WO2018221922A1 - Composition pour la prévention et le traitement de maladies musculaires, contenant un extrait de coptidis rhizoma, et son utilisation - Google Patents

Composition pour la prévention et le traitement de maladies musculaires, contenant un extrait de coptidis rhizoma, et son utilisation Download PDF

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WO2018221922A1
WO2018221922A1 PCT/KR2018/006065 KR2018006065W WO2018221922A1 WO 2018221922 A1 WO2018221922 A1 WO 2018221922A1 KR 2018006065 W KR2018006065 W KR 2018006065W WO 2018221922 A1 WO2018221922 A1 WO 2018221922A1
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cancer
muscle
ethanol
water
mixed solvent
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PCT/KR2018/006065
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Korean (ko)
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류재하
이혜진
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숙명여자대학교산학협력단
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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • 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
    • A61K31/66—Phosphorus compounds
    • 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)
    • A61K36/71—Ranunculaceae (Buttercup family), e.g. larkspur, hepatica, hydrastis, columbine or goldenseal
    • A61K36/718—Coptis (goldthread)
    • 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

Definitions

  • the present invention relates to a composition for the prophylaxis and treatment of muscle-related diseases containing the extract of the yellow lotus and its use.
  • Muscle regeneration includes atony, muscular atrophy, muscular dystrophy, muscle degeneration, myopathy, muscular atrophy, ataxia, cachexia and sarcopenia It has been noted for the purpose of overcoming skeletal muscle degenerative diseases such as. ( J Cachexia Sarcopenia Muscle. 2015 , 6 , 197)
  • muscle loss can also be caused by the progression of aging and various chronic diseases. As aging progresses, some of the newly created skeletal muscles are replaced with fibrous tissues, resulting in sarcopenia, which reduces the body's skeletal muscle mass and strength. In addition, muscle loss also occurs in chronic diseases in which the incidence increases with age, such as hypertension, impaired glucose tolerance, and diabetes, obesity, dyslipidemia, atherosclerosis, and cardiovascular disease. ( Pharmacol Res. 2015 , 99 , 86).
  • muscle In muscle, assimilation and catabolism are balanced and regulate muscle production. In this context, many biological signaling processes are regulated in muscle cells. If the signaling response that induces synthesis is activated rather than the degradation of muscle proteins, the synthesis of muscle proteins is increased, leading to increased muscle size (hypertrophy) or increased muscle fiber (hyperplasia) resulting in excessive muscle production ( Br) J Pharmacol . 2008, 154 , 557).
  • Muscle induction factors induce muscle protein synthesis by activating the phosphatidylinositol-3 kinase (PI3K) / Akt pathway in muscle cells and thereby phosphorylating downstream proteins.
  • PI3K phosphatidylinositol-3 kinase
  • the activity of mTOR (mammalian target of rapamycin) by PI3K / Akt signaling is recognized as a central growth signaling mechanism that integrates various growth signals in cells.
  • Activation of mTOR activates two subtargets, 4EBP1 (4E-binding protein) and p70S6K (phosphorylated 70-kDa ribosomal S6 kinase), which induce muscle protein synthesis to increase muscle mass ( Br J Pharmacol. 2008, 154 , 557; Mech Ageing Dev. 2010, 131, 202).
  • myogenic cell differentiation and muscle formation are regulated by a variety of factors (5. Cell Mol Life Sci . 2013 , 70 , 4117).
  • myoD initiates the expression of specific genes related to muscle differentiation, inducing mesenchymal stem cells to differentiate into myoblasts.
  • Myogenin and myosin heavy chain (MHC) which are regulated by MyoD, induce fusion of myoblasts, leading to the formation of myotubes and muscle fibers.
  • the muscle fibers formed through this process are bundled to finally form muscles ( Cell Mol Life Sci. 2013 , 70 , 4117; 6. Brain Dev. 2013 , 35, 349)
  • myo D myogenic factor
  • myogenin myogenin
  • Mrf-4 myogenin and Mrf-4.
  • MyoD and Myf-5 are transcription factors specifically expressed in myogenic lineage and play an important role in initiating myoblast differentiation.
  • Myo D induces the expression of myogenic proteins such as MHC and myogenin through binding to non-muscular specific factors such as E protein, Mef-2 family proteins and transcriptional cofactors.
  • Coptis japonica Makino Is a perennial herb, Coptis japonica Makino, and Coptis kinensis. chinensis ), etc., is used as a medicinal plant, and its constituents are berberine, magnoflorine, coptisine, consoine, palmin, and yate. Ingredients such as jateorrhizine are known, and anti-cancer activity and anti-microbial action have been reported (Humyang Metabolism Dictionary, Younglimsa, 1998 , 490-493).
  • the inventors also note that the methanol extract of rhubarb and six compounds isolated therefrom, namely magnoflorine, jatrohizzine, epiberberine, 8-hydroxy-7,8-dihydrocorptycin (8-hydroxy-7,8-dihydrocoptisine), palmatine and berberine have been shown to activate p38 MAPK and Akt signaling systems that are activated during myoblast differentiation ( Heyjin Lee, et al, Isoquinoline alkaloids from Coptis japonica stimulate the myoblast differentiation via p38 MAP- kinase and Akt signaling pathway, Bioorg.Med. Chem. Lett., 27 (6), 1401-1404 (2017), 2017-3-15 .
  • the inventors of the present invention show a stronger therapeutic effect than conventionally known rhubarb extracts and compounds isolated therefrom in order to increase the possibility of development as a new product or a health functional food with stronger therapeutic activity in terms of the therapeutic effect of more muscle-related diseases.
  • (1) to examine the degree of MHC expression in C2C12 cells differentiated through Western blotting analysis for (1) myoblast differentiation efficacy evaluation (Experimental Example 1) of the extract of the present invention The sulfur fraction butanol fraction of the present invention was identified as the most potent MHC expression composition, and (2) the effect of MHC on the myoblast differentiation (Experimental Example 2) increased the expression of MHC by the rhubarb extract and the cylindrical form (cylinder).
  • the present invention aims to develop a natural product therapeutic agent that is more excellent for improving muscle diseases.
  • An object of the present invention to provide a pharmaceutical composition for the prevention and treatment of muscle-related diseases containing (a) crude extracts available in a solvent mixture of water and ethanol or (b) purified products separated therefrom as an active ingredient.
  • the present invention provides a dietary supplement for the prevention and improvement of muscle-related diseases containing (a) crude extracts available in a mixed solvent of water and ethanol or (b) purified products separated therefrom as an active ingredient.
  • Humilies as defined herein are characterized by comprising rhizomes, stems, outpost leaves, preferably rhizomes or outposts of homogeneous plants such as Coptis japonica Makino, Coptis chinensis , etc. .
  • the crude extracts available for (a) water and ethanol mixed solvents as defined herein are 1 to 100% (v / v) water and ethanol mixed solvents, more preferably 30 to 100% (v / v) water and ethanol mixed Characterized in that the extract is soluble in a solvent, more preferably 70-100% (v / v) water and ethanol mixed solvent.
  • (b) purified from the crude extracts soluble in the mixed solvent of water and ethanol is a non-polar purified from the crude extracts of the present invention only soluble in hexane, ethyl ether, methylene chloride, chloroform, or ethyl acetate solvent. Extract fractions soluble in the solvent selected from the remaining water, methanol, ethanol, butanol or a mixed solvent thereof after removing the extract fractions soluble in the solvent,
  • ethyl ether solvent is added and fractionated in a conventional process to remove the ethyl ether solvent fraction to obtain an acid treated layer.
  • CJBU purified product isolated from the crude extract available in a mixture of water and ethanol containing a) alkaloid;
  • the total weight of purified water (CJBU) (100 parts by weight, w / w), 0.10 to 0.50 parts by weight of magnoflorine, 0.20 to 0.50 parts by weight of jatrohizzine, epi-berberine ( epi-berberine 0.80 to 2.0 parts by weight, 1.80 to 3.6 parts by weight of 8-hydroxy-7,8-dihydrocoptisine, 2.70 to 4.7 parts by weight of palmatine , 10.40 to 15.40 parts by weight of berberine, even more preferably, 0.20 to 0.40 parts by weight of magnoflorine, 0.25 to 0.45 parts by weight of jatrohizzine, epi-berberine 1.00 to 1.80 parts by weight, 8-hydroxy-7,8-dihydrocoptisine 2.00 to 2.80 parts by weight, palmatine 3.00 to 4.20 parts by weight, berberine 11.40 To 13.40 parts by weight.
  • CJBU purified water
  • Acid treatment in the first step of the production method is a strong acid, such as hydrochloric acid, sulfuric acid in the range of 0.5 M to 10 M, preferably in the range of 1 M to 5 M; Or a weak acid aqueous solution such as acetic acid and formic acid.
  • a strong acid such as hydrochloric acid, sulfuric acid in the range of 0.5 M to 10 M, preferably in the range of 1 M to 5 M; Or a weak acid aqueous solution such as acetic acid and formic acid.
  • the alkaline solution is neutralized by adjusting the pH to 7 to 14, preferably 8 to 11 by treating a strong base such as sodium chloride or sodium hydroxide or a weak base such as aqueous ammonia or sodium bicarbonate. It is done.
  • a strong base such as sodium chloride or sodium hydroxide or a weak base such as aqueous ammonia or sodium bicarbonate. It is done.
  • Muscle-related disorders as defined herein include atony, muscular atrophy, muscular dystrophy, muscle degeneration, myopathy, muscular atrophy, ataxia, cachexia and sarcopenia At least one muscle disease selected from the group consisting of muscle specific diseases caused by senile muscular atrophy or cancer, more specifically, muscular atrophy due to senile muscular atrophy or cancer, muscular dystrophy, Muscle degeneration, myopathy, muscular dystrophy, ataxia, ataxia, cachexia, sarcopenia and muscular loss.
  • the present invention also provides an agent for the prevention and treatment of muscle-related diseases caused by cancer diseases containing (a) crude extracts available in a mixed solvent of water and ethanol, or (b) purified products separated therefrom. .
  • the present invention provides a cachexia therapeutic agent containing (a) crude extracts soluble in a mixed solvent of water and ethanol or (b) purified products separated therefrom as an active ingredient.
  • the present invention is a prevention of muscle-related diseases caused by cancer disease (a) crude extracts soluble in a mixed solvent of water and ethanol or (b) purified from this and a combination of conventional anticancer agents as an active ingredient And therapeutic pharmaceutical compositions, anticancer adjuvant or health functional food.
  • Existing anticancer agents as defined herein include cyclophosphamide, methotrexate, 5-fluorouracil, doxorubicin, mustine, vicristine, procarba Procarbazine, prednisolone, bleomycin, vinblastine, dacarbazine, etoposide, cisplatin, epirubicin, cispool Cisplatin, capecitabine, oxaliplatin and the like.
  • lung cancer non-small cell Sex lung cancer, small cell lung cancer
  • thymic cancer kidney cancer, bladder cancer, prostate cancer, testicular cancer, ovarian cancer, cervical cancer, sarcoma, gastrointestinal stromal tumor (
  • Extracts of the present invention can be obtained by the preparation method as follows.
  • (A) crude extract soluble in water and ethanol mixed solvent of the present invention or (b) purified product separated therefrom may be prepared as follows.
  • the crude extract of (a) water and ethanol mixed solvent of the present invention of the present invention (1) after washing and rinsing the dried yellow lotus, water containing purified water, methanol, ethanol, butanol and the like having a lower carbon number of 1 to 4
  • a solvent selected from an alcohol or a mixed solvent thereof, preferably water and an ethanol mixed solvent, more preferably 30 to 100% ethanol is mixed several times and then at a temperature of 30 to 150 ⁇ , preferably 50 to 100 ⁇ . Obtained by repeatedly performing about 1 to 20 times, preferably 2 to 10 times, ultrasonic extraction, hot water extraction, room temperature extraction or reflux extraction, preferably reflux extraction for 30 minutes to 48 hours, preferably 1 hour to 12 hours.
  • the extract can be filtered, concentrated under reduced pressure, and dried to obtain the crude extract of the present invention.
  • the purified product separated from the crude extract available in the mixed solvent of water and ethanol of the present invention (a) after the acid treatment of the crude extract of the present application, added with an ethyl ether solvent and fractionated in a conventional process Removing the ethyl ether solvent fraction to obtain an acid treatment layer; (b) a second step of removing the ethyl ether solvent fraction of step 1 and neutralizing by adding an alkaline solution to the remaining acid treated layer to obtain a neutralized filtrate; (c) a third step of further fractionating the neutralized filtrate of step 2 by using a chloroform or ethyl acetate solvent in a conventional process to remove the chloroform or ethyl acetate solvent soluble fraction to obtain further purified product; (d) a large amount of quaternary isoquinoline (isoquinoline) obtained through the fourth step process of adding butanol to the further purified product to obtain a purified product separated from the crude extract available in the mixed solvent of water
  • the present inventors performed Western blotting to evaluate the differentiation-promoting effect of (1) myoD transcriptional activity measuring the myoD transcriptional activity of the sample extract obtained by the above production method, differentiated C2C12 cells MHC expression was increased most strongly in (Experimental Example 1), (2) Effect on myoblast differentiation (Experimental Example 2) through the induction of myogenic cell differentiation through the experiment of cylindrical type ( The number of cylinder-shaped multinucleated myotubes was increased, and protein expression of MHC and myoD was increased and myostatin expression was suppressed.
  • the present invention is for the prevention and treatment of muscle-related diseases containing (a) crude extracts soluble in a solvent mixture of water and ethanol obtained by the above method or (b) purified products separated therefrom as an active ingredient.
  • composition of the present invention comprises 0.01 to 99% by weight of the extract or purified based on the total weight of the composition.
  • composition as described above is not necessarily limited thereto, and may vary according to the condition of the patient and the type and extent of the disease.
  • compositions comprising extracts or tablets of the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions.
  • compositions comprising extracts or tablets according to the present invention may be prepared in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, oral formulations, external preparations, suppositories, and sterile injectable solutions, respectively.
  • Carriers, excipients, and diluents that may be formulated in a form may include, but are not limited to, 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, magnesium stearate and mineral oil.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may contain at least one or more excipients in at least one excipient, cotton, starch, calcium carbonate, and sucrose. It is prepared by mixing sucrose or lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium styrate talc are also used.
  • Oral liquid preparations include suspending agents, liquid solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories.
  • the non-aqueous solvent and suspending agent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used.
  • As the base of the suppository witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
  • Preferred dosages of the extracts or tablets of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art.
  • the extract is preferably administered at 0.01 mg / kg to 10 g / kg, preferably 1 mg / kg to 1 g / kg per day. Administration may be administered once a day or may be divided several times. Therefore, the above dosage does not limit the scope of the present invention in any aspect.
  • composition of the present invention can be administered to mammals such as mice, live mice, livestock, humans, etc. by various routes. All modes of administration can be anticipated, for example, by oral and rectal or intravenous methods.
  • the present invention also provides a treatment for senile muscular dystrophy or cachexia containing (a) crude extracts soluble in a mixed solvent of water and ethanol, or (b) purified products separated therefrom, as an active ingredient. Or to improve the weight change or appetite recovery in combination therapy of cachexia, provides a combination therapy using the composition of the present invention rather than administration of anticancer agent alone in cachexia treatment.
  • the present invention also provides a treatment for treating patients with muscle-related diseases, comprising administering (a) crude extracts available in a mixed solvent of water and ethanol or tablets isolated therefrom to muscle-related diseases patients. Provide a method.
  • the present invention also provides the use of crude extracts (a) crude extracts soluble in water and ethanol mixed solvents or (b) purified from them for the preparation of a medicament for the treatment of muscle-related diseases.
  • the present invention provides a health functional food for the prevention and improvement of muscle-related diseases containing (a) crude extracts soluble in a mixed solvent of water and ethanol or (b) purified products separated therefrom as an active ingredient. do.
  • health functional food refers to physical or mental functionality by adding the extract of the present invention to a conventional specimen for preventing or ameliorating a target disease in a human body or a mammal according to Korean Health Functional Food Act No. 6723. It includes a functional food to improve the functionality such as.
  • the extract comprises 0.01 to 95% by weight, preferably 1 to 80% by weight relative to the total weight of the composition.
  • composition of the present invention can be used as a major component or additive and adjuvant in the manufacture of various dietary supplements and dietary supplements for the purpose of preventing or treating muscle-related diseases.
  • a health functional food in the form of a pharmaceutical dosage form such as powders, granules, tablets, capsules, pills, suspensions, emulsions, syrups or tea bags, leaching tea, health drinks for the purpose of preventing and improving muscle-related diseases. Manufacturing and processing are possible.
  • composition of the present invention may be a health food for the purpose of treating and ameliorating muscle-related diseases.
  • a health functional food in the form of a pharmaceutical dosage form such as powders, granules, tablets, capsules, pills, suspensions, emulsions, syrups or tea bags, leaching tea, health drinks for the purpose of preventing and improving muscle-related diseases. Manufacturing and processing are possible.
  • the present invention also provides a dietary supplement for the prevention and improvement of muscle-related diseases containing (a) crude extracts available in a mixed solvent of water and ethanol, or (b) purified products separated therefrom as active ingredients. .
  • the functional beverage composition of the present invention is not particularly limited to other ingredients except for containing the extracts or purified products as essential ingredients in the indicated proportions, and may contain various flavors or natural carbohydrates as additional ingredients, such as ordinary drinks.
  • natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose and the like; And conventional sugars such as polysaccharides such as dextrin, cyclodextrin, and sugar alcohols such as xylitol, sorbitol, and erythritol.
  • natural flavoring agents such as, tauumatin, stevia extract (e.g., Rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used.
  • the proportion of the natural carbohydrate is generally about 1-20 g, preferably about 5-12 g per 100 ml of the composition of the present invention.
  • the present invention also provides a food or food additive for the prevention and improvement of muscle-related diseases containing (a) crude extracts soluble in a mixed solvent of water and ethanol or (b) purified products separated therefrom as an active ingredient. do.
  • the extract or purified product may be added as it is or used with other food or food ingredients, and may be appropriately used according to a conventional method.
  • the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes, etc., includes all of the health food in the conventional sense.
  • the composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring and neutralizing agents (such as cheese, chocolate), pectic acid and salts thereof, alginic acid and its Salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like.
  • the compositions of the present invention may also contain pulp for the production of natural fruit juices and fruit juice beverages and vegetable beverages. These components can be used independently or in combination. The proportion of such additives is not so critical but is generally selected from the range of 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.
  • the extract or purified product of the present invention may be added to food or beverages for the purpose of preventing muscle-related diseases.
  • the amount of the extract in the food or beverage can be added in 0.01 to 15% by weight of the total food weight
  • the health beverage composition is added in a ratio of 0.02 to 5 g, preferably 0.3 to 1 g based on 100 ml Can be.
  • MHC expression in differentiated C2C12 cells as a result of Western blotting to evaluate the differentiation efficacy of myoblasts measuring myoD transcriptional activity of the rhubarb extract or purified product of the present invention was most strongly increased, and (2) the number of cylinder-shaped multinucleated myotubes expressing MHC through myoblast differentiation was investigated through experiments on the effect on myoblast differentiation. Increased protein expression of MHC and myoD and inhibited myostatin expression. It was also confirmed that myoD transcriptional activity was also significantly increased (3) to induce myoblast differentiation by activating p38MAPK and Akt signaling processes, and (4) the effect of the sample was independent of cell viability.
  • the sample of the present invention recovers the expression of MHC and myoD, confirming that it has muscle regeneration ability, The composition was found to be useful as a pharmaceutical composition or health functional food for the prevention and treatment of muscle diseases.
  • the above experimental results indicate that the sample of the present invention is atony, muscular atrophy, muscular dystrophy, muscle degeneration, muscular dystrophy, muscular dystrophy, ataxia, cachexia and senile muscular dystrophy.
  • muscle diseases such as (sarcopenia)
  • the composition is useful as a pharmaceutical composition for preventing and treating muscle diseases, health functional foods and health supplements.
  • Figure 1 is an HPLC chromatogram of the butanol fraction of the present invention, wherein 1: Magnoflorine, 2: Jatrohizzine, 3: Epi-berberine, 4: 8-hydroxy-7,8-dihydrocopticine ( 8-hydroxy-7,8-dihydrocopisine, 5: palmatine, 6: berberine respectively);
  • Figure 2 is a diagram showing the cytotoxicity to C2C12 cells of the butanol fraction of the present invention.
  • Example 2 The dried ethanol extract obtained in Example 1 was suspended in 900 mL of distilled water 2M hydrochloric acid aqueous solution, fractionated with diethyl ether according to a conventional fractionation method in the art, and the remaining hydrochloric acid layer was separated using 20% ammonia water.
  • HPLC conditions performed for the component analysis of the sample of the Example are as follows.
  • C2C12 cells which are mouse myocytes, were treated with ethanol extract, butanol fractionation layer, and chloroform fractionation layer, respectively.
  • MHC expression a marker of differentiation, was used to evaluate the differentiation-promoting effect of myoblasts ( Bioorg Med Chem Lett. 2017, 15, 27).
  • Each sample was added to obtain cell lysates in differentiated cells and the protein expression level of MHC was assessed by western blot.
  • C2C12 myoblasts (Cat # CRL-1771, ATCC) were treated with differentiation medium (2% horse serum-containing) by treatment of ethanol extract, butanol fraction, and chloroform fraction (1, 10, 100 ng / mL) of nasturtium. Differentiation was induced by treatment on DMEM, Cat # 11965-084, Gibco) for 3 days.
  • myoD-responsive gene and luciferase fused to C2C12 cells which are mouse myocytes (MyoD-responsive reporter 4RTK-luc) was inserted, and myoD transcriptional activity was analyzed by measuring the change in luciferase activity according to the extract treatment to evaluate the differentiation promoting effect of myoblasts ( J. Biol. Chem 2004, 279 , 30966).
  • MyoD transcriptional activity of a reporter vector MyoD-responsive reporter 4RTK-luciferase construct (provided by Prof. Kyu-Woon Bae, College of Pharmacy, Sookmyung Women's University)) containing four E-box sites fused to a thymidine kinase promoter was used to evaluate.
  • a myoD expression vector and a reporter vector (MyoD-responsive reporter 4RTK-luc) in mouse source cell line C2C12 considered as a model cell for skeletal muscle growth study (Sookmyung Women's University College of Pharmacy) Prof. Kyu-Woon Bae) was transfected ( PLoS One . 2017 , 12 , e0175271). Twenty four hours after transfection, cells were treated with rhubarb extract (1, 10, 100 ng / mL) and further incubated for 24 hours. The cultured cells were washed twice with phosphate buffered saline solution, lysed with reporter lysis buffer (Cat #.
  • the source cells were differentiated in the differentiation medium to which each sample was added, and immunofluorescence staining was performed.
  • Each differentiation medium was removed, washed twice with phosphate buffered saline, and fixed for 20 minutes with 4% paraformaldehyde. Again washed with phosphate buffered saline twice, and treated with 0.1% tritonX-100 for 20 minutes. After washing twice with phosphate buffered saline, blocking in 5% horse serum solution, mouse anti-MHC (MAB4470, R & D systems) was added and reacted at 4 ° C for 12 hours.
  • MAB4470 mouse anti-MHC
  • MHC expression was analyzed using Alexa Flouor 568-bound secondary anti-mouse (A-21144, MicoProbes) and DAPI (D9542, Sigma). Immunfluorescence results of MHC-positive myotubes were visualized in red and DAPI-labeled nuclei were visualized in blue.
  • Table 2 shows that when luciferase activity in control cells is set to 1, 2.6 times when treated with 1 ng / mL nasal fraction, but 3.1 times when treated with 10 ng / mL, and 100 ng / mL were treated. The time was increased by 1.5-fold, respectively, and it was confirmed that the rye-butanol fraction increased MyoD transcriptional activity in a concentration-dependent manner.
  • Table 3 shows the effects on the MHC, myoD and myostatin expressions of L. butanol fractions.
  • Treatment of myoblasts with different concentrations of 1, 10, and 100 ng / mL was used to differentiate them into myotube fibers for 3 days.
  • the cells were harvested to obtain differentiation markers of MHC and myoD, and myostatin, a differentiation inhibitor. Protein expression was analyzed by Western blotting assay.
  • L. butanol fractions not only increase the expression of MHC and myoD in differentiated C2C12 cells, but also strongly reduce the expression of myostatin, a differentiation inhibitor. It was.
  • Akt proteinkinase B, PKB signaling system with p38 MAPK is known to be a very important mechanism for myoblast differentiation ( Mol. Biol. Cell. 2010 , 21, 2399). The method described in the literature was conducted to determine whether it affects the p38 MAPK signaling system during myoblast differentiation.
  • p38 mitogen-activated protein kinase plays an important role in MyoD activation.
  • Akt along with p38 MAPK, induces muscle-specific gene expression in muscle cells ( Int J Mol Med . 2015 , 36 , 1073) are known to play a very important role in muscle differentiation.
  • the protein concentration of the cell eluate obtained by centrifugation at 14000 rpm for 30 minutes was measured by BCA method, and 25 ⁇ g of protein extract was electrophoresed and transferred to PVDF to obtain a blot.
  • the primary rabbit-antiphosphorylated p38 MAPK (Cat # 9211, Cell Signaling Technology) or primary rabbit-antiphosphorylated Akt (Cat # 9271, Cell Signaling Technology) and its anti-rabbit secondary antibody (goat anti-rabbit IgG-HRP, sc-2004, SantaCruz) were reacted to measure the expression of phosphorylated p38 MAPK or phosphorylated Akt.
  • the primary rabbit-antigen was analyzed to analyze the expression levels of total p38 MAPK and total Akt, which is the respective loading control.
  • p38 MAPK Cat # 9212, Cell Signaling Technology
  • primary rabbit-anti Akt Cat # 9272, Cell Signaling Technology
  • the present inventors treated C2C12 cells with a butanol fraction at a concentration of 1, 10, 100 ng / mL and measured cell viability using the MTT assay.
  • Living cells are well soluble by mitochondrial reductase due to their mitochondrial function and yellow MTT (3- (4,5-dimethylthiazol-2-yl) -2-5-diphenyltetrazolium bromide) is insoluble and purple. It can be converted to the formazan reactant, which measures the intrinsic absorbance (560 nm) of this purple reactant and estimates that the higher the value, the more live cells.
  • MTT assay approximately 2 X 10 3 myoblasts (C2C12) were dispensed in 96 well plates, incubated for 24 hours, and then MTT solution (0.5 mg / mL in phosphate buffered saline, Cat # M2128, Sigma) 100 ul Add and leave for 3 hours in a 37 ° C CO 2 incubator. After removing the medium and adding 100 ul of dimethyl sulfoxide (DMSO) to each well for 5 minutes, the absorbance of each well was measured at 560 nm using a MicroMax Microplate Multimode Reader (Promega).
  • DMSO dimethyl sulfoxide
  • Cell viability (%) for butanol fraction was expressed as 100 X (absorbance-blank of control treated cells) / (control absorbance-blank), where blank is the absorbance of wells containing only DMSO without cells. Therefore, as the viability of the cell decreases, the toxicity of the sample increases.
  • myoblasts C2C12
  • DMEM differentiation medium with 2% horse serum added 24 hours later to differentiate into myotubes.
  • a new differentiation medium was replaced every 3 days.
  • Samples were cultured for 3 hours with butanol fractions at 1, 10, and 100 ng / mL concentrations in differentiated myotube cells, and then cultured with CT-26 mouse colorectal cancer cells at 20% final concentration for 3 days. Further differentiated.
  • Differentiated and sampled cells were prepared by washing with phosphate buffered saline. The prepared cells were then subjected to Western blotting to see protein expression of muscle differentiation factors and immunofluorescence staining to confirm MHC expression.
  • MHC and myoD protein expression in each cell group was analyzed by Western blotting, and when MHC expression in control (NC) root canal cells without colon cancer culture was set to 1, only muscle cell line culture was used to treat muscle loss. MHC expression was rapidly decreased by 0.6-fold in the root canal cell group, but 1.0-fold, 10-fold ng / mL was 1.2-fold, and 100-ng / mL was 1.1-fold when 1 ng / mL of nasturtium butanol fraction was treated. MHC expression levels were restored in the non-induced control root canal cell group.
  • myoD an important transcription factor for muscle differentiation
  • / mL increased 1.3-fold and 100 ng / mL increased 0.9-fold to restore myoD expression levels in the control myotubes.
  • Both MHC and myoD showed the highest expression at 10 ng / mL and the pattern of expression increase with sample concentration ( Table 6 ) .
  • MHC expression was evaluated at the level of red fluorescence in the root canal cells to which the colon cancer culture was added, to confirm the muscle regeneration ability reduced by the cancer cell line culture, and the DAPI read staining.
  • the formation of cylinder-shaped multinucleated myotubes was evaluated. Muscle differentiation was significantly inhibited by cancer cell line culture compared with control (NC) myotubes without colon cancer cell culture.
  • Treatment of butanol fractions of the USSR recovered the number of multinucleated MHC-positive cells reduced by the culture of cancer cell lines. It is demonstrated that muscle regeneration by the sample of the present invention is effectively promoted.
  • NC CT26-CM One 10 100 MHC / pan-cadherin (fold) 1.0 0.6 1.0 1.2 1.1 myoD / pan-cadherin (fold) 1.0 0.8 0.9 1.3 0.9
  • the above ingredients are mixed and filled in an airtight cloth to prepare a powder.
  • tablets are prepared by tableting according to a conventional method for preparing tablets.
  • the above ingredients are mixed and filled into gelatin capsules to prepare capsules.
  • the amount of the above ingredient is prepared per ampoule (2 ml).
  • each component is added to the purified water to dissolve it, the lemon flavor is added appropriately, the above components are mixed, purified water is added, the whole is adjusted to 100 ml by the addition of purified water, and then filled in a brown bottle.
  • the solution is prepared by sterilization.
  • Vitamin A Acetate 70 ⁇ g
  • Vitamin E 1.0 mg
  • Vitamin B1 0.13 mg
  • Vitamin B2 0.15 mg
  • Vitamin B6 0.5 mg
  • Vitamin B12 0.2 ⁇ g
  • Vitamin C 10 mg
  • Nicotinamide 1.7 mg
  • composition ratio of the said vitamin and mineral mixture was mixed and comprised in the preferable embodiment the component suitable for a healthy food, you may change arbitrarily the compounding ratio considered that it does not deviate from the mind and range of this invention.
  • Citric Acid 1000 mg
  • Plum concentrate 2 g
  • composition ratio is a composition that is relatively suitable for the preferred beverage in a preferred embodiment
  • compounding ratio may be arbitrarily modified according to regional and ethnic preferences such as demand hierarchy, demand country, and usage.
  • the sample of the present invention recovers MHC and myoD expression and has muscle regeneration ability.
  • the composition is useful as a pharmaceutical composition or health functional food for the prevention and treatment of muscle diseases.
  • the results of the above experiments showed that the extract of the present invention was used in the extract of the present invention, atony, muscular atrophy, muscular dystrophy, muscle degeneration, muscular dystrophy, muscular atrophy, atrophy, cachexia and senile muscle.
  • the composition can be applied as a pharmaceutical composition, health functional food, and health supplement for preventing and treating muscle diseases.

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Abstract

La présente invention concerne une composition pour prévenir et traiter des maladies musculaires, contenant un extrait de Coptidis rhizoma (ou une fraction butanol de celui-ci) en tant que principe actif. Une fraction de Coptidis rhizoma de la présente invention a été confirmée comme étant utilisable en tant qu'agent thérapeutique ou adjuvant pour des maladies musculaires par l'intermédiaire de diverses expériences et analogues, ce qui peut être utilisée dans une composition pharmaceutique pour la prévention et le traitement de maladies musculaires, dans un aliment fonctionnel de santé, dans un aliment complémentaire de santé, et analogues.
PCT/KR2018/006065 2017-06-01 2018-05-29 Composition pour la prévention et le traitement de maladies musculaires, contenant un extrait de coptidis rhizoma, et son utilisation WO2018221922A1 (fr)

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KR102162787B1 (ko) 2019-04-19 2020-10-07 건국대학교 글로컬산학협력단 사비넨을 유효성분으로 함유하는 근원세포에서 근관세포로의 분화 촉진용 조성물
KR102424758B1 (ko) * 2020-08-11 2022-07-26 숙명여자대학교산학협력단 오리나무 추출물을 함유하는 골격근 근육관련 질환의 예방 및 치료용 조성물

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US20040097534A1 (en) * 2001-07-10 2004-05-20 Eugenbio Inc. Composition for the protection and regeneration of nerve cells containing berberine derivatives
KR20140102867A (ko) * 2013-02-15 2014-08-25 한국 한의학 연구원 황련해독탕 또는 이의 유산균 발효물을 포함하는 암전이 억제용 조성물

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US20040097534A1 (en) * 2001-07-10 2004-05-20 Eugenbio Inc. Composition for the protection and regeneration of nerve cells containing berberine derivatives
KR20140102867A (ko) * 2013-02-15 2014-08-25 한국 한의학 연구원 황련해독탕 또는 이의 유산균 발효물을 포함하는 암전이 억제용 조성물

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Title
LEE, HEYJIN ET AL.: "Isoquinoline alkaloids from Coptis japonica stimulate the myoblast differentiation via p38 MAP-kinase and Akt signaling pathway", BIOORGANIC & MEDICINAL CHEMISTRY LETTERS, vol. 27, no. 6, 4 February 2017 (2017-02-04), pages 1401 - 1404, XP055637926, DOI: 10.1016/j.bmcl.2017.02.003 *
LIZUKA, NORIO ET AL.: "Anticachectic effects of the natural herb Coptidis rhizoma and berberine on mice bearing colon 26/clone 20 adenocarcinoma", INTERNATIONAL JOURNAL OF CANCER, vol. 99, no. 2, 11 April 2002 (2002-04-11), pages 286 - 291, XP055637910, DOI: 10.1002/ijc.10338 *
TE-CHUN YANG ET AL.: "Alkaloids from Coptis chinensis root promote glucose uptake in C2C12 myotubes", FITOTERAPIA, vol. 93, 18 January 2014 (2014-01-18), pages 239 - 244, XP055637930, DOI: 10.1016/j.fitote.2014.01.008 *

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