방 및 개선용 식품 조성물의 제조방법.A method for preparing a food composition for preventing and improving non-alcoholic fatty liver disease, comprising the step of extracting nutmeg.
- S1) 육두구를 추출하는 단계, 및S1) extracting nutmeg, andS2) 상기에서 수득한 육두구 추출물로부터 하기 화학식 1의 화합물 또는 이의 약학적으로 허용되는 염을 분리하는 단계를 포함하는 비알코올성 지방간 질환의 예방 또는 개선용 식품 조성물의 제조방법.S2) A method of preparing a food composition for preventing or improving non-alcoholic fatty liver disease, comprising the step of isolating the compound of Formula 1 or a pharmaceutically acceptable salt thereof from the nutmeg extract obtained above.[화학식1][Formula 1]
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