WO2023101420A1 - Composition pour la prévention ou le traitement de l'obésité, de la stéatose hépatique ou de la stéatohépatite comprenant un extrait de momordica charantia ou de l'acide hydroférulique dérivé de momordica charantia en tant que principe actif - Google Patents

Composition pour la prévention ou le traitement de l'obésité, de la stéatose hépatique ou de la stéatohépatite comprenant un extrait de momordica charantia ou de l'acide hydroférulique dérivé de momordica charantia en tant que principe actif Download PDF

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WO2023101420A1
WO2023101420A1 PCT/KR2022/019212 KR2022019212W WO2023101420A1 WO 2023101420 A1 WO2023101420 A1 WO 2023101420A1 KR 2022019212 W KR2022019212 W KR 2022019212W WO 2023101420 A1 WO2023101420 A1 WO 2023101420A1
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Prior art keywords
fatty liver
composition
obesity
preventing
liver
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PCT/KR2022/019212
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English (en)
Korean (ko)
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김은경
범미기
최영진
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동아대학교 산학협력단
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Publication of WO2023101420A1 publication Critical patent/WO2023101420A1/fr

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K20/00—Accessory food factors for animal feeding-stuffs
    • A23K20/10—Organic substances
    • A23K20/111—Aromatic compounds
    • 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
    • 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
    • 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
    • A23L33/30—Dietetic or nutritional methods, e.g. for losing weight
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • 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
    • A23V2200/324—Foods, ingredients or supplements having a functional effect on health having an effect on the immune system
    • 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
    • A23V2200/332—Promoters of weight control and weight loss

Definitions

  • the present invention provides a composition for the prevention or treatment of obesity, fatty liver or steatohepatitis, containing bitter gourd extract or hydroferulic acid derived from bitter gourd as an active ingredient, which can be usefully used throughout the industry related to obesity, fatty liver or steatohepatitis. In particular, it can be applied to food compositions for improvement and the pharmaceutical industry.
  • fatty liver refers to a case where the degree of fat deposited in the liver is 5% or more of the weight of the liver, and neutral fat (triglyceride, TG) is accumulated in hepatocytes.
  • Fatty liver occurs when fat ingested through food is not properly processed, and a research team led by Professor Su Lim at Seoul National University Bundang Hospital said, “If inflammation from fatty liver worsens, it goes beyond steatohepatitis to liver-specific complications such as liver cirrhosis and liver cancer, as well as cardiovascular disease and It is important to break this link because it can lead to chronic diseases such as diabetes and lead to death.”
  • bitter gourd Momordica charantia
  • the stem wraps around objects with tendrils, and the leaves are largely divided into 5-7 pieces. The tendrils and leaves meet.
  • Flowers blooming in July-August run one by one in the axil and are yellow.
  • the fruit is unusual, with a long round shape about 7 to 9 cm long, with small lump-shaped projections sprouting full of fruits, and when ripe, they are cracked to reveal seeds wrapped in red flesh.
  • Bitter gourd contains a lot of insulin, so it has an excellent effect on type 2 diabetes.
  • effects such as anticancer effect, fatigue relief, bronchial strengthening, seaweed action, and diet.
  • nothing is known about the preventive or therapeutic effect of hydroferulic acid isolated from the water extract of bitter melon, obesity, fatty liver or steatohepatitis.
  • An object of the present invention is to provide a food composition for the prevention and improvement of obesity or fatty liver containing bitter gourd leaf extract as an active ingredient.
  • Another object of the present invention is to provide a feed composition for preventing or improving obesity or fatty liver containing bitter gourd leaf extract as an active ingredient.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating obesity or fatty liver containing bitter melon leaf extract as an active ingredient.
  • Another object of the present invention is to provide a method for treating obesity or fatty liver comprising administering the pharmaceutical composition to a subject.
  • an object of the present invention is to provide a food composition for preventing or improving obesity, fatty liver or steatohepatitis, containing hydroferulic acid as an active ingredient.
  • Another object of the present invention is to provide a feed composition for preventing or improving obesity, fatty liver or steatohepatitis, containing hydroferulic acid as an active ingredient.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating obesity, fatty liver or steatohepatitis, containing hydroferulic acid as an active ingredient.
  • Another object of the present invention is to provide a method for treating obesity, fatty liver or steatohepatitis comprising administering the pharmaceutical composition to a subject.
  • the extract may be extracted with at least one solvent selected from water, C1 to C4 alcohol, ethyl acetate, chloroform and hexane, but is not limited thereto.
  • the fatty liver may be alcoholic fatty liver or non-alcoholic fatty liver, but is not limited thereto.
  • the composition may have an antioxidant effect, but is not limited thereto.
  • the composition may inhibit lipid accumulation, but is not limited thereto.
  • the composition may increase alanine aminotransferase (ALT), alkaline phosphatase (ALP), and aspartate aminotransferase (AST), but is not limited thereto.
  • ALT alanine aminotransferase
  • ALP alkaline phosphatase
  • AST aspartate aminotransferase
  • the composition may reduce serum cholesterol (TC), triglyceride (TG) and low-density lipoprotein cholesterol (LDL) in the blood and increase high-density lipoprotein cholesterol (HDL). It is not limited.
  • the composition comprises adipose triglyceride lipase (ATGL), triacylglycerol hydrolase (TGH), hormone sensitive lipase (HSL), carnitine palmitoyl transferase 1 (CPT-1) and pig isolate It may be to increase the activity of protein 3 (UCP 3), but is not limited thereto.
  • ATGL adipose triglyceride lipase
  • TGH triacylglycerol hydrolase
  • HSL hormone sensitive lipase
  • CPT-1 carnitine palmitoyl transferase 1
  • pig isolate It may be to increase the activity of protein 3 (UCP 3), but is not limited thereto.
  • the composition comprises CCAAT/enhancer binding protein (C/EBP ⁇ ), regulatory factor activated receptor ⁇ (PPAR- ⁇ ), cholesterol 7 ⁇ -hydroxylase (CYP7A1), acyl-CoA synthase (ACS), acetyl coenzyme A carboxylase (ACC) and fatty acid synthase (FAS) activity may be reduced, but is not limited thereto.
  • C/EBP ⁇ CCAAT/enhancer binding protein
  • PPAR- ⁇ regulatory factor activated receptor ⁇
  • CYP7A1 cholesterol 7 ⁇ -hydroxylase
  • ACS acyl-CoA synthase
  • ACC acetyl coenzyme A carboxylase
  • FAS fatty acid synthase
  • the present invention is to provide a feed composition for preventing or improving obesity or fatty liver containing bitter melon leaf extract as an active ingredient.
  • the present invention is to provide a pharmaceutical composition for the prevention or treatment of obesity or fatty liver containing bitter gourd leaf extract as an active ingredient.
  • the present invention is to provide a method for preventing or treating obesity or fatty liver comprising administering the pharmaceutical composition to a subject.
  • fatty liver or steatohepatitis containing hydroferulic acid isolated from bitter gourd leaf extract as an active ingredient.
  • the fatty liver may be obesity, fatty liver or steatohepatitis, but is not limited thereto.
  • the composition may inhibit lipid accumulation, but is not limited thereto.
  • the composition reduces the serum LDL / VLDL ratio, serum triglycerides (TG), serum free fatty acids (FFAs) and serum cholesterol (TC), and increases high-density lipoprotein cholesterol (HDL). It may be, but is not limited thereto.
  • the composition may be to inhibit liver fibrosis, but is not limited thereto.
  • the composition may reduce the expression of ⁇ -SMA, COL1A1 and COL1A2, but is not limited thereto.
  • the composition may inhibit inflammation in the liver, but is not limited thereto.
  • the composition may reduce the expression level of F4/80, CD68, Gas6, MerTK, ERK, p-ERK and TGF- ⁇ , but is not limited thereto.
  • the composition may reduce the expression level of IL-1 ⁇ , TNA- ⁇ , MCP-1, but is not limited thereto.
  • the present invention provides a feed composition for preventing or improving obesity, fatty liver or steatohepatitis, comprising hydroferulic acid as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating obesity, fatty liver or steatohepatitis, comprising hydroferulic acid as an active ingredient.
  • the present invention provides a method for preventing or treating obesity, fatty liver or steatohepatitis, comprising administering the pharmaceutical composition to a subject.
  • the extract of bitter gourd or hydroferulic acid derived from bitter gourd of the present invention is excellent in preventing or treating obesity, fatty liver or steatohepatitis, so it can be usefully used throughout the health functional food industry or pharmaceutical industry.
  • Figure 1 is (a) pictures of the four parts of bitter melon (MCF), roots (MCR), shoots (MCS) and leaves (MCL) used in the experiment, (b) a schematic diagram of 3T3-L1 cell differentiation experiments and (c) It is a diagram of the in vivo study schedule.
  • MCF bitter melon
  • MCR roots
  • MCS shoots
  • MCL leaves
  • Figure 2 is for the antioxidant and anti-obesity effects of bitter gourd composition (a) DPPH radical scavenging activity, (b) reducing power, (c) a picture of adipocytes and (d) a diagram showing the percentage of fat accumulation compared to the untreated group.
  • Figure 3 is a graph showing a graph analyzed by HPLC of (a) water extract, (b) ethanol extract and (c) hexane extract of bitter gourd leaves.
  • Figure 4 is HFD-induced mouse bitter gourd extract (MCLW) treatment group (a) liver and fat cell weight, (b) biochemical parameters, (c) liver metabolism-related factors, (d) cholesterol amount and (e) It is a diagram showing the amount of serum leptin measured.
  • MCLW mouse bitter gourd extract
  • Figure 5 is a diagram showing (a) radiographs, (b ⁇ c) micrographs and graphs of changes in fat mass and fat cell size according to the diet of HFD-induced mice.
  • FIG. 6 is a diagram showing the histological analysis of the livers of HFD-induced mice fed different diets.
  • Figure 7 is a real-time PCR (Real time PCR) measured the amount of mRNA expression in mouse liver (a) ATGL, (b) TGH, (c) HSL, (d) UCP3, (e) CPT-1, (f) ) C/EBP- ⁇ , (g) PPAR- ⁇ , (h) FAS, (i) ACS, and (j) CYP7A1 are respectively measured and shown.
  • Figure 8 is a mouse sample Western blot (Western blot) to measure the amount of protein (a) Western blot picture, (b) adiponectin (Adiponectin), (c) p-AMPK / AMPK, (d) C / EBP ⁇ , (e) PPAR- ⁇ , (f) SREBP-1c and (g) FAS are graphs.
  • FIG. 9 is a schematic diagram showing the fat metabolism pathway of HFD mice.
  • 10 is a schematic diagram showing a simple animal experiment design.
  • FIG. 11 is a diagram showing the effect of reducing fat in liver tissue according to the administration of hydroferulic acid according to the present invention.
  • FIG. 12 is a diagram showing the results of blood and adipose tissue analysis according to the administration of hydroferulic acid of the present invention.
  • FIG. 13 is a diagram showing the effect of inhibiting fat deposition and liver fibrosis according to administration of hydroferulic acid according to the present invention.
  • FIG. 14 is a diagram showing the results of measuring the expression level of liver fibrosis-related factors in liver tissue according to administration of hydroferulic acid according to the present invention.
  • FIG. 15 is a diagram showing the results of measurement of the expression level of inflammation-related factors (F4/80, CD68, IL-1 ⁇ , TNA- ⁇ , MCP-1) in liver tissue according to administration of hydroferulic acid according to the present invention.
  • FIG. 16 is a diagram showing the results of measuring the expression level of inflammation-related factors (Gas6, MerTK, ERK, p-ERK, TGF- ⁇ ) in liver tissue according to administration of hydroferulic acid according to the present invention.
  • the present invention is to provide a health functional food composition for preventing or improving obesity or fatty liver containing bitter gourd leaf extract or hydroferulic acid as an active ingredient.
  • the present invention is to provide a feed composition for preventing or improving obesity or fatty liver containing bitter melon leaf extract or hydroferulic acid as an active ingredient.
  • the present invention is to provide a pharmaceutical composition for the prevention or treatment of obesity or fatty liver containing bitter gourd leaf extract or hydroferulic acid as an active ingredient.
  • the present invention provides a method for treating obesity and fatty liver comprising administering the pharmaceutical composition to a subject.
  • the present invention provides a food composition for preventing or improving obesity, fatty liver or steatohepatitis, comprising bitter gourd leaf extract or hydroferulic acid as an active ingredient.
  • hydroferulic acid may be derived from bitter gourd, preferably isolated from water extract of bitter gourd, but is not limited thereto.
  • momordica charantia used in the present invention is a vegetable or medicinal plant belonging to the Cucurbitaceae family and mainly grows in tropical or subtropical regions, and mainly uses fruits for food. is called bitter gourd or bitter melon. Young fruits are green, and when more mature, they turn orange, and as time passes, the flesh bursts to expose the red arils, and the seeds are generally brown. In Japan, China, and Southeast Asia, immature fruits are mainly cultivated for food, but young leaves are also used for food.
  • the obesity may include obesity-related diseases such as diabetes, fatty liver, hyperlipidemia, arteriosclerosis, and their complications in addition to obesity.
  • the fatty liver may be alcoholic fatty liver or non-alcoholic fatty liver, preferably non-alcoholic fatty liver.
  • non-alcoholic fatty liver used in the present invention refers to a disease caused by accumulation of excess energy in the form of triglycerides in the liver due to bad lifestyles such as lack of exercise and high-calorie meals. If left untreated, it can progress from hepatitis, liver cirrhosis to liver cancer, but until now there is no suitable treatment, so treatment through exercise and improvement in dietary life is mainly performed.
  • the non-alcoholic fatty liver includes various types of liver diseases ranging from simple non-alcoholic fatty liver with only fat and almost no liver cell damage, chronic non-alcoholic steatohepatitis with severe and persistent liver cell damage, and cirrhosis.
  • composition may inhibit lipid accumulation, but is not limited thereto.
  • the composition may reduce serum LDL / VLDL ratio, serum triglycerides (TG), serum free fatty acids (FFAs) and serum cholesterol (TC), and increase high-density lipoprotein cholesterol (HDL), but is limited thereto. it is not going to be
  • composition may be to inhibit liver fibrosis, but is not limited thereto.
  • composition may reduce the expression of ⁇ -SMA, COL1A1 and COL1A2, but is not limited thereto.
  • composition may inhibit inflammation in the liver, but is not limited thereto.
  • composition may reduce the expression level of F4/80, CD68, Gas6, MerTK, ERK, p-ERK and TGF- ⁇ , but is not limited thereto.
  • composition may reduce the expression level of IL-1 ⁇ , TNA- ⁇ , MCP-1, but is not limited thereto.
  • the food composition of the present invention may contain various flavoring agents or natural carbohydrates as additional ingredients like conventional food compositions.
  • natural carbohydrates examples include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides such as conventional sugars such as dextrins, cyclodextrins, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol.
  • natural flavoring agents thaumatin
  • stevia extracts eg, rebaudioside A, glycyrrhizin, etc.
  • synthetic flavoring agents sacharin, aspartame, etc.
  • the food composition of the present invention can be formulated in the same way as the pharmaceutical composition and used as a functional food or added to various foods.
  • Foods to which the composition of the present invention can be added include, for example, beverages, meat, chocolate, foods, confectionery, pizza, ramen, other noodles, gum, candy, ice cream, alcoholic beverages, vitamin complexes and health supplements, etc. there is
  • the food composition includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and salts thereof, in addition to extracts that are active ingredients. , alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like.
  • the food composition of the present invention may contain natural fruit juice and fruit flesh for preparing fruit juice beverages and vegetable beverages.
  • the functional food composition of the present invention can be prepared and processed in the form of tablets, capsules, powders, granules, liquids, pills and the like.
  • 'health functional food composition' refers to a food manufactured and processed using raw materials or ingredients having useful functionality for the human body according to Health Functional Food Act No. 6727, and the structure and function of the human body It refers to intake for the purpose of obtaining useful effects for health purposes such as regulating nutrients or physiological functions.
  • the food composition for improvement of the present invention may include conventional food additives, and the suitability as a food additive is determined in accordance with the General Rules of the Code of Food Additives and General Test Methods approved by the Ministry of Food and Drug Safety, unless otherwise specified. It is judged according to the standards and standards for Examples of the items listed in the 'Food Additive Code' include, for example, chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid; natural additives such as persimmon pigment, licorice extract, crystalline cellulose, kaoliang pigment, and guar gum; and mixed preparations such as sodium L-glutamate preparations, noodle-added alkali preparations, preservative preparations, and tar color preparations.
  • chemical compounds such as ketones, glycine, calcium citrate, nicotinic acid, and cinnamic acid
  • natural additives such as persimmon pigment, licorice extract,
  • a food composition in the form of a tablet is obtained by granulating a mixture obtained by mixing the active ingredient of the present invention with excipients, binders, disintegrants, and other additives in a conventional manner, and then adding a lubricant or the like and compression molding the mixture. can be directly compression molded.
  • the food composition in the form of a tablet may contain a flavoring agent and the like, if necessary.
  • hard capsules can be prepared by filling a mixture in which the active ingredient of the present invention is mixed with additives such as excipients in a normal hard capsule, and soft capsules contain the active ingredient of the present invention and additives such as excipients.
  • the soft capsule may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary.
  • the food composition in the form of a pill may be prepared by molding a mixture of the active ingredient of the present invention mixed with an excipient, a binder, a disintegrant, etc. by a conventionally known method, and may be coated with sucrose or other coating agent if necessary, or The surface can also be coated with a material such as starch or talc.
  • a food composition in granular form may be prepared by a conventionally known method from a mixture of an excipient, a binder, a disintegrant, etc. of the active ingredient of the present invention in granular form, and may contain a flavoring agent, a flavoring agent, etc., if necessary. there is.
  • the present invention provides a feed composition for preventing or improving obesity, fatty liver or steatohepatitis, comprising bitter gourd leaf extract or hydroferulic acid as an active ingredient.
  • the feed composition of the present invention replaces conventional antibiotics and suppresses the growth of harmful food pathogens, improving the health of animals, improving the weight gain and quality of livestock, and increasing milk production and immunity.
  • the feed composition of the present invention may be prepared in the form of fermented feed, formulated feed, pellet form and silage.
  • the fermented feed can be prepared by fermenting organic matter by adding various microorganisms or enzymes other than the peptide of the present invention, and the formulated feed can be prepared by mixing several types of general feed with the peptide of the present invention.
  • Feed in the form of pellets can be prepared by applying heat and pressure to the formulated feed, etc. in a pellet machine, and silage can be prepared by fermenting green feed with the microorganism according to the present invention.
  • Wet fermented feed collects and transports organic matter such as food waste, mixes excipients for sterilization and moisture control in a certain ratio, and ferments for more than 24 hours at a temperature suitable for fermentation, so that the moisture content is about 70%. It can be made by adjusting.
  • the fermented dry feed can be prepared by further subjecting the wet fermented feed to a drying process to adjust the moisture content to about 30% to 40%.
  • the feed composition of the present invention may further include components added to conventional feed.
  • ingredients added to such feeds may include grain powder, meat powder, and pulses.
  • the grain powder may use at least one selected from rice flour, wheat flour, barley flour, and corn flour.
  • meat powder at least one selected from chicken, beef, pork, and ostrich meat may be used as a powdered meat powder.
  • at least one selected from soybeans, kidney beans, peas, and black beans may be used as the pulse.
  • the feed composition of the present invention in addition to grain powder, meat powder, and pulses, which are ingredients added to conventional feeds mentioned above, at least one selected from nutrients and minerals may be added to increase the nutritional properties of the feed, and the feed quality In order to prevent the degradation of antifungal agents, antioxidants, anticoagulants, emulsifiers, and at least one selected from a binder may be included.
  • the present invention provides a pharmaceutical composition for preventing or treating obesity, fatty liver or steatohepatitis, comprising bitter gourd leaf extract or hydroferulic acid as an active ingredient.
  • treatment means that the symptoms of obesity, fatty liver or steatohepatitis-related diseases are improved or beneficially changed by administration of a pharmaceutical composition containing hydroferulic acid as an active ingredient according to the present invention. means all actions.
  • the term "administration" means introducing a pharmaceutical composition containing a predetermined substance, that is, hydroferulic acid according to the present invention, as an active ingredient to a subject by any suitable method.
  • the pharmaceutical composition of the present invention may be administered by intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, intrapulmonary administration, intrarectal administration, etc., but intravenous administration It is preferably administered internally, subcutaneously or orally.
  • intravenous administration is preferred, and when used for prevention or treatment of fatty liver or steatohepatitis, subcutaneous administration is preferred.
  • the pharmaceutical composition of the present invention may further include an adjuvant in addition to the active ingredient.
  • an adjuvant in addition to the active ingredient.
  • any one may be used without limitation, but, for example, Freund's complete adjuvant or incomplete adjuvant may be further included to increase immunity.
  • the pharmaceutical composition according to the present invention may be prepared in the form of incorporating the active ingredient into a pharmaceutically acceptable carrier.
  • the pharmaceutically acceptable carrier includes carriers, excipients and diluents commonly used in the pharmaceutical field.
  • Pharmaceutically acceptable carriers usable in the pharmaceutical composition of the present invention include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • the pharmaceutical composition of the present invention may be formulated and used in the form of oral formulations such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations, suppositories or sterile injection solutions according to conventional methods, respectively. .
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations contain at least one or more excipients such as starch, calcium carbonate, sucrose, lactose, and gelatin in addition to active ingredients. It can be prepared by mixing etc. In addition to simple excipients, lubricants such as magnesium stearate and talc may also be used.
  • Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, syrups, etc.
  • compositions for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations and suppositories.
  • Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents.
  • Witepsol, Tween 61, cacao paper, laurin paper, glycerogelatin, and the like may be used as a base for suppositories.
  • the dosage of the pharmaceutical composition according to the present invention is selected in consideration of the age, weight, sex, and physical condition of the subject. It is obvious that the concentration of the active ingredient included in the pharmaceutical composition can be variously selected depending on the subject, and is preferably included in the pharmaceutical composition at a concentration of 0.01 to 5,000 ⁇ g/ml. If the concentration is less than 0.01 ⁇ g/ml, pharmacological activity may not appear, and if the concentration exceeds 5,000 ⁇ g/ml, toxicity to the human body may be exhibited.
  • the pharmaceutical composition may be formulated in various oral or parenteral dosage forms.
  • dosage forms for oral administration include tablets, pills, hard and soft capsules, solutions, suspensions, emulsifiers, syrups, and granules.
  • these formulations contain diluents (e.g. lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/or glycine), lubricants (e.g. silica, talc, stearic acid and its magnesium or calcium salts and/or or polyethylene glycol) may be further included.
  • the tablet may contain a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and in some cases starch, agar, alginic acid or a disintegrant or effervescent mixture, such as its sodium salt, and/or absorbents, colorants, flavors, and sweeteners.
  • a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and/or polyvinylpyrrolidine, and in some cases starch, agar, alginic acid or a disintegrant or effervescent mixture, such as its sodium salt, and/or absorbents, colorants, flavors, and sweeteners.
  • the formulation may be prepared by conventional mixing, granulating or coating methods.
  • a typical formulation for parenteral administration is an injection formulation, and water, Ringer's solution, isotonic physiological saline or suspension may be used as a solvent for the injection formulation.
  • Sterile fixed oils of the above injectable preparations may be used as a solvent or suspension medium, and any bland fixed oil may be used for this purpose, including mono- and di-glycerides.
  • the formulation for injection may use a fatty acid such as oleic acid.
  • Fresh bitter gourd fruits, roots, stems and leaves (Shandong, China, Fig. 1) were air-dried at room temperature (RT) in the shade for 2 weeks, and then the samples were pulverized into powder. Powder samples were stored at room temperature prior to extraction. Thereafter, extracts were prepared from the powder using various solvents (distilled water, 70% ethanol and 95% hexane). For extraction efficiency, 100 g of powders of fruits, roots, stems and leaves were separately immersed in a 2 L standard flask containing 1 L solvent (3 different flasks using different solvents for each plant part).
  • the flask containing the soaked plant powder was sealed with a cotton stopper and aluminum foil and placed on a shaker at room temperature for 1 hour (distilled water), 2 hours (70% ethanol) and 6 hours (95% hexane). This process was repeated 3 times for maximum extraction. After repeated extraction three times, the extract was concentrated at 60° C. using a rotary evaporator, filtered, and lyophilized for 5 days.
  • mice 4-week-old C57BL/6 male mice were purchased from the Nara Bio Animal Center (Nara Biotech, Seoul, Korea) and acclimatized for 1 week in the experimental facility, and then housed in groups of 4 mice each inside a transparent plastic cage equipped with an Aspen chip. Standard mouse chow diet and untreated tap water were supplied under clean pathogen-free conditions. The animal environment was appropriately controlled with a 12-hour dark period at 20 to 21 °C and a relative humidity of 40 to 45%. All experiments were approved by the Animal Care Committee of Konkuk University Animal Hospital, and the experiments related to the present invention (KU17089) were designed to minimize the number and pain of animals used.
  • DPPH 2,2-diphenyl-1-picrylhydrazyl
  • 3T3-L1 cells obtained from the Korean Cell Line Bank (Seoul, Korea) were cultured at 37 °C in 5% humidified DMEM medium containing 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S). were incubated under atmospheric conditions of CO 2 .
  • FBS heat-inactivated fetal bovine serum
  • P/S penicillin/streptomycin
  • the culture medium was replaced with DMEM containing 1% P/S and 10% FBS for 2 days, and then the medium was replaced every 2 days until Day 8, when adipocytes acquire intracellular lipid droplets.
  • Bitter melon leaf water extract (MCLW) (1 mg/mL) and bitter gourd leaf ethanol extract (MCLE) (1 mg/mL) were administered 4 times every 2 days from day 0 to day 6.
  • Control cells were composed of the same medium composition without bitter gourd extract.
  • the effect of the extract on fat accumulation was related to the size of differentiated lipid droplets in 3T3-L1 preadipocytes.
  • DXA dual energy X-ray absorptiometry
  • a full-body scanner InAlyzer dual X-ray absorptiometry, Medikors, Gyeonggi-do, Korea. Livers were cut into two pieces for paraffin and frozen sectioning. Liver and epididymal adipose tissue was dissected for histological examination, buffered in 10% neutral formalin, and embedded in paraffin. 4 ⁇ m paraffin sections were stained with Hematoxylin and Eosin (H&E). Samples were imaged under a microscope at 200 ⁇ . Using frozen sections (10 ⁇ m with OCT compound), lipid accumulation was confirmed by Oil Red O staining. Slices were imaged under a microscope at 200 ⁇ and the size of adipocytes was recorded.
  • H&E Hematoxylin and Eosin
  • Oil-Red O is a lysochromdia crude dye used to stain neutral triglycerides in frozen parts and some lipoproteins in lipids and paraffin parts.
  • Adipocyte differentiation was induced for 8 days by culturing 3T3-L1 cells in a 12-well plate as described above.
  • Oil Red O cell lipid index was measured on the 8th day after rinsing the cells with physiological saline, fixing with 10% formaldehyde solution, and reacting at room temperature with 0.7 g of isopropyl alcohol per 200 mL of Oil Red O solution for 60 minutes at RT. . After staining the lipid droplets, they were rinsed with distilled water and dried to remove the staining solution.
  • Lipid droplets in 3T3-L1 adipocytes were observed under a microscope. Finally, the stain remaining on the cells was eluted with isopropanol, and the absorbance was measured at 520 nm using a microplate reader.
  • MCLW bitter melon leaf water extract
  • MCLE bitter melon leaf ethanol extract
  • bitter melon leaf water extract MCLW
  • bitter gourd leaf ethanol extract MCLE
  • a Vion UPLCTM system (Vion, Waters, Milford, MA, USA) was used and LC conditions were optimized within 9 minutes on an Acquity UPLC BEH C18 column (2.1 ⁇ 100 mm, 1.7 ⁇ m; Waters, Milford, MA, USA). The temperature was set at 55°C. The flow rate was set at 0.35 mL/min. The mobile phase was used by adding 0.1% acetonitrile (ACN) to water (FA) containing 0.1% formic acid. The spectrometer was used with electrospray ionization (ESI) in negative mode.
  • ACN acetonitrile
  • FA water
  • spectrometer was used with electrospray ionization (ESI) in negative mode.
  • MS operating conditions are: capillary voltage 2.5 kV, sample cone 20 V, ion source temperature 200 °C, desolvation temperature 400 °C, cone gas 30 L/h, desolvation gas 900 L/h, scan time 0.2 seconds , a scan range of 50 to 1500 m/z, and a collision energy lamp of 10 to 30 eV (50 to 1000 m/z) were used.
  • MarkerLynx software (Waters, Milford, MA, USA) collected MS data sets analyzed by UPLC-Q-TOF MS (Waters, Milford, MA, USA), and was used for normalization and alignment. All spectra were normalized with internal standards (Terfenadine [M + H] and Zidovudine [M - H]).
  • HMDB human metabolome database
  • METLIN database Metalin.scripps.edu
  • Chemspider www.chemspider.com
  • the chemical composition of various bitter gourd extracts was analyzed using UPLC-Q-TOF MS.
  • 3 shows an ion flow chromatogram using the extract.
  • the compounds of each extract mainly flavonoids, triterpenes and phenolic acids, were measured and the results are shown in Table 1.
  • the result of analyzing bitter melon leaf water extract (MCLW) is shown in FIG. 3a. It was found that the content of total flavonoids and total phenols in the extract increased as the polarity of the solvent increased.
  • bitter melon leaf water extract contained hydroferulic acid, Kaempferol 3-glucuronide and ( It was found to contain ⁇ )-30,40-methylenedioxy-5,7-dimethylepicatechin (Fig. 3, Table 1).
  • triterpenoid saponins are mainly concentrated in bitter melon leaf ethanol extract (MCLE).
  • Table 3 shows the physiological activity results of bitter gourd leaf hexane extract (MCLH).
  • mice were randomly divided into two groups to confirm the effect of the bitter melon leaf extract.
  • HFD 60% fat, 20% carbohydrate, 20% protein, total 5.24 kcal/g
  • the three groups treated for 4 weeks were mice fed a high-fat diet treated with 0.9% saline, mice fed an oral dose of 200 mg/kg/d of a mixture of high-fat diet and bitter gourd leaf extract (MCLW), and mice fed a high-fat diet+ Mice with an oral dose of 60 mg/kg/d in combination with Orlistat are grouped.
  • Orlistat was used as a positive control. Mice were fasted for one day after 12 weeks and then used in the next experiment (Fig. 1c). Blood samples were collected for further analysis.
  • Blood samples were collected using cardiac puncture, serum was separated by centrifugation (3000 rpm for 20 minutes) and stored at -80°C until further analysis.
  • Blood urea nitrogen (BUN), alanine aminotransferase (ALT), alkaline phosphatase (ALP), and aspartate aminotransferase (AST) were measured using an automated analyzer (Abaxis VETSVAN VS2 Chemistry Analyzer, Union City, CA, USA). ) and serum albumin (ALB).
  • a rapid blood lipid analyzer (OSANG Healthcare Lipid Pro, Anyang, Korea) was used to measure serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL), high-density lipoprotein cholesterol (HDL), and triglyceride (TG).
  • Serum leptin was quantified using an ELISA leptin kit (Merck, Darmstadt, Germany).
  • the weight of white adipose tissue was significantly reduced in the group treated with bitter gourd leaf extract (MCLW) or orlistat (FIG. 4b).
  • MCLW bitter gourd leaf extract
  • orlistat FIG. 4b
  • blood biochemical analysis was performed to confirm the effect of bitter gourd leaf water extract (MCLW) on biochemical parameters.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • ALP alkaline phosphatase
  • BUN blood urea nitrogen
  • TC serum total cholesterol
  • TG triglyceride
  • LDL low-density lipoprotein cholesterol
  • High-density lipoprotein cholesterol (HDL) levels in the bitter gourd leaf water extract (MCLW) or orlistat group were increased compared to the high-fat diet group.
  • concentration of leptin in the bitter gourd leaf extract (MCLW) and orlistat groups showed a very significant decrease compared to the high-fat diet group (FIG. 4e). Lipids and leptin were found to be closely related to the expression of plasma adipokines to regulate energy intake. According to the above results, it is suggested that serum leptin plays an important role in reducing adipose tissue.
  • bitter gourd leaf water extract MCLW
  • DXA dual-energy X-ray absorptiometry
  • MCLW bitter gourd leaf water extract
  • Orlistat supplementation significantly reduced hepatic adipocyte area in the bitter gourd leaf water extract (MCLW) or Orlistat group (FIG. 6).
  • Adipose tissue (epididymis, subcutaneous, viscera, and scapula) was weighed, and epididymal fat and liver were collected, frozen in liquid nitrogen, and stored at -80 °C for further examination. For histological analysis, sections of epididymal fat and liver tissue were fixed in 10% formaldehyde. Total RNA was collected from mouse liver and epididymal adipose tissue and subjected to qPCR. PCR reaction is AccuPower PCR was performed using PreMix (Bioneer, Chungwon, Korea) and VeritiTM 96-Well Thermal Cycler (Applied Biosystems, Foster City, CA, USA). The primers used in this study were synthesized by Innotek (Daejeon, Korea) (Table 4). GAPDH was selected as an internal reference in qPCR and relative quantification of gene expression was determined with the 2 - ⁇ Ct method.
  • Frozen mouse liver and epididymal adipose tissue were homogenized in liquid nitrogen. Tissues were lysed in RIPA lysis buffer containing 1% PI for western blot analysis. Cell lysates were incubated on ice for 1 hour and homogenates were centrifuged at 13,000 rpm for 20 minutes at 4°C. Western blotting was performed as previously described.
  • Adenylate-activated protein kinase K (AMPK), adipose triglyceride lipase (ATGL), triacylglycerol hydrolase (TGH), hormone-sensitive lipase (HSL), carnitine palmitoyl transferase I (CPT- I) and porcine isolate protein 3 (UCP3) mRNA and protein levels were measured.
  • AMPK Adenylate-activated protein kinase K
  • ATGL adipose triglyceride lipase
  • TGH triacylglycerol hydrolase
  • HSL hormone-sensitive lipase
  • CPT- I carnitine palmitoyl transferase I
  • UCP3 porcine isolate protein 3
  • mRNA levels Fig. 7a-e
  • AMPK adiponectin and adenylate-activated protein kinase K
  • MCLW bitter gourd leaf extract
  • PPAR ⁇ lipogenic enzyme and regulatory factor activated receptor ⁇
  • CYP7A1 cholesterol 7 ⁇ -hydroxylase
  • ACS acyl-CoA synthetase
  • acetyl coenzyme A carboxylase The anti-obesity effect was analyzed by measuring mRNA and protein concentrations for ACC (ACC) and fatty acid synthase (FAS).
  • bitter melon leaf extract prepared by using distilled water as a solvent was used.
  • mice 9-week-old C57BL/6 male mice were purchased from the Nara Bio Animal Center (Nara Biotech, Seoul, Korea) and acclimatized for one week in the experimental facility, and housed in groups of 4 mice each inside a transparent plastic cage with an Aspen chip attached. Mice were randomly divided into two groups and supplied with a standard mouse chow diet and untreated tap water under clean pathogen-free conditions.
  • the WD group was allowed to freely drink 23.1g/L d-fructose (Sigma-Aldrich, F0127) and 18.9g/L d-glucose (Sigma-Aldrich, G8270) dissolved in water to increase sugar intake.
  • 23.1g/L d-fructose Sigma-Aldrich, F0127
  • 18.9g/L d-glucose Sigma-Aldrich, G8270
  • 0.2 ⁇ l (0.32 ⁇ g/g) of CCl4 (Sigma-Aldrich, 289116-100ML) was intraperitoneally injected once a week.
  • WD + Silymarin administration group 10 mg/kg of Silymarin was orally administered once a day using a zone.
  • the amount of feed consumed was recorded every day, and the daily intake of each mouse was not restricted, and the weight of the mice was measured every day to search for anti-obesity and anti-inflammatory effects of hydroferulic acid.
  • epididymal fat, subcutaneous fat, brown fat, visceral fat, and liver tissue were separated and weighed with surgical instruments from which RNAse had been removed. C and the remaining part was stored in 10% formaldehyde for H&E staining.
  • the animal environment was appropriately controlled with a 12-hour dark period at 20 to 21 °C and a relative humidity of 40 to 45%. All experiments were approved by the Animal Care Committee of Konkuk University Animal Hospital, and the experiments related to the present invention (KU17089) were designed to minimize the number and pain of animals used.
  • Hydroferulic acid was isolated from the water extract of bitter gourd leaves showing high antioxidant activity.
  • a Vion UPLCTM system (Vion, Waters, Milford, MA, USA) was used and LC conditions were optimized within 9 minutes on an Acquity UPLC BEH C18 column (2.1 ⁇ 100 mm, 1.7 ⁇ m; Waters, Milford, MA, USA). The temperature was set at 55°C. The flow rate was set at 0.35 mL/min. The mobile phase was used by adding 0.1% acetonitrile (ACN) to water (FA) containing 0.1% formic acid. The spectrometer was used with electrospray ionization (ESI) in negative mode.
  • MS operating conditions are: capillary voltage 2.5 kV, sample cone 20 V, ion source temperature 200 °C, desolvation temperature 400 °C, cone gas 30 L/h, desolvation gas 900 L/h, scan time 0.2 seconds , a scan range of 50 to 1500 m/z, and a collision energy lamp of 10 to 30 eV (50 to 1000 m/z) were used.
  • MarkerLynx software (Waters, Milford, MA, USA) collected MS data sets analyzed by UPLC-Q-TOF MS (Waters, Milford, MA, USA), and was used for normalization and alignment. All spectra were normalized with internal standards (Terfenadine [M + H] and Zidovudine [M - H]).
  • mice in each experimental group were measured, and liver tissue was removed and histological changes in liver tissue weight were analyzed.
  • the body weight of the experimental animals was measured once a week. After 6 weeks of administration, the mice were bled and exsanguinated under ether anesthesia, and then the liver was removed and the weight of the liver was measured.
  • Liver tissues isolated from mice were fixed in 10% formalin for more than one day, formalin washed in running water for more than 12 hours, and then treated with 60% etahnol, 70% ethanol, 80% ethanol, 90% ethanol, 95% ethanol, and 100% ethanol, respectively. Dehydrated for 1 hour. After clearing in xylene three times for 1 hour each, infiltration process was carried out twice for 1 hour in paraffin. After the embedding process, the tissue was cut to a thickness of about 4 ⁇ m, placed on a slide, dried, and then hematoxylin-eosin staining was performed. The slide was dried, mounting medium was dropped, and cover glass was covered to finish. Histological changes of the liver tissue were confirmed under a microscope. The results are shown in FIG. 11 .
  • liver of the NASH group showed a bright pink color with considerable progress in fat deposition.
  • -H&E tissue staining photograph fat deposited in liver tissue ward was confirmed.
  • the hydroferulic acid (HFA) intake group showed a concentration-dependent decrease in liver tissue weight, liver weight relative to body weight, and the size and number of fat globules in liver tissue.
  • TG serum triglycerides
  • FFAs serum free fatty acids
  • TC serum cholesterol
  • LDL/VLDL serum LDL/VLDL
  • liver tissue was obtained by harvesting, weighing, and freezing in liquid nitrogen, serum samples were obtained by collecting blood samples using cardiac puncture, and separating serum by centrifugation (3000 rpm for 20 minutes). obtained. Liver tissue and serum samples were stored at -80°C until further analysis. Serum triglycerides (TG), serum free fatty acids (FFAs), serum total cholesterol (TC), low-density lipoprotein cholesterol (LDL), ultra-low-density lipoprotein ( VLDL) and high-density lipoprotein cholesterol (HDL) were measured. The results are shown in FIG. 12 .
  • FFAs serum free fatty acids
  • TC serum total cholesterol
  • LDL low-density lipoprotein cholesterol
  • VLDL ultra-low-density lipoprotein
  • HDL high-density lipoprotein cholesterol
  • TG serum triglycerides
  • FFAs serum free fatty acids
  • TC serum cholesterol
  • LDL/VLDL serum LDL/VLDL decreased concentration-dependently in the hydroferulic acid intake group compared to the non-alcoholic fatty liver induction group.
  • triglycerides (TG), free fatty acids (FFAs), and LDL/VLDL in adipose tissue decreased, while HDL increased.
  • liver tissue was extracted from animal models of each experimental group, and the degree of fat deposition (Oil Red O) and liver fibrosis (Masson, Sirius Red) were analyzed.
  • frozen liver tissue for Oil Red O staining was cut to a thickness of 4 ⁇ m, the specimen was fixed on a slide, stained with Oil Red O (Sigma, St. Louis, MO) reagent, and fat in the liver was examined using an optical microscope. Calmness was observed.
  • Oil Red O Sigma, St. Louis, MO
  • a tray containing slides to which slices of liver tissue isolated from mice were cut into about 4 ⁇ m were attached was immersed in 100% ethanol for 3 minutes, 95% ethanol for 3 minutes, and 70% ethanol for 3 minutes in order. After soaking in Weigert's hematoxylin working solution for 10 minutes, rinse the solution in running water for 10 minutes, repeat soaking in distilled water for 5 times, and soak in Ponceau S acid fuchsin solution for 15 minutes, then soak in distilled water for 5 times and remove. .
  • Sirius Red staining was performed by immersing a tray containing slides to which slices of about 4 ⁇ m slices of liver tissue isolated from mice were attached were sequentially immersed in 100% ethanol for 3 minutes, 95% ethanol for 3 minutes, and 70% ethanol for 3 minutes, followed by Sirius Red staining. After soaking in the working solution for 30 minutes, immersing in distilled water 5 times and taking it out was repeated. After repeating the immersion in 95% ethanol and 100% ethanol three times in sequence and taking out, the samples were dipped in xylene twice for 3 minutes to remove paraffin. After drying the tissue at room temperature, mounting medium prepared in the ratio of Map balsam (3) : xylene (1) was dropped, and cover glass was covered to finish.
  • liver tissue staining results showed that fat deposition and liver fibrosis increased in the non-alcoholic fatty liver induction group, but fat deposition and liver fibrosis were suppressed in a concentration-dependent manner in the case of the hydroferulic acid intake group.
  • ⁇ -SMA immunohistochemical staining was performed manually using a wet chamber.
  • 4 ⁇ m-thick paraffin sections were prepared, attached to slides, sufficiently dried in a slide dryer at 60oC, hydrated with deparaffinization and alcohol in xylene, and immunohistochemical staining was performed using a wet chamber.
  • Tissue sections were washed with water and treated with 3% hydrogen peroxidase for 10 minutes to block the activity of endogenous peroxidase, washed with Tris buffer saline, and then transferred to block solution to block non-specific reactions and allowed to act for 60 minutes.
  • each was reacted with an ⁇ -SMA monoclonal antibody as a primary antibody overnight, washed appropriately with Tris buffer saline, and reacted for 2 h using HRP-polymer. After that, the color was developed with DAB for 5 minutes, counterstained with Gill's hematoxylin, dehydrated, and sealed with mount through transparency.
  • the liver was lysed using LIPA buffer, and proteins were extracted and quantified.
  • the quantified protein samples were loaded onto an SDS-acrylamide gel, electrophoresed, and transferred to a membrane. After blocking the membrane with blocking buffer for 2 hours at room temperature, it was shaken overnight at 4°C using ⁇ -SMA and COL1A1 monoclonal antibodies. After washing with TBST for 10 minutes, the secondary antibody was shaken at room temperature for about 1 hour. After washing with TBST (or PBST) for 10 minutes, ECL solution was sprayed on the membrane, and the protein expression level was measured using chemi-doc.
  • ⁇ -SMA staining results related to fibrosis in liver tissue showed that the expression was decreased in a concentration-dependent manner in the hydroferulic acid intake group compared to the non-alcoholic fatty liver induction group.
  • the expression of COL1A1 and COL1A2 was decreased in a concentration-dependent manner.
  • immunohistochemical staining for CD68, F4/80, and MerTK was performed manually using a wet chamber. The process is to prepare paraffin sections with a thickness of 4 ⁇ m, attach them to slides, dry them sufficiently in a slide dryer at 60 o C, hydrate them with deparaffinization and alcohol in xylene, and then perform immunohistochemical staining using a wet chamber. did Tissue sections were washed with water and treated with 3% hydrogen peroxidase for 10 minutes to block the activity of endogenous peroxidase, and washed with Tris buffer saline. In order to block the next non-specific reaction, it was transferred to Block solution and allowed to act for 60 minutes.
  • CD68 monoclonal antibody, F4/80 monoclonal antibody, and MerTK monoclonal antibody were reacted overnight as primary antibodies, washed with Tris buffer saline, and reacted for 2 hours using HRP-polymer. After that, the color was developed with DAB for 5 minutes, counterstained with Gill's hematoxylin, dehydrated, transparent, and sealed with mount.
  • RT-PCR Reverse Transcription-Polymerase Chain Reaction
  • qPCR was performed using 1 ⁇ g of reverse-transcribed cDNA in an Accupower PCR Premix (Bioneer Co., Korea) tube. was performed and expression of IL-1 ⁇ , TNA- ⁇ , MCP-1, and TGF- ⁇ mRNA was performed.
  • the liver was lysed using LIPA buffer, and proteins were extracted and quantified.
  • the quantified protein samples were loaded onto an SDS-acrylamide gel, electrophoresed, and transferred to a membrane. After blocking the membrane with blocking buffer for 2 hours at room temperature, it was shaken overnight at 4°C using Gas6, MerTK, ERK, p-ERK, and TGF- ⁇ monoclonal antibodies. After washing with TBST for 10 minutes, the secondary antibody was shaken at room temperature for about 1 hour. After washing with TBST (or PBST) for 10 minutes, ECL solution was sprayed on the membrane, and the protein expression level was measured using chemi-doc.
  • FIG. 15 As shown in FIG. 15, as a result of measuring the expression levels of F4/80, CD68, Gas6, MerTK, ERK, p-ERK, and TGF- ⁇ related to inflammation in liver tissue, the hydroferulic acid intake group compared to the non-alcoholic fatty liver induction group. It was found that the expression of the inflammation-related factors was inhibited in a concentration-dependent manner (FIGS. 15A and 16). In addition, as a result of checking the expression level of inflammation-related factors in liver tissue RNA, it was confirmed that the expression of IL-1 ⁇ , TNA- ⁇ , MCP-1, etc. was reduced.

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Abstract

La présente invention concerne une composition efficace pour prévenir, améliorer ou traiter l'obésité ou la stéatose hépatique, comprenant un extrait de feuilles de Momordica charantia ou de l'acide hydroférulique dérivé de Momordica charantia en tant que principe actif. La composition peut être utilisée efficacement dans les industries alimentaires ou pharmaceutiques.
PCT/KR2022/019212 2021-11-30 2022-11-30 Composition pour la prévention ou le traitement de l'obésité, de la stéatose hépatique ou de la stéatohépatite comprenant un extrait de momordica charantia ou de l'acide hydroférulique dérivé de momordica charantia en tant que principe actif WO2023101420A1 (fr)

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FAN MEIQI, LEE JAE-IN, RYU YOUNG-BAE, CHOI YOUNG-JIN, TANG YUJIAO, OH MIRAE, MOON SANG-HO, LEE BOKYUNG, KIM EUN-KYUNG: "Comparative Analysis of Metabolite Profiling of Momordica charantia Leaf and the Anti-Obesity Effect through Regulating Lipid Metabolism", INTERNATIONAL JOURNAL OF ENVIRONMENTAL RESEARCH AND PUBLIC HEALTH, vol. 18, no. 11, pages 5584, XP093070675, DOI: 10.3390/ijerph18115584 *
KOLMAR BNH CO., LTD.: "Development of hypoglycemic health functional food using bitter melon(Momordica charantia)", RESEARCH ON MASS PRODUCTION PROCESS DEVELOPMENT AND COMMERCIALIZATION OF MOMORDICA CHARANTIA EXTRACT, 2017, pages 1 - 311, XP009546810 *
MA YUNCI, CHEN KAI, LV LIN, WU SHAOYU, GUO ZHIJIAN: "Ferulic acid ameliorates nonalcoholic fatty liver disease and modulates the gut microbiota composition in high-fat diet fed ApoE−/− mice", BIOMEDICINE & PHARMACOTHERAPY, ELSEVIER, FR, vol. 113, 1 May 2019 (2019-05-01), FR , pages 108753, XP093070678, ISSN: 0753-3322, DOI: 10.1016/j.biopha.2019.108753 *

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