WO2020080641A1 - Composition pour la prévention et le traitement de maladies liées à l'ampk, comprenant un extrait de gynostemma longipes vk1 ou un composé isolé à partir de celui-ci en tant qu'ingrédient actif - Google Patents

Composition pour la prévention et le traitement de maladies liées à l'ampk, comprenant un extrait de gynostemma longipes vk1 ou un composé isolé à partir de celui-ci en tant qu'ingrédient actif Download PDF

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WO2020080641A1
WO2020080641A1 PCT/KR2019/008089 KR2019008089W WO2020080641A1 WO 2020080641 A1 WO2020080641 A1 WO 2020080641A1 KR 2019008089 W KR2019008089 W KR 2019008089W WO 2020080641 A1 WO2020080641 A1 WO 2020080641A1
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compound
ramnopyranosyl
dione
glucopyranoside
lamnopyranosyl
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Korean (ko)
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오원근
팜하탄텅
허정무
트란반온
흐앙둑트롱
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서울대학교산학협력단
하노이 약학대학교
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23K—FODDER
    • A23K10/00—Animal feeding-stuffs
    • A23K10/30—Animal feeding-stuffs from material of plant origin, e.g. roots, seeds or hay; from material of fungal origin, e.g. mushrooms
    • 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
    • A23K20/163—Sugars; Polysaccharides
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • 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
    • A23L33/125—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives containing carbohydrate syrups; containing sugars; containing sugar alcohols; containing starch hydrolysates
    • 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
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/42—Cucurbitaceae (Cucumber family)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • 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
    • 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
    • A23V2200/316—Foods, ingredients or supplements having a functional effect on health having an effect on regeneration or building of ligaments or muscles
    • 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 is Gnosthema longifose VK1 ( Gynostemma longipes VK1) relates to a composition for preventing or treating AMPK-related diseases comprising an extract or a compound isolated therefrom as an active ingredient.
  • AMPK (5'-AMP-activated protein kinase) is activated by a decrease in the energy state of the cells (the ratio of AMP / ATP and ADP / ATP), such as the nutritional status or movement of cells.
  • Activation of AMPK affects various physiological functions such as glucose transport, fatty acid synthesis, and cholesterol biosynthesis by regulating phosphorylation of various other enzymes involved in cell energy metabolism. It is known (Hardie, DG, 2007).
  • the effect on AMPK activation is related to target organs such as the liver, muscles, fats and pancreas, which are closely related to the regulation of energy metabolism. When AMPK is activated in the liver, it inhibits the synthesis of fatty acids and cholesterol and promotes the oxidation of fatty acids.
  • AMPK When AMPK is activated in skeletal muscle, it promotes oxidation and sugar absorption of fatty acids, and fat cells inhibit fat breakdown and fat production. In addition, activation of AMPK in pancreatic ⁇ cells is known to promote insulin secretion, and many studies have been conducted as major drug targets for metabolic diseases in relation to the role of AMPK (Ha Joo Heon, et al. 2010).
  • AMPK is involved in glucose uptake in muscle cells promoted by exercise, and AMPK is a sensor that generally monitors energy levels in cells. ) It turns out to play a role. In exercise or hunger, muscle cells, hepatocytes, and fat cells inhibit the synthesis of fat, glycogen, etc. in order to supply the necessary energy, and play a role to generate the energy required by the body through fat decomposition from storage materials (fat). Perform.
  • AMPK is also known as an intracellular signaling substance of leptin and adiponectin secreted from adipocytes.
  • adiponectin is estimated to have a high relationship with insulin resistance due to obesity, as it is measured at a lower concentration in the blood of an obese person compared to a normal-weight person, and accordingly, AMPK activator is a great drug for obesity It is possible as a target (Hardie DG, 2007 (1)).
  • the muscle is the tissue that has the most amount in the human body, and in order to maintain the functional ability of the human body and prevent metabolic diseases, it is essential to secure an adequate muscle mass.
  • the muscles are largely divided into smooth muscle, cardiac muscle, and skeletal muscle. Skeletal muscle is a major tissue that promotes skeletal movement, maintains breathing, and occupies a significant portion of our body. And primary peripheral tissues that use glucose and fatty acids important for the prevention of type 2 diabetes (Fu, X., et al., 2015).
  • AMPK consists of catalytic ⁇ -subunits ( ⁇ 1 and ⁇ 2), modulatory ⁇ -subunits ( ⁇ 1 and ⁇ 2) and ⁇ -subunits ( ⁇ 1, ⁇ 2 and ⁇ 3).
  • Skeletal muscles which make up a significant portion of our body, induce energy imbalances through the use of ATP during exercise, thereby increasing intracellular AMP and ADP concentrations.
  • the binding of ADP to AMP in the ⁇ -subunit causes a structural change that activates the AMPK enzyme up to 10 times through an allosteric mechanism.
  • This structural change also triggers phosphorylation of catalytic ⁇ -subunit 172 threonine ( ⁇ -Thr172) by the upper gene LKB1 (liver kinase B1) and protects dephosphorylation by protein phosphatase, thereby activating 100 Increases fold.
  • AMPK is activated up to 1000 times or more.
  • AMPK is also activated by the rise of the concentration of Ca 2 + cells with ⁇ -Thr172 phosphorylation catalyzed by CaMKK ⁇ (calcium / calmodulin-dependent protein kinase kinase ⁇ ).
  • CaMKK ⁇ calcium / calmodulin-dependent protein kinase kinase ⁇
  • Skeletal muscles which make up a large portion of the human body, do not divide, are composed of multinucleated muscle fibers, and are made during the embryo formation process. After the embryo formation process is over, muscles are formed by the process of postnatal growth or myogenesis. In particular, it has been reported that muscle differentiation for muscle regeneration occurs when muscles are damaged by muscle frostbite, sprains, bruises, and the like. In muscle differentiation, satellite cells are activated first, and activated satellite cells are differentiated into myoblasts (Morgan, J.E., et al., 2003). Differentiated myoblasts proliferate and fusion occurs between myoblasts and develop into myotubes. These root canal cells gather to form muscle fibers, and the muscle fibers form a bundle to finally form muscles.
  • AMPK ⁇ 1 is a dominant isoform in satellite cells, and Warburg-like glycolysis through atypical shh (sonic hedgehog) signaling of muscle cells during muscle regeneration. It has been shown to promote muscle regeneration by enhancing it.
  • muscle differentiation such as aging or differentiation from satellite cells to myoblasts, myoblast division, muscle atrophy, myopathy, muscle injury, muscular dystrophy
  • myoblasts myoblast division
  • muscle atrophy myopathy
  • muscle injury muscular dystrophy
  • Various muscle disorders and diseases can occur, such as dystrophy, sarcopenia, myoneural conductive disease and nerve injury (Bonaldo, P., et al., 2013; Wagatsuma , A., et al., 2014).
  • the sarcopenia may be caused by cachexia.
  • Cachexia is a type of chronic wasting complex syndrome and refers to a high level of systemic weakness seen in the end stages of cancer, tuberculosis, and hemophilia. In particular, it occurs with chronic diseases such as malignant tumors, chronic obstructive pulmonary disease, and chronic heart failure, and weight loss, loss of muscle mass and body fat, and inflammatory reactions accompanied by anorexia.
  • Muscle loss due to cachexia is manifested as a complex syndrome caused by continuous reduction of skeletal muscle mass and functional impairment. Acute muscle loss symptoms, unlike aging with progressive and slow muscle mass loss, and muscle loss disorders due to muscle differentiation disorders This appears. The difference in physiological characteristics also shows a difference in prevention and treatment. Therefore, even if there are signs of muscle loss, depending on the cause of the occurrence, treatment according to the characteristics of cachexia, aging, and muscle differentiation disorders is necessary (Ryu Seung-wan, 2017).
  • Gynostemma longipes is a perennial vine plant belonging to the Cucurbitaceae family that grows in Vietnam and southern China, and is a perennial plant that grows in Ulleungdo and southern regions of Korea, Vietnam, Japan, and China. Gynostemma pentaphyllum ) has a similar external appearance.
  • Gynostemma pentaphyllum is a vine plant whose stem grows entangled around it with other tendrils and entangles it and uses it as a "stone car” to dry the leaves. Stone others are called rhizomes or outposts, and have been traditionally used for respiratory and circulatory system diseases such as expectorant, bronchitis, phlegm, poison, poisoning, and seawater. The leaves beside the stone are coarse and have multi-celled white hairs on both sides, but soon disappear and the lobules are usually 5 but 3-7.
  • Zinostema rongipes is characterized by having 7 to 9 leaves relatively more than stones, and in Vietnam, it is relatively difficult to distinguish it from the shape of leaves and leaves.
  • Gnosthema rongipes is characteristically more leafy than dolce, and is characterized by a pubescent on the stem, and a bitter taste than dolce.
  • Ginosthema rongipes CYWu ( Gynostemma) gylongiposide II-III and gypentoside A were separated from longipes CYWu (Anh PT, et al., 2015).
  • Ginostema rongipes VK1 is a new variety discovered by the present inventors, showing morphological differences from other plants of the genus genome ( Gynostemma spp.), And has a single-base polymorphism different from the known genome lonegipes. It was confirmed that the chemical composition differs not only from the genus geno genome but also from the same geno genome Rongipes in terms of the chemical composition components (Korea Registered Patent No. 1811048).
  • the present inventors separated compounds from the extract in the process of studying the extract of Ginosthema rongipes VK1, and these extracts of Gnosthema rongipes VK1 and the compounds separated therefrom were various such as glucose transfer, fatty acid synthesis, cholesterol biosynthesis, etc. It was possible to complete the present invention by confirming that it is possible to treat muscle disease, obesity, diabetes or metabolic syndrome by activating AMPK involved in physiological action and, in addition, activating AMPK in fat cells and muscle cells.
  • Korean Registered Patent No. 1811048 discloses the extract of Ginosthema rongipes VK1 and a compound isolated therefrom, but related to the AMPK activation and muscle regeneration effect or metabolic syndrome of the present invention as a prevention or treatment effect of cognitive dysfunction No prophylactic or therapeutic effect on the disease is described and implied at all.
  • Korean Patent Publication No. 2014-0108621 discloses an extract of Gynostemma pentaphyllum containing a phenoside compound, and Korean Registered Patent No.
  • 0945382 discloses an extract of Gynostemma pentaphyllum , but the genos of the present invention Theme Rongipes VK1 ( Gynostemma longipes VK1) The extract and the compound isolated therefrom and the effect related to AMPK activation thereof are not described, and thus are different from the present invention.
  • China Patent No. 103238741 describes Ginoostemma longipes , but the compound of the present invention and its AMPK activation effect are not described at all, and thus are different from the present invention.
  • the object of the present invention is Gnosthema longifes VK1 ( Gynostemma longipes VK1) to provide a composition for the prevention or treatment of AMPK-related diseases comprising an extract or a compound isolated therefrom as an active ingredient.
  • the present invention is 3 ⁇ , 20 (S) -dihydroxydamar-24-en-12,23-dione-3-O- ⁇ -L-ramnopyranosyl- (1 ⁇ 2)-[ ⁇ of formula 1 -L-lamnopyranosyl- (1 ⁇ 3)]- ⁇ -D-glucopyranoside (3 ⁇ , 20 (S) -dihydroxydammar-24-en-12,23-dione-3-O- ⁇ -L- rhamnopyranosyl- (1 ⁇ 2)-[ ⁇ -L-rhamnopyranosyl- (1 ⁇ 3)]- ⁇ -D-glucopyranoside) (Compound 1), 3 ⁇ , 20 (S) -dihydroxydamar-24-ene- 12,23-Dione-3-O- ⁇ -L-ramnopyranosyl- (1 ⁇ 2)-[ ⁇ -L-ramnopyranosyl- (1 ⁇ 3)]- ⁇ -D-glucopyranosyl-20- O
  • the genostema longifose VK1 extract is a group consisting of water, C1-4 lower alcohol, C1-4 acetic ester, acetone, and methylethylketone. It may be an extract extracted with one or more solvents selected from.
  • the present invention is 3 ⁇ , 20 (S) -dihydroxydamar-24-en-12,23-dione-3-O- ⁇ -L-ramnopyranosyl of the formula (1)-(1 ⁇ 2)- [ ⁇ -L-Lamnopyranosyl- (1 ⁇ 3)]- ⁇ -D-glucopyranoside (Compound 1), 3 ⁇ , 20 (S) -dihydroxydamar-24-en-12,23- Dione-3-O- ⁇ -L-lamnopyranosyl- (1 ⁇ 2)-[ ⁇ -L-ramnopyranosyl- (1 ⁇ 3)]- ⁇ -D-glucopyranosyl-20-O- ⁇ - D-glucopyranoside (Compound 2), 3 ⁇ , 20 (S) -dihydroxydamar-24-en-12,23-dione-3-O- ⁇ -L-lamnopyranosyl- (1 ⁇ 2 )-[(4-O-acetyl) -
  • the compound is preferably 3 ⁇ , 20 (S) -dihydroxydamar-24-en-12,23-dione-3-O- ⁇ -L-ramnopyranosyl- (1 ⁇ 2)-[ ⁇ - L-lamnopyranosyl- (1 ⁇ 3)]- ⁇ -D-glucopyranoside (Compound 1), 3 ⁇ , 20 (S) -dihydroxydamar-24-en-12,23-dione-3 -O- ⁇ -L-lamnopyranosyl- (1 ⁇ 2)-[(4-O-acetyl) - ⁇ -L-ramnopyranosyl- (1 ⁇ 3)]- ⁇ -D-glucopyranoside ( Compound 3), Zipentonoside A (Compound 4), 3 ⁇ , 20 (S) -dihydroxydamar-24-en-12,23-dione-3-O- ⁇ -L-lamnopyranosyl- (1 ⁇ 2) - ⁇ -D-glucopyranoside (
  • the composition can activate AMPK.
  • the AMPK-related disease may be muscle disease, obesity, diabetes or metabolic syndrome.
  • the muscle diseases include muscle atrophy, myopathy, muscle injury, muscular dystrophy, myasthenia, sarcopenia, myoneural conductive disease, In the group consisting of dermatomyositis, diabetic amyotrophy, nerve injury, amyotrophic lateral sclerosis (ALS), cachexia, and degenerative muscle diseases Can be selected.
  • the cachexia is cancer, AIDS (acquired immune deficiency syndrome, AIDS), celiac disease, chronic obstructive pulmonary disease (COPD), multiple sclerosis, rheumatoid arthritis, Chronic heart failure, congestive heart failure, uremia, tuberculous, crohn's disease, untreated or severe type 1 diabetes, anorexia nervosa and hormonal deficiency It may be a wasting syndrome.
  • the present invention is a genothema extracted by using at least one selected from the group consisting of water, C1 ⁇ 4 lower alcohol, C1 ⁇ 4 acetic acid ester, acetone and methyl ethyl ketone as a solvent.
  • the present invention is also a novel compound 3 ⁇ , 20 (S) -dihydroxydamar-24-en-12,23-dione-3-O- ⁇ -L-lamnopyranosyl having a chemical structure represented by the following Chemical Formula 2.
  • (1 ⁇ 2) [ ⁇ -L-lamnopyranosyl- (1 ⁇ 3)]- ⁇ -D-glucopyranosyl-20-O- ⁇ -D-glucopyranoside (Compound 2)
  • the present invention is 3 ⁇ , 20 (S) -dihydroxydamar-24-en-12,23-dione-3-O- ⁇ -L-lamnopyranosyl- (1 ⁇ 2)-[ ⁇ in Chemical Formula 1 -L-lamnopyranosyl- (1 ⁇ 3)]- ⁇ -D-glucopyranoside (Compound 1), 3 ⁇ , 20 (S) -dihydroxydamar-24-en-12,23-dione- 3-O- ⁇ -L-ramnopyranosyl- (1 ⁇ 2)-[ ⁇ -L-ramnopyranosyl- (1 ⁇ 3)]- ⁇ -D-glucopyranosyl-20-O- ⁇ -D- Glucopyranoside (Compound 2), 3 ⁇ , 20 (S) -dihydroxydamar-24-en-12,23-dione-3-O- ⁇ -L-lamnopyranosyl- (1 ⁇ 2)- [(4-O-acetyl) - ⁇ -
  • the extract of Gnosthema rongipes VK1 is an extract obtained by extracting one of one or more solvents selected from the group consisting of water, C1-4 lower alcohols, C1-4 acetic acid esters, acetone and methyl ethyl ketone.
  • solvents selected from the group consisting of water, C1-4 lower alcohols, C1-4 acetic acid esters, acetone and methyl ethyl ketone.
  • the acetic acid ester of C1-4 is low toxicity of the Ministry of Food and Drug Safety "Guidelines for Residual Solvent Solvent Standards" Classified as a solvent, methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, isobutyl acetate, etc.
  • the solvent is preferably one or more solvents selected from the group consisting of water and lower alcohols of C1 to 4, more preferably ethanol, and most preferably 70% [v / v] ethanol.
  • the solvent may be added to a weight of 2 to 500 times the weight of Ginosthema Rongipes VK1.
  • the Ginosthema longifose VK1 extract contains one or more solvents selected from the group consisting of water, C1-4 lower alcohol, C1-4 acetic acid ester, acetone, and methyl ethyl ketone.
  • the extracted extract may be a fraction obtained by diluting the concentrated concentrate and diluting the diluted liquid by adding it to the ion exchange resin and eluting with an organic solvent.
  • the diluent can be made by adding distilled water to the concentrate of the Ginosthema longifose VK1 extract, and the distilled water can add 5 to 200 times the weight of the concentrate.
  • the ion exchange resin may be selected from Diaion HP-20, SP825, AXT204, XAD1600T, MN200, SP70, SP710, and the like. Preferred is diaion HP-20, SP70 or SP710, most preferably a synthetic adsorbent among aromatic unsubstituted resins that can be used in the United States FDA for food and pharmaceuticals (CFR173.62, Code of Federal Regulations Title 21 ), SP70 or SP710.
  • the organic solvent may be C1-4 lower alcohol, C1-4 acetic acid ester, and acetone.
  • the lower alcohol of C1-4 may be methanol, alcohol, ethanol, propanol, isopropanol, butanol, etc.
  • the acetic ester of C1-4 is methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, isobutyl acetate Etc. It is preferably ethanol and acetone, and most preferably 95% [v / v] ethanol.
  • the present invention is 3 ⁇ , 20 (S) -dihydroxydamar-24-en-12,23-dione-3-O- ⁇ -L-ramnopyranosyl of the formula (1)-(1 ⁇ 2)- [ ⁇ -L-Lamnopyranosyl- (1 ⁇ 3)]- ⁇ -D-glucopyranoside (Compound 1), 3 ⁇ , 20 (S) -dihydroxydamar-24-en-12,23- Dione-3-O- ⁇ -L-lamnopyranosyl- (1 ⁇ 2)-[ ⁇ -L-ramnopyranosyl- (1 ⁇ 3)]- ⁇ -D-glucopyranosyl-20-O- ⁇ - D-glucopyranoside (Compound 2), 3 ⁇ , 20 (S) -dihydroxydamar-24-en-12,23-dione-3-O- ⁇ -L-lamnopyranosyl- (1 ⁇ 2 )-[(4-O-acetyl) -
  • Compounds 1 to 19 of Chemical Formula 1 may be separated from the extract of Genostema longifose VK1.
  • Compounds 1 to 19 of Formula 1 isolated from the extract of Genostema rongipes VK1 can be obtained by fractionating the extract of genostema rongipes VK1 by chromatography, and the chromatography is diaion HP-20 column chromatography (diaion HP-20 column chromatography, silica gel column chromatography, reverse phase silica gel column chromatography, RP-18 column chromatography, LH-20 column Chromatography (LH-20 column chromatography), preparative reversed-phase high performance chromatography, medium pressure liquid chromatography, high-performance liquid chromatography, HPLC) and the like.
  • the compound of the present invention may be synthesized according to a conventional method in the art, and may be prepared with a pharmaceutically acceptable salt.
  • the AMPK-related disease may be muscle disease, obesity, diabetes or metabolic syndrome, but is not limited thereto.
  • the muscle disease is a disease that may occur due to deficiency of muscle cells, abnormal reduction, or dysfunction of muscle cells, muscle atrophy, myopathy, muscle injury, muscular dystrophy, Myasthenia, sarcopenia, myoneural conductive disease, dermatomyositis, diabetic amyotrophy, nerve injury, amyotrophic lateral sclerosis , ALS), cachexia, degenerative muscle diseases, and the like.
  • the cachexia is also called wasting syndrome, and is characterized by weight loss accompanied by anorexia, muscle mass and body fat reduction, and inflammatory reactions.
  • These cachexia are cancer, AIDS (acquired immune deficiency syndrome, AIDS), celiac disease, chronic obstructive pulmonary disease (COPD), multiple sclerosis, rheumatoid arthritis, Chronic heart failure, congestive heart heart failure, uremia, tuberculous, crohn's disease, untreated or severe type 1 diabetes, anorexia nervosa, hormonal deficiency, etc. It may appear, but is not limited to this.
  • the degenerative muscle disease is that the muscle is continuously destroyed, Duchenne muscular dystrophy, Baker's muscular dystrophy, Limb-girdle muscular dystrophy, Congenital muscular dystrophy, Facioscapulohumeral muscular dystrophy, myotonic dystrophy, oculopharyngeal muscular atrophy, distal muscular dystrophy, emery-dreifuss muscular dystrophy, etc. have.
  • the diabetes may be type 1 diabetes or type 2 diabetes. It is preferably type 2 diabetes.
  • the metabolic syndrome refers to a set of abnormal conditions such as increased body fat, increased blood pressure, elevated blood sugar, and abnormal lipids in the blood, which increase the risk of cerebrovascular diseases and diabetes, and can be improved through treatment of diabetes and reducing body fat. have.
  • composition for preventing or treating AMPK-related diseases including the genostema longifes VK1 extract or a compound isolated therefrom, can activate AMPK.
  • the 'AMPK (5'-AMP-activated protein kinase)' of the present invention is activated by a decrease in a cell's energy state, such as a nutritional state or exercise of a cell, and activation of AMPK is involved in various energy metabolism of cells. It regulates phosphorylation of enzymes and affects various physiological functions such as glucose migration, fatty acid synthesis, and cholesterol biosynthesis.
  • AMPK Activation of the AMPK inhibits the synthesis of fatty acids and cholesterol, promotes fatty acid oxidation and sugar absorption, and promotes muscle regeneration.
  • Activation of the AMPK can increase fatty acid oxidation by phosphorylating and deactivating acetyl-CoA carboxylase (ACC), an enzyme required for fatty acid synthesis.
  • ACC acetyl-CoA carboxylase
  • composition for preventing or treating AMPK-related diseases including the genostema rongipes VK1 extract or a compound isolated therefrom, may be a pharmaceutical composition.
  • the pharmaceutical composition may include the genostema rongipepes VK1 extract or a compound isolated therefrom and a pharmaceutically acceptable carrier.
  • Each of the pharmaceutical compositions may be formulated and used in the form of an oral dosage form such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, external preparation, suppository, and sterile injectable solution according to a conventional method.
  • an oral dosage form such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, external preparation, suppository, and sterile injectable solution according to a conventional method.
  • Carriers, excipients and diluents that can be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, cellulose , Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations include at least one excipient in the genostema rongipes VK1 extract of the present invention or a compound isolated therefrom. It is prepared by mixing starch, calcium carbonate, sucrose or lactose, gelatin, etc. Also, lubricants such as magnesium stearate and talc are used in addition to simple excipients.
  • Liquid preparations for oral use include suspensions, intravenous solutions, emulsions, syrups, etc.
  • various excipients such as wetting agents, sweeteners, fragrances, and preservatives, can be included.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories.
  • Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.
  • As a base for suppositories witepsol, macrogol, tween 61, cacao butter, laurin butter, and glycerogelatin may be used.
  • the dosage of the pharmaceutical composition will vary depending on the age, gender, weight of the subject to be treated, the specific disease or pathology to be treated, the severity of the disease or pathology, the route of administration and the judgment of the prescriber. Dosage determination based on these factors is within the level of those skilled in the art, and generally, dosages range from 0.1 to 1000 mg / kg / day. A more preferable dosage is 0.5 to 500 mg / kg / day. Administration may be administered once a day, or may be divided into several times. The above dosage does not limit the scope of the present invention in any way.
  • the pharmaceutical composition can be administered to various mammals, such as rats, livestock, companion animals, humans. All modes of administration can be expected, for example, oral, rectal or intravenous, intramuscular, subcutaneous, intramucosal mucosal or intracranial injection.
  • the pharmaceutical composition may be used to prevent or treat obesity, diabetes, or metabolic syndrome by promoting AMPK in muscle cells, thereby promoting fatty acid burning and storage of glucose into muscle and fat.
  • the pharmaceutical composition may also be used to prevent or treat muscle disease by inducing muscle regeneration by promoting the proliferation of myoblasts and activation of AMPK, an important signaling material for muscle regeneration.
  • composition for preventing or treating AMPK-related diseases comprising the extract of Ginosthema longifes VK1 or a compound isolated therefrom may be a health functional food composition.
  • the health functional food composition may include the ginosthema longifes VK1 extract or a compound separated therefrom, and a food additive that is food-acceptable.
  • the ginosthema longifose VK1 extract or a compound isolated therefrom may be included as 0.001 to 100% by weight in the health functional food composition of the present invention.
  • the health functional food composition of the present invention includes tablets, capsules, pills or liquids, etc., and foods that can be added to the Ginosthema rongipes VK1 extract of the present invention or a compound separated therefrom are, for example For example, there are various foods, beverages, gums, teas, vitamin complexes, and health functional foods.
  • composition for preventing or treating AMPK-related diseases comprising the extract of the genostema rongipes VK1 of the present invention or a compound isolated therefrom as an active ingredient is muscle disease, obesity, diabetes or metabolic syndrome Provided is a composition for improving animal feed.
  • the animal feed composition may include feed additives.
  • the feed additive of the present invention corresponds to the supplementary feed under the feed management method.
  • the type of animal feed is not particularly limited, and a feed commonly used in the art may be used.
  • Non-limiting examples of the feed vegetable feed, such as grains, muscles, food processing by-products, algae, fiber, pharmaceutical by-products, fats and oils, starches, peels or grain by-products;
  • animal feeds such as proteins, inorganics, fats and oils, minerals, single cell proteins, animal planktons, and food. These may be used alone or in combination of two or more.
  • the present invention also relates to a method for separating the compounds 1 to 19 of Formula 1, wherein the genostema rongipes VK1 is water, C1-4 lower alcohol, C1-4 acetic acid ester, acetone and methyl ethyl ketone.
  • 3 ⁇ , 20 (S) -dihydroxydamar-24-en-12,23-dione-3-O- ⁇ -L- of Chemical Formula 1 by directly crystallizing the fraction of the second step or adding it to column chromatography.
  • the Gnosthema rongipepes VK1 extract of step 1 can be concentrated to obtain a concentrate, and the obtained concentrate can be diluted and added to the ion exchange resin of step 2.
  • the ion exchange resin in the second step may be selected from diaion HP-20, SP825, AXT204, XAD1600T, MN200, SP70, SP710, and the like.
  • diaion HP-20, SP70 or SP710 most preferably a synthetic adsorbent among aromatic unsubstituted resins that can be used in the United States FDA for food and pharmaceuticals (CFR173.62, Code of Federal Regulations Title 21 ), SP70 or SP710.
  • the organic solvent in the second step may be C1-4 lower alcohol, C1-4 acetic acid ester, and acetone.
  • the lower alcohol of C1-4 may be methanol, alcohol, ethanol, propanol, isopropanol, butanol, etc.
  • the acetic ester of C1-4 is methyl acetate, ethyl acetate, propyl acetate, isopropyl acetate, butyl acetate, isobutyl acetate Etc. It is preferably ethanol and acetone, and most preferably 95% [v / v] ethanol.
  • the three-step column chromatography is diaion HP-20 column chromatography, silica gel column chromatography, reverse phase silica gel column chromatography, RP-18 column chromatography, LH-20 column chromatography, preparative reversed-phase high performance chromatography, medium pressure liquid chromatography (medium pressure liquid chromatography), high-performance liquid chromatography (HPLC), and the like.
  • the present invention is Gnosthema longifose VK1 ( Gynostemma longipes VK1) relates to a composition for the prevention or treatment of AMPK-related diseases comprising an extract or a compound isolated therefrom as an active ingredient, wherein the Ginosthema rongipes VK1 extract or compounds isolated therefrom move glucose, synthesize fatty acids, It was confirmed that AMPK, which is involved in various physiological functions such as muscle regeneration, is activated, and promotes proliferation of myoblasts as well as sugar absorption into adipocytes.
  • composition of the present invention can be used to develop a therapeutic agent for AMPK-related diseases such as muscle disease, obesity, diabetes, metabolic syndrome, etc., which have excellent effects, health functional foods for improvement, and animal feed.
  • AMPK-related diseases such as muscle disease, obesity, diabetes, metabolic syndrome, etc., which have excellent effects, health functional foods for improvement, and animal feed.
  • Figure 1 is a result of confirming the cytotoxicity of the compounds isolated from the extract of the genostema Rongipes VK1 of the present invention, (A) and (C) the toxicity in adipocytes 3T3-L1 cells, (B) and ( D) shows toxicity in C2C12 cells, myoblasts. (A) and (B) show cytotoxicity when compounds 10-14 are treated at a concentration of 40 ⁇ M, and (C) and (D) show cytotoxicity according to the treatment concentration of compound 19 (10, 20, 30 ⁇ M). Giving.
  • Figure 2 is a result of confirming the effect of promoting the AMPK activation (phosphorylation) and ACC inactivation (phosphorylation) of the ginosthema Rongipes VK1 extract of the present invention or compounds isolated therefrom,
  • (A) is a Ginosthema Rongipes VK1 extract
  • (B) shows the results of confirming whether AMPK activation and ACC inactivation of Compounds 1 to 19 separated from the extract of Genostema rongipepes VK1.
  • Figure 3 is a result of confirming the effect of promoting the myoblast proliferation of the extract or a compound isolated therefrom of the present invention genostema rongipepes VK1, (A) and (B) is a muscle according to the treatment of the genostema rongipepes VK 1 extract The results confirming the effect of promoting blast growth through MTT (A) and WST (B) assays, (C) shows the effect of stimulating the growth of myoblasts of compounds 1-9 isolated from the extract of Genostema rongipepes VK1 by MTT and WST It shows the results confirmed through the assay.
  • Figure 4 shows the results of confirming whether or not to promote cell proliferation of cancer cells (MCF-7, MDA-MB) of compound 1 isolated from Zinosthema rongipepes VK1 extract or the present invention.
  • Figure 6 is a result of confirming the effect of promoting sugar absorption in the adipocytes of the present invention ginosthema Rongipes VK1 extract or compounds 10-19 isolated therefrom, (A) is the fluorescent derivative of glucose, 2-NBDG fat The result of confirming the fluorescence image due to intracellular absorption, and (B) shows the result of measuring the fluorescence intensity in the cell.
  • Figure 7 is a result of confirming the potential of the glucose transporter (GLUT4) in the adipocytes of the present invention ginosthema Rongipes VK1 extract or compounds 10, 11, 13 and 17 isolated therefrom, (A) adipocytes The result of confirming the amount of GLUT4 in the plasma membrane as a band, (B) shows the result of digitizing the band.
  • GLUT4 glucose transporter
  • Ginoostemma rongipes VK1 ( Gynostemma) of the present invention longipes VK1) is collected from Ha Giang, Vietnam (GPS 23 ° 08'34.9 "N 105 ° 25'42.6” E) in March and September 2016. Based on the morphological characteristics, it was confirmed that it was Ginosthema rongipes VK1.
  • HPLC High-performance liquid chromatography
  • Extraction solvent Extract amount (%) of extract containing compound 1 of the present invention
  • Compound 1 content of the present invention (%) Distilled water 16.5 1.27 30% ethanol 18.5 6.6 50% ethanol 17.5 12.2 70% ethanol 14.0 16.5 95% ethanol 11.5 10.3 100% methanol 11.5 9.7 n-hexane 8 0
  • Acetone 4 1.3
  • 20% [v / v] ethanol suspension is adsorbed by passing through SP70 resin, 500 ml of distilled water equivalent to 100 times the weight of the extract concentrate, 20% [v / v] ethanol, 40% [v / v] ethanol, 60% [v / v] ethanol, 80% [v / v] ethanol and 95% [v / v] ethanol were sequentially treated to elute adsorbents to secure fractions. Finally, acetone was used as a solvent for adsorption. The material was eluted.
  • Extract or fraction Content of compound 1 of the present invention (%) Configuration Concentration (mg / ml) Injection volume ( ⁇ l) 70% ethanol extract One 10 14.0 Distilled water fraction One 10 0 20% ethanol fraction One 10 0 40% ethanol fraction One 10 1.2 60% ethanol fraction One 10 17.2 80% ethanol fraction One 10 47.7 95% ethanol fraction One 10 8.6
  • the extract was obtained by using 70% [v / v] ethanol of the Gnosthema longifose VK1 cultivated and collected in March, and the obtained extract was concentrated. Thereafter, the concentrate was suspended in 20% [v / v] ethanol and added to the SP70 resin, followed by washing the resin with 30% [v / v] ethanol from which the compound 1 of the present invention did not flow out, and then 95%.
  • the C9 fraction was subjected to RP-C18 column chromatography under a concentration gradient condition of methanol: distilled water (1: 1 ⁇ 1: 0 [v / v]) to obtain 8 small fractions (C9-1 to C9-8). ).
  • the GLS is a reverse phase silica gel (RP-18) under a gradient condition of methanol: distilled water (1: 1 ⁇ 1: 0 [v / v]). Six fractions were obtained using column chromatography (GLS-M1 to GLS-M6).
  • the GLS-M4 fraction was added to RP-C18 column chromatography, followed by isotonitrile: distilled water (1: 1 [v / v]) isocratic, HPLC at 2 ml / min flow rate (column: Optima Pak C 18 ) Compound 10 (6.5 mg) was isolated.
  • the GLS-M6 fraction is 100% [v / v] methanol isocratic conditions, Sephadex LH-20 column chromatography and isotonitrile: distilled water (6: 4 [v / v]) isocratic, flow rate 2 ml / min As a condition, HPLC (Optima Pak C 18 ) was continuously performed to separate Compound 11 (22.5 mg) and Compound 19 (15.5 mg).
  • the GLP-N6 fraction was applied to Sephadex LH-20 column chromatography using 100% [v / v] methanol as a solvent, followed by acetonitrile: distilled water (7:20 [v / v]) isocratic conditions. HPLC was performed to separate compound 12 (6.1 mg), compound 13 (17.0 mg) and compound 14 (12.0 mg).
  • the GLP-N11 fraction was subjected to RP-C18 column chromatography under acetonitrile: distilled water (1: 1 ⁇ 0: 1 [v / v]) concentration gradient to obtain three small fractions (GLP-N11-1 to GLP). -N11-3).
  • the GLP-N11-2 small fraction was subjected to HPLC (column: Optima Pak C 18 ) under isotonitrile: distilled water (3:10 [v / v]) isocratic, 2 ml / min flow rate conditions to obtain Compound 15 (5.5). Mg), Compound 17 (6.0 mg) and Compound 18 (11.2 mg) were isolated.
  • Example 3-1 3 ⁇ , 20 (S) - Dihydroxydamar -24-yen-12,23- Dione -3-O- ⁇ -L- Rhamno Pyranosyl- (1 ⁇ 2)-[ ⁇ -L-ramnopyranosyl- (1 ⁇ 3)]- ⁇ -D-glucopyranoside (Compound 1).
  • Example 3-2 3 ⁇ , 20 (S) -dihydroxydamar-24-en-12,23-dione-3-O- ⁇ -L-ramnopyranosyl- (1 ⁇ 2)-[ ⁇ -L-ramnopyranosyl- (1 ⁇ 3)]- ⁇ -D-glucopyranosyl-20-O- ⁇ -D-glucopyranoside (Compound 2).
  • Example 3-3 3 ⁇ , 20 (S) -dihydroxydamar-24-en-12,23-dione-3-O- ⁇ -L-ramnopyranosyl- (1 ⁇ 2)-[(4-O-acetyl)- ⁇ -L-ramnopyranosyl- (1 ⁇ 3)]- ⁇ -D-glucopyranoside (Compound 3).
  • Example 3-5 3 ⁇ , 20 (S) - Dihydroxydamar -24-yen-12,23- Dione -3-O- ⁇ -L- Rhamno Pyranosyl- (1 ⁇ 2) - ⁇ -D-glucopyranoside (Compound 5).
  • Example 3-8 3 ⁇ , 20 (S) , 25- Trihydroxydamaran -12,23- Dione -3-O- ⁇ -L- Rhamno Pyranosyl- (1 ⁇ 2)-[ ⁇ -L-ramnopyranosyl- (1 ⁇ 3)]- ⁇ -D-glucopyranoside (Compound 8).
  • Example 3-13 3 ⁇ -hydroxy-22,23,24,25,26,27-hexanordamaran-12,20-dione-3-O- ⁇ -L-ramnopyranosyl- (1 ⁇ 2)-[ ⁇ -L -Ramnopyranosyl- (1 ⁇ 3)]- ⁇ -D-glucopyranoside (Compound 13).
  • Example 3-14 3 ⁇ -hydroxy-22,23,24,25,26,27-hexanordamaran-12,20-dione-3-O- ⁇ -L-ramnopyranosyl- (1 ⁇ 2)-[ ⁇ -L -Ramnopyranosyl- (1 ⁇ 3)]-[ ⁇ -L-ramnopyranosyl- (1 ⁇ 6)]- ⁇ -D-glucopyranoside (Compound 14).
  • Example 3-15 3 ⁇ , 20 (S), 25-trihydroxydamaran-12,23-dione-3-O- ⁇ -L-ramnopyranosyl- (1 ⁇ 2)-[ ⁇ -L-ramnopyranosyl- (1 ⁇ 3)]-[ ⁇ -L-ramnopyranosyl- (1 ⁇ 6)]- ⁇ -D-glucopyranoside (Compound 15).
  • Example 3-16 3 ⁇ -hydroxydamar-20,24-diene-12,23-dione-3-O- ⁇ -L-ramnopyranosyl- (1 ⁇ 2)-[ ⁇ -L-ramnopyranosyl- (1 ⁇ 3 )]-[ ⁇ -L-lamnopyranosyl- (1 ⁇ 6)]- ⁇ -D-glucopyranoside (Compound 16).
  • Example 3-17 3 ⁇ , 20 (S) , 25- Trihydroxydamar -23 (E) -en-12-one-3-O- ⁇ -L-ramnopyranosyl- (1 ⁇ 2)-[ ⁇ -L-ramnopyranosyl- (1 ⁇ 3)]- ⁇ -D- Glucopyranoside (Compound 17).
  • Example 3-18 3 ⁇ , 20 (S), 25-trihydroxydamar-23 (E) -en-12-one-3-O- ⁇ -L-lamnopyranosyl- (1 ⁇ 2)-[ ⁇ -L-lamno Pyranosyl- (1 ⁇ 3)]-[ ⁇ -L-ramnopyranosyl- (1 ⁇ 6)]- ⁇ -D-glucopyranoside (Compound 18).
  • myoblast C2C12 mouse skeletal muscle cell
  • adipocyte 3T3 -L1 cells were used.
  • the C2C12 cells and 3T3-L1 cells were DMEM (Dulbecco's modified Eagle's medium, Gibco) containing 10% fetal bovine serum (FBS), 100 U / mL penicillin, and 100 ⁇ g / mL streptomycin. ) The medium was cultured in a 37 ° C, 5% CO 2 incubator.
  • MTT (3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide) assay was performed to confirm the cytotoxicity of the compound isolated from the Ginosthema rongipes VK1 of the present invention.
  • C2C12 cells or 3T3-L1 cells cultured in Example 4 were dispensed in 96-well plates and cultured for 24 hours.
  • Compounds 1-19 isolated in Example 2 were treated to 40 ⁇ M and further cultured for 24 hours. .
  • 20 ⁇ l of 2 mg / ml MTT solution was added to each well and reacted for 4 hours.
  • the formed formazan precipitate was dissolved with 100 ⁇ l of DMSO (dimethyl sulfoxide) and dissolved at 550 nm. Absorbance was measured, and the results are shown in FIGS. 1 (A) and 1 (B).
  • the concentration of the compound 19 is treated to be 10 ⁇ M, 20 ⁇ M, and 30 ⁇ M to confirm whether or not it is cytotoxic, and the results are shown in FIGS. 1 (C) and 1 (C). Shown.
  • AMPK activation promotes muscle regeneration in the muscle regeneration process.
  • AMPK is phosphorylated and activated in skeletal muscle, it promotes oxidation and sugar absorption of fatty acids, and activated AMPK phosphorylates and deactivates acetyl-CoA carboxylase (ACC), an enzyme required for fatty acid synthesis. Increases fatty acid oxidation.
  • ACC acetyl-CoA carboxylase
  • the C2C12 cells cultured in Example 4 were dispensed into a 6-well plate and cultured until the cells were about 70-80% full.
  • the culture medium was removed and 2% horse serum (Gibco) was added to DMEM medium (differentiation medium) and cultured to induce differentiation into root canal cells. At this time, it was replaced with a new medium for differentiation every two days until the root canal cells are produced.
  • Example 2 During the process of separating the compound of Example 2 into the differentiated myotube, 70% [v / v] ethanol was added to the SP70 resin after extracting the bronchiose bronchipes VK1 extract, and 30% [v / v] ethanol was used. After washing the resin, the 95% [v / v] ethanol fraction obtained by eluting with 95% [v / v] ethanol was 10 ⁇ g / ml, 20 ⁇ g / ml, 40 ⁇ g / ml, in Example 2 Compounds 1-18 treated with 20 ⁇ M, compound 19 treated with 10 ⁇ M, and incubated for 30-60 minutes.
  • Aicar (5-aminoimidazole-4-carboxamide ribonucleotide), known as an AMPK activator, was treated as a positive control to secure cells.
  • Western blot was performed using the obtained cells.
  • the cells treated with each sample are collected, washed with phosphat buffered saline (PBS), and cell lysis buffer (50 mM Tris-HCl (pH 7.6), 120 mM NaCl, 1 mM EDTA, 0.5%. NP-40, 50 mM NaF) was added to lyse the cells and centrifuged to obtain proteins in the supernatant. The obtained supernatant protein was separated through SDS-PAGE (sodium dodecyl polyacrylamide gel electrophoresis) and transferred to a polyvinylidenefluoride (PVDF) membrane.
  • PVDF polyvinylidenefluoride
  • Example 2 of (A) As shown in FIG. 2, when the 95% [v / v] ethanol fraction of Example 2 of (A) was treated by concentration, it was confirmed that phosphorylation of AMPK increased according to the concentration of the fraction treatment. In addition, when the compounds 1 to 19 of Example 2 of (B) were treated, as shown in the results of quantifying the protein expression band (the upper figure of (B)) and the protein expression band (the lower graph of (B)). , It was confirmed that phosphorylation of AMPK and ACC in cells treated with each compound is increased in comparison with cells that have not been treated with anything.
  • the Ginosthema rongipepes VK1 extract of the present invention and the compounds isolated therefrom induce and activate AMPK phosphorylation in muscle-associated cells in which differentiation from myoblasts to myotubes is induced, and ACC is inactivated to regenerate muscle It was found that it promotes.
  • the effect of promoting myoblast proliferation by using the MTT assay and the water-soluble tetrazolium salt-1 (WST-1) assay to confirm the muscle regeneration effect of the ginosthema rongipepes VK1 extract of the present invention or compounds isolated therefrom was confirmed.
  • the compounds 1 to 18 of Example 2 were treated to be 20 ⁇ M, compound 19 was 10 ⁇ M, and cultured for 48 hours, followed by MTT assay or WST-1 assay. Did.
  • Zinosthema rongipepes VK1 extract of the present invention or the compounds isolated therefrom promotes the proliferation of myoblasts, and sufficiently predicts that muscle regeneration can be induced by promoting the proliferation of these myoblasts.
  • the Zinosthema rongipepes VK1 extract of the present invention or the compounds isolated therefrom promotes the proliferation of myoblasts, and sufficiently predicts that muscle regeneration can be induced by promoting the proliferation of these myoblasts.
  • Example 7-2 Myoblast Confirm the effect of promoting specific growth
  • the above-described method was performed using cancer cell lines MCF-7 and MDA-MB cells in order to confirm whether the effect of promoting the myoblast proliferation of the genostema rongipepes VK1 extract or the compound isolated therefrom occurs specifically on myoblasts. It was performed in the same manner as in the MTT assay method of Example 7-1. At this time, the 95% [v / v] ethanol fraction of Example 2 was treated with 10 ⁇ g / ml, and Compound 1 of Example 2 was treated with 10 ⁇ M, followed by cultivation for 48 hours. It is shown in FIG. 4.
  • BrdU 5-bromo-2′-deoxyuridine
  • C2C12 cells cultured in Example 4 are dispensed into a cell culture dish containing a cover slide, and 20 ⁇ g / ml of the 95% [v / v] ethanol fraction of Example 2 or the above Example 20 ⁇ M of compound 1 of 2 was treated and cultured for 8 hours. At this time, DMSO-treated cells were used as a control. After incubation, 50 ⁇ M of BrdU labeling solution (Sigma-Aldrich, USA) was treated at 37 ° C. for 2 hours and cells were washed with PBS after 2 hours.
  • BrdU labeling solution Sigma-Aldrich, USA
  • the washed cells were treated with 4% formaldehyde at room temperature for 15 minutes to fix the cells, washed with PBS, added Triton X-100 buffer solution and treated at room temperature for 20 minutes to increase cell permeability, and then 2N HCl The solution was treated on ice for 20 minutes. The 2N HCl solution was removed and phosphate / citric acid buffer solution (pH 7.4) was treated at room temperature for 10 minutes, cells were washed with Triton X-100 buffer solution, and the anti-BrdU antibody was treated overnight.
  • the anti-BrdU antibody was removed, and the secondary antibody bound with fluorescein isothiocyanate (FITC) capable of recognizing the anti-BrdU antibody was treated for 1 hour, and then DAPI (4 ', 6-diamidino-2-phenylindole) Cell nuclei were stained by treatment. After fixing the cell-attached cover slide with a mounting solution, cell images were observed with a fluorescence microscope (Olympus ix70 Fluorescence Microscope, Olympus Corporation, JPN), and the results are shown in FIG. 5 (A).
  • FITC fluorescein isothiocyanate
  • Example 4 the C2C12 cells cultured in Example 4 were dispensed into a 36 mm cell culture dish, and the compound 1 of Example 2 was treated to 5 ⁇ M, 10 ⁇ M, 20 ⁇ M and cultured for 8 hours. At this time, cells treated with nothing were used as a normal group, and cells treated with DMSO were used as a control. After incubation, 50 ⁇ M of BrdU labeling solution was treated at 37 ° C. for 2 hours, and after 2 hours, cells were washed with PBS and cells were collected using trypsin.
  • Example 9-1 Confirm the effect of promoting sugar absorption
  • glucose uptake of adipocytes of the ginosthema rongipes VK1 extract having the AMPK activation effect of the present invention or a compound isolated therefrom (glucose uptake) It was confirmed whether to promote.
  • 2-NBDG (2- (N- (7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino) -2-deoxyglucose
  • a fluorescent analogue of glucose (Invtrogen, USA ) 50 ⁇ M was treated in all wells, and 100 nM insulin (positive control) or 20 ⁇ M of compounds 1-18 of Example 2 and 10 ⁇ M of compound 19 were treated in each well and incubated for 1 hour.
  • DMSO treatment was used as a control.
  • genostema rongipepes VK1 extract of the present invention or a compound isolated therefrom exhibits the effect of treating obesity, diabetes or metabolic syndrome by promoting sugar absorption of fat cells.
  • Metabolic syndrome refers to insulin resistance in which glucose absorption into peripheral tissues is resistant. In obesity and metabolic syndrome, weight loss is known to reduce insulin resistance and promote glucose uptake into storage tissue. In addition, promoting the absorption of glucose into peripheral tissues is an essential mechanism of action for removing glucose in the blood, which is a prerequisite for the management of type 2 diabetes.
  • Glucose transporter 4 (GLUT4) is a major glucose transporter that is activated in response to insulin, and the translocation of adipocytes to the plasma membrane is essential.
  • the plasma membrane protein of the adipocytes was separated and subjected to Western blot in order to confirm whether the effect of translocation of GLUT4 in adipocytes of the genothema longifes VK1 extract of the present invention or the compound isolated therefrom was performed.
  • the protein concentration of the obtained plasma membrane fraction was quantified using a BCA protein analysis kit (Bio-Rad Laboratories, Inc., USA). After the protein quantification result was used to make the amount of protein in the plasma membrane fraction the same, it was separated by size through 12% SDS-PAGE, and then transferred to the PVDF membrane. The membrane was blocked with a blocking solution containing 5% skim milk, and then the antibody against GLUT4 was treated for 2 hours. At this time, the antibody was treated with Na + / K + ATPase ⁇ 1 as a loading control. After 2 hours, the secondary antibody for each antibody was treated, and protein expression was confirmed using ECL (Amersham), and the results are shown in FIG. 7.
  • the genostema rongipepes VK1 extract of the present invention or a compound isolated therefrom promotes absorption of glucose through the potential of GLUT4, thereby reducing insulin resistance in obesity, diabetes or metabolic syndrome, thereby reducing the sugar content in the blood. By doing this, it was found that obesity, diabetes or metabolic syndrome can be treated.
  • the extract of Ginosthema longifose VK1 the appropriate amount of vitamin mixture, 70 ⁇ g of vitamin A acetate, 1.0 mg of vitamin E, 0.13 mg of vitamin B1, 0.15 mg of vitamin B2, 0.5 mg of vitamin B6, 0.2 ⁇ g of vitamin B12, vitamin C 10 mg, 10 ⁇ g of biotin, 1.7 mg of nicotinamide, 50 ⁇ g of folic acid, 0.5 mg of pantothenate, appropriate amount of inorganic mixture, 1.75 mg of ferrous sulfate, 0.82 mg of zinc oxide, 25.3 mg of magnesium carbonate, 15 mg of potassium phosphate, 2nd 55 mg of calcium phosphate, 90 mg of potassium citrate, 100 mg of calcium carbonate, and 24.8 mg of magnesium chloride were mixed into granules, but can be prepared by modifying them into various formulations depending on the application.
  • the composition ratio of the above-mentioned vitamin and mineral mixture may be arbitrarily modified, and the above ingredients may be mixed and prepared

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Abstract

La présente invention concerne une composition pour la prévention ou le traitement de maladies liées à l'AMPK, comprenant un extrait de Gynostemma longipes VK1 ou un composé isolé à partir de celui-ci en tant qu'ingrédient actif. On a découvert que l'extrait de Gynotarma longipes VK1 ou le composé isolé à partir de celui-ci active l'AMPK impliquée dans diverses activités physiologiques telles que le transport de glucose, la synthèse d'acides gras et la régénération musculaire, et favorise la prolifération des myoblastes ainsi que l'absorption de glucose dans les adipocytes. Par conséquent, on s'attend à ce que la composition de la présente invention puisse être utilisée pour développer un agent thérapeutique ou un médicament vétérinaire, un aliment fonctionnel améliorant la santé ou un aliment pour animaux pour des maladies liées à l'AMPK, telles que des maladies musculaires, l'obésité, le diabète et le syndrome métabolique, avec d'excellents effets.
PCT/KR2019/008089 2018-10-17 2019-07-02 Composition pour la prévention et le traitement de maladies liées à l'ampk, comprenant un extrait de gynostemma longipes vk1 ou un composé isolé à partir de celui-ci en tant qu'ingrédient actif WO2020080641A1 (fr)

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KR10-2018-0123598 2018-10-17
KR1020180123598A KR102143342B1 (ko) 2018-10-17 2018-10-17 지노스테마 론기페스 vk1 추출물 또는 이로부터 분리한 화합물을 유효성분으로 포함하는 ampk 관련 질환 예방 또는 치료용 조성물

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WO2020080641A1 true WO2020080641A1 (fr) 2020-04-23

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CN115368426B (zh) * 2021-08-17 2023-07-18 遵义医科大学 三萜类化合物及其制备方法与平喘用途

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