CN113995133A - Composition for improving, preventing or treating muscular atrophy comprising Potentilla tormentilla extract or Polygonum tinctorium extract - Google Patents
Composition for improving, preventing or treating muscular atrophy comprising Potentilla tormentilla extract or Polygonum tinctorium extract Download PDFInfo
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- CN113995133A CN113995133A CN202110711194.6A CN202110711194A CN113995133A CN 113995133 A CN113995133 A CN 113995133A CN 202110711194 A CN202110711194 A CN 202110711194A CN 113995133 A CN113995133 A CN 113995133A
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- muscle
- polygonum tinctorium
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- muscle atrophy
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Abstract
The present invention relates to a composition for improving, preventing or treating muscle atrophy, and more particularly, to a composition for improving, preventing or treating muscle atrophy, which comprises an extract of potentilla chinensis or an extract of polygonum tinctorium. The composition containing Potentilla chinensis extract or Polygonum tinctorium extract as effective component can inhibit the over-expression of ubiquitin ligase Atrogin1 and MuRF1, and inhibit the activity of muscle atrophy regulating factor Forkhead box O, thereby improving muscle atrophy. The composition comprising Potentilla tormentilla extract or Polygonum tinctorium extract as an active ingredient inhibits myostatin, which is involved in inhibiting the growth of muscle tissue, and can enhance muscle, increase the size of myotube and inhibit the activity of histone deacetylase by increasing follistatin which is an inhibitor of myostatin. The composition comprising tormentum chinensis extract or Polygonum tinctorium extract as an active ingredient exerts more excellent effects than suberoylanilide hydroxamic acid, ursolic acid and creatine monohydrate, which have muscle atrophy improving and muscle strengthening effects.
Description
Technical Field
The present invention relates to a composition for improving, preventing or treating muscle atrophy, and more particularly, to a composition for improving, preventing or treating muscle atrophy, which comprises an extract of potentilla chinensis or an extract of polygonum tinctorium.
Background
Muscle atrophy (muscle atrophy) refers to a reduction in skeletal muscle mass resulting from a reduction in the number of myofibrils in a muscle due to a reduction in the size of muscle cells. Skeletal muscle accounts for 40% of body weight, and skeletal muscle has increased proteolysis and decreased protein synthesis, resulting in muscle atrophy. In addition, the phenomenon of rapid decrease in muscle mass and muscle force due to muscle atrophy is called sarcopenia. Muscle atrophy is caused by inactivity, denervation, aging, and abuse of steroids.
The adrenal cortex secretes steroid hormones, and glucocorticoid (glucocorticoid) is involved in the regulation of immune response and glucose, fat, and anti-inflammatory response, but long-term administration causes side effects such as muscle atrophy causing a decrease in muscle mass and diabetes. The administration of steroids selectively affects type ii muscles, which consist of fast-twitch muscle fibers, and patients receiving steroid therapy lose the ability to produce maximal muscle strength rather than the ability to alter muscle endurance.
If muscular atrophy and sarcopenia caused by aging or steroid hormones are left alone, the muscle mass can be reduced to 60% at most, and the function of the muscle is also significantly lost. When muscle mass is reduced, basal metabolism itself is reduced, and thus, the incidence of diabetes is increased along with the increase in blood glucose. Also, fractures, falls, depression, obesity, disability, and even death may result. Therefore, there is a need for a study for improving, preventing or treating muscle atrophy and a solution thereof.
Documents of the prior art
Patent document
Patent document 1: korean patent No. 10-1671713 (granted date: 10/27 in 2016) discloses a pharmaceutical composition for internal use for the prevention and treatment of muscle atrophy, comprising flavan derivatives derived from broussonetia papyrifera as an active ingredient.
Disclosure of Invention
In the present invention, it is intended to develop and provide a composition capable of improving muscle atrophy using Potentilla chinensis extract or Polygonum tinctorium extract.
The present invention provides a food composition for ameliorating muscular atrophy, which is characterized by containing a potentilla extract as an active ingredient.
The present invention also provides a food composition for ameliorating muscle atrophy, which comprises a Polygonum tinctorium extract as an active ingredient.
The present invention also provides a pharmaceutical composition for preventing or treating muscle atrophy, which comprises an extract of potentilla chinensis as an active ingredient.
Also, the present invention provides a pharmaceutical composition for preventing or treating muscle atrophy, characterized by comprising a Polygonum tinctorium extract as an active ingredient.
Also provided is an animal pharmaceutical composition for preventing or treating muscular dystrophy, which is characterized by containing an extract of Potentilla chinensis as an active ingredient.
Also, provided is an animal pharmaceutical composition for preventing or treating muscle atrophy, characterized by containing a Polygonum tinctorium extract as an active ingredient.
Also provided is a feed composition for ameliorating muscular atrophy, which is characterized by containing a potentilla extract as an active ingredient.
Also, provided is a feed composition for ameliorating muscle atrophy, characterized by containing a Polygonum tinctorium extract as an active ingredient.
The composition of the present invention comprising the potentilla chinensis extract or the polygonum tinctorium extract as an active ingredient inhibits overexpression of Atrogin1 and MuRF1, which are ubiquitin ligases, inhibits the activity of the muscle atrophy regulatory factor Forkhead box (FOX) O, and thus exerts an effect of improving muscle atrophy.
Also, the composition of the present invention comprising potentilla chinensis extract or polygonum tinctorium extract as an effective ingredient inhibits myostatin (myostatin), which is involved in inhibiting the growth of muscle tissue, and can strengthen muscle by increasing follistatin (follistatin), which is an inhibitor of myostatin, increase the size of myotubes (myotubes), and inhibit the activity of Histone Deacetylases (HDACs).
The composition of the present invention containing a potentilla extract or a polygonum tinctorium extract as an active ingredient exhibits more excellent effects than conventionally known suberoylanilide hydroxamic acid (SAHA, HDAC inhibitor), ursolic acid (ursolic acid), and creatine monohydrate (creatine monohydrate) having muscle atrophy improving and muscle strengthening effects.
Drawings
Fig. 1 is a graph showing the pathogenesis of muscle atrophy according to the action of the muscle atrophy transcription factor FOXO of glucocorticoid (glucoorticoide) and ubiquitin ligase (MuRF1, Atrogin 1).
Fig. 2 is a bar graph of the results confirming the effect of the tormentum chinensis extract or the polygonum tinctorium extract of the present invention on mRNA expression of muscle atrophy factors (FOXO3A, FOXO1, MuRF1, atrogin-1) after treatment of differentiated muscle cells with dexamethasone.
Fig. 3 is a bar graph of results confirming the effect of potentilla chinensis extract of the present invention on mRNA expression of muscle growth related factors (myostatin, follistatin) after treatment of differentiated muscle cells with dexamethasone.
Fig. 4 is a bar graph of the results confirming the effect of the tormentum chinensis extract or the polygonum tinctorium extract of the present invention on the protein expression of the muscle atrophy factors (FOXO3A, FOXO1, MuRF1, atrogin-1) after treatment of differentiated muscle cells with dexamethasone.
Fig. 5 is a bar graph showing the results of confirming the effect of the cinquefoil extract or the polygonum tinctorium extract of the present invention on mRNA expression and protein expression of muscle atrophy factors (FOXO3A, FOXO1, MuRF1, atrogin-1) in differentiated muscle cells.
Fig. 6 is a microscopic view for confirming the effect of the tormentum chinensis extract of the present invention on the diameter of myotubes after treating differentiated muscle cells with dexamethasone.
Fig. 7 is a bar graph of results of studying the binding ability of the tormentum chinensis extract or polygonum tinctorium extract of the present invention to FOXO3a, MuRF1, and the promoter of Atrogin1 to Glucocorticoid Receptor (GR), FOXO3a, and RNA polymerase ii (Pol ii) in differentiated muscle cells.
Fig. 8 is a schematic diagram showing the activity mechanism of Histone Deacetylases (HDACs).
Fig. 9 is a bar graph of the results of studying the effect of the tormentum chinensis extract or the polygonum tinctorium extract of the present invention on the activity of Histone Deacetylase (HDAC) 6 in differentiated muscle cells.
Detailed Description
Muscle atrophy (muscle atrophy) refers to a decrease in skeletal muscle mass due to a decrease in myocyte size, resulting in a decrease in the number of myofibrils in the muscle, due to fasting, inactivity, denervation, aging, and abuse of steroids, among other reasons. The adrenal cortex secretes steroid hormones, and glucocorticoid (glucocorticoid) is involved in the regulation of immune response and glucose, fat, and anti-inflammatory response, but causes muscular atrophy and side effects such as diabetes when administered for a long time.
Foxo (forkhead box o) acts as a muscle atrophy transcription factor when the Glucocorticoid Receptor (GR) is activated, while MuRF1 and Atrogin1 act as ubiquitin ligases (ubiquitin ligases) specifically expressed in muscle cells. When their expression is increased, muscle proteins are ubiquitinated (ubiquitination) and proteasomes (proteosomes) are dependently decomposed, resulting in muscle loss. The relevant mechanism is shown in fig. 1.
If muscular atrophy and sarcopenia caused by aging or steroid hormones are left alone continuously, the muscle mass can be reduced to 60% at most, the function of the muscle is obviously lost, and fracture, fall injury, depression, obesity, disability and even death can be caused. Therefore, there is a need for a study for improving, preventing or treating muscle atrophy and a solution thereof. Suberoylanilide hydroxamic acid (SAHA, HDAC inhibitor), ursolic acid (ursolic acid), creatine monohydrate (creatine monohydrate) have been used in the past to improve muscle atrophy and strengthen muscles. The present invention is intended to develop and provide a substance that exerts a very excellent effect in the improvement of muscle atrophy as compared with the existing commercially available substances.
The present invention provides a food composition for ameliorating muscular atrophy, which is characterized by containing a potentilla extract as an active ingredient. The present invention also provides a food composition for ameliorating muscle atrophy, which comprises a Polygonum tinctorium extract as an active ingredient. The present invention also provides a pharmaceutical composition for preventing or treating muscle atrophy, which comprises an extract of potentilla chinensis as an active ingredient. Also, the present invention provides a pharmaceutical composition for preventing or treating muscle atrophy, characterized by comprising a Polygonum tinctorium extract as an active ingredient. Also provided is an animal pharmaceutical composition for preventing or treating muscular dystrophy, which is characterized by containing an extract of Potentilla chinensis as an active ingredient. Also, provided is an animal pharmaceutical composition for preventing or treating muscle atrophy, characterized by containing a Polygonum tinctorium extract as an active ingredient. Also provided is a feed composition for ameliorating muscular atrophy, which is characterized by containing a potentilla extract as an active ingredient. Also, provided is a feed composition for ameliorating muscle atrophy, characterized by containing a Polygonum tinctorium extract as an active ingredient.
Potentilla refers to root or whole herb with heel of Tianqing Di Bai (Potentilla chinensis) belonging to Rosaceae as perennial herb. It has antidotal effect, and can be used for treating hemorrhagic diseases, dysentery, hemoptysis, inflammation, furuncle, etc., and relieving pain of muscle or joint, and is mainly used as medicinal material. Collected between spring and autumn, dried in the sun, decocted or made into powder for administration.
Polygonum tinctorium (Indigo) is an annual plant belonging to the Polygonaceae family of the Polygonales of dicotyledonous plants, and has been grown as a dye resource in the past. The leaves of Polygonum tinctorium contain a pigment called indigo as a blue dye. It has effects of clearing heat, removing toxic substance, and relieving swelling, and can be used for treating common cold, jaundice, dysentery, hematemesis, and various inflammations.
In the invention, the potentilla chinensis extract or the polygonum tinctorium extract can be obtained by the following method: solvent extraction by adding water or an organic solvent; or ultra-high pressure (supercritical) extraction. In this case, the above organic solvent may employ an extraction solvent selected from the group consisting of: such as anhydrous or hydrous lower alcohols having 1 to 4 carbon atoms, propylene glycol, butylene glycol, glycerin, acetone, ethyl acetate, chloroform, butyl acetate, diethyl ether, methylene chloride, hexane, and mixtures thereof.
In this case, it is preferable that the Potentilla chinensis extract is extracted by a solvent extraction method in which 70% ethanol is added in an amount of 10 times (by volume) based on the weight of the whole herb of Potentilla chinensis, and the Polygonum tinctorium extract is extracted by a solvent extraction method in which 70% ethanol is added in an amount of 10 times (by volume) based on the weight of the whole herb of Polygonum tinctorium. More preferably, in the solvent extraction method, the extraction solution is obtained at room temperature after immersion at 50 to 70 ℃ for 22 to 26 hours, respectively, and extracted by applying ultrasonic waves for about 3 hours. In this case, when it is more or less than the above range of the extraction time and the extraction temperature, the yield and activity of the extract may be decreased.
On the other hand, it was confirmed from the following experiments that the potentilla chinensis extract or polygonum tinctorium extract of the present invention inhibits overexpression of Atrogin1 and MuRF1, which are ubiquitin ligases, and inhibits the activity of the muscle atrophy regulatory factor FOXO. Also, the Potentilla tormentilla extract or Polygonum tinctorium extract of the present invention inhibits myostatin (myostatin), which is involved in inhibiting the growth of muscle tissue, increases follistatin (follistatin), which is an inhibitor of myostatin, and increases the size of myotubes (myotube). Furthermore, the potentilla chinensis extract or the polygonum tinctorium extract reduces the binding capacity of the promoter of FOXO3a with glucocorticoid receptor and RNA polymerase (polymerase) II, reduces the binding capacity of the promoters of MuRF1 and Atrogin1 with FOXO3a and RNA polymerase II, and reduces the activity of Histone deacetylase (Histone deacetylase). From this, it was confirmed that the potentilla extract or the polygonum tinctorium extract of the present invention is effective for ameliorating muscular atrophy or ameliorating and inhibiting muscle growth.
In another aspect, the food composition of the present invention may be any one selected from the group consisting of: such as, but not limited to, meats, grains, caffeine beverages, general drinks, chocolate, breads, snacks, cookies, pizza, jellies, noodles, chewing gums, ice creams, alcoholic beverages, wine, vitamin complexes, and other health foods.
In another aspect, the pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier, diluent or excipient. Carriers, excipients, or diluents that may be used include: lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl paraben, propyl paraben, talc, magnesium stearate, and mineral oil, and one or more selected from them can be used. When the therapeutic agent and the prophylactic agent are drugs, they may further contain fillers, anticoagulants, lubricants, wetting agents, perfumes, emulsifiers, preservatives, and the like.
On the other hand, the dosage forms of the pharmaceutical composition and the animal pharmaceutical composition of the present invention may be prepared in a preferred form according to the method of use, and particularly, the dosage forms are preferably prepared using a method well known in the art to rapidly, continuously or delay release of the active ingredient after administration to a mammal. Specific dosage forms may include any one selected from the group consisting of: PLASTERS (PLASTERS), GRANULES (cereals), EMULSIONS (lotiones), Liniments (LINIMENTS), LEMONADES (LEMONADES), AROMATIC water (AROMATIC WATERS), POWDERS (POWDERS), SYRUPS (syrupes), OPHTHALMIC OINTMENTS (opthalmic ointements), SOLUTIONS (liquoids AND solubles), AEROSOLS (AEROSOLS), EXTRACTS (extractants), ELIXIRS (ELIXIRS), OINTMENTS (ointements), FLUIDEXTRACTS (fluidextrts), EMULSIONS (EMULSIONS), suspensions (suspensions), DECOCTIONS (DECOCTIONS), infusion SOLUTIONS (INFUSIONS), eye drops (oculars), suppositories (suppositions), INJECTIONS (INFUSIONS), PILLS (SPIRITS), gels (CATAPLSMA), Caps (CAPSULES), Cremes (CREAMS), lozenges (lozenges), soft AND hard gelatin CAPSULES (gelatine CAPSULES).
On the other hand, for the pharmaceutical composition and the animal drug composition of the present invention, the administration dose is preferably determined in consideration of the administration method, the age, sex and weight of the subject, the severity of the disease, and the like. For example, the composition can be orally administered at 0.000001mg/kg (body weight) to 100mg/kg (body weight) at least once per day based on Potentilla chinensis extract or Polygonum tinctorium extract as an active ingredient. However, the above administration dose is merely exemplary and may be changed according to the state of the user and the prescription of a doctor.
On the other hand, in the present invention, "containing as an active ingredient" means that the potentilla extract or the polygonum tinctorium extract as the ingredient of the present invention has the effect of improving, preventing or treating the muscular atrophy required in the present invention, and may further contain other ingredients as auxiliary ingredients.
On the other hand, the feed composition of the present invention may contain other components in addition to the above-mentioned potentilla extract or polygonum tinctorium extract. For example, corn, soybean meal, palm, wheat (wheat flour), and the like, as well as vitamins, minerals, and the like may be included, and a growth promoting substance for promoting the growth of an animal or an enhancing substance for enhancing the immunity of an animal may be included as a functional substance.
Hereinafter, the contents of the present invention will be described in more detail by the following examples or experimental examples. However, the scope of the present invention is not limited to the following examples and experimental examples, and includes modifications of equivalent technical ideas.
Example 1: preparation of Potentilla chinensis extract
A Potentilla chinensis extract is extracted by adding 10 times (by volume) of an extraction solvent (70% ethanol) based on the weight of the whole Potentilla chinensis, immersing at 50-70 ℃ for 22-26 hours, obtaining an extract at room temperature, and applying ultrasonic waves for about 3 hours. The process can be repeated 2-3 times. Then, the extract was filtered, concentrated under reduced pressure, and dried to obtain a 70% ethanol extract.
Example 2: preparing Polygonum tinctorium extract
The Polygonum tinctorium extract is extracted by adding 10 times (volume) of an extraction solvent (70% ethanol) based on the weight of the whole plant of Polygonum tinctorium, soaking at 50-70 deg.C for 22-26 hours, obtaining an extract at room temperature, and applying ultrasonic waves for about 3 hours. The process can be repeated 2-3 times. Then, the extract was filtered, concentrated under reduced pressure, and dried to obtain a 70% ethanol extract.
Experimental example 1: evaluating the effect of Potentilla chinensis extract or Polygonum tinctorium extract on improving muscular atrophy (muscle atrophy)
In this experimental example, the following experiment was performed to confirm whether or not the muscle atrophy was improved when treated with the potentilla extract (example 1) or the polygonum tinctorium extract (example 2).
1) Cell culture and induced differentiation into myotube cells
L6 mouse myoblasts (myoblasts) were obtained from ATCC (American Type Culture Collection, Manassas, Va., USA), and supplemented with 10% FBS and 0.5% antibiotic-antifungal (antibiotic-antifungal) in DMEM (Dulbecco's Modified Eagle's medium)At a temperature of 37 ℃ in 5% CO2Culturing in a constant temperature reactor. To differentiate myoblasts into myotube cells (myotube), DMEM differentiation medium containing 2% horse serum was replaced every 2 days for cells in a 100% cell culture (confluent) state. After 5-7 days, myotube (myotube) formation was observed under a microscope and treatment with Dexamethasone (DEXA, 20nmol/L) was used to induce inhibition of differentiation to induce muscle atrophy.
2) Evaluation of mRNA expression levels of muscle atrophy factor and muscle growth-related factor after treatment of differentiated muscle cells with dexamethasone
Cells induced muscle atrophy by treating differentiated muscle cells (L6) with dexamethasone were treated with Potentilla chinensis extract (example 1) or Polygonum tinctorium extract (example 2), and gene expression patterns of FOXO3A, FOXO1, MuRF-1, atrogin-1, and muscle growth-related factors myostatin (myostatin) and follistatin (follistatin) were observed as muscle atrophy factors. For comparison, commercially available drugs known to improve muscle atrophy and muscle enhancement, such as suberoylanilide hydroxamic acid (SAHA, HDAC inhibitor), ursolic acid (ursolic acid), creatine monohydrate (creatine monohydrate) were used in the experiment.
After inoculating LG6 mouse myoblasts into 6-well plates, differentiation was performed using DMEM medium supplemented with 2% horse serum, followed by treatment with Dexamethasone (dexamehasone, DEXA, 20nmol/L) and each extract for 8 hours. Cells were recovered and RNA extracted using TRIzol reagent (Invitrogen, Carlsbad, Calif. (CA)) was quantified using a spectrophotometer, and cDNA was synthesized using the RevertAID first Strand cDNA Synthesis kit (Fermentas, Futasai Biotech, Inc.) for 3. mu.g of RNA. qRT-PCR was performed by ABI Prism 7500sequence detection System (ABI Prism 7500sequence evaluation System) (Applied Biosystems, USA; Foster City, Calif.) using 4. mu.L of cDNA, specific primers andand a Sybergreen master mix (Takara Bio Inc.). For the reaction results, by calculation 2-ΔΔCtValues to compare expression levels.
The genes used were atrogin-1/MAFbx, MuRF1, FOXO3A, FOXO1, myostatin (myostatin) and follistatin (follistatin). When Glucocorticoid Receptor (GR) is activated, fox (forkhead box) O acts as a muscle atrophy transcription factor, while atrogin-1 and MuRF (muscle-specific ring finger protein) 1 act as ubiquitin ligases (ubiquitin ligases) specifically expressed in muscle cells. When its expression is increased, muscle proteins are ubiquitinated (ubiquitination), and proteasomes (proteosomes) are dependently decomposed, resulting in muscle loss. The above expression amount of the target mRNA is expressed as a ratio (ratio) normalized by GAPDH (normalization).
Results of confirming the effect of tormentum chinensis extract (example 1) or polygonum tinctorium extract (example 2) on mRNA expression of muscle atrophy factors (FOXO3A, FOXO1, MuRF1, atrogin-1), as shown in fig. 2, it was confirmed that tormentum chinensis extract and polygonum tinctorium extract reduced mRNA expression of FOXO3A, FOXO1, MuRF1, atrogin-1 in a concentration-dependent manner and were more effective than suberoylanilide hydroxamic acid (SAHA), ursolic acid (ursalic acid), creatine monohydrate (creatine monohydrate).
Furthermore, the effect of potentilla chinensis extract on myostatin (growth differentiation factor 8) that inhibits muscle growth and on mRNA expression of follistatin (follistatin) that inhibits myostatin and increases muscle cells was confirmed. As a result, as shown in fig. 3, it was confirmed that cinquefoil extract decreased the expression of myostatin and increased the expression of follistatin. From this, it was confirmed that the potentilla extract or the Polygonum tinctorium extract is effective for improving muscle atrophy and inhibiting muscle growth.
3) Evaluation of protein expression of muscle atrophy factor after treatment of differentiated muscle cells with dexamethasone
The cells induced muscle atrophy by treating differentiated muscle cells (L6) with dexamethasone were treated with Potentilla chinensis extract (example 1) or Polygonum tinctorium extract (example 2), and protein expression patterns of FOXO3A, FOXO1, MuRF-1, and atrogin-1 as muscle atrophy factors were observed.
Differentiated L6 cells were treated with Dexamethasone (Dexamethasone, DEXA, 20nmol/L) and each extract for 2 hours and homogenized with lysis buffer. Then, the supernatant was recovered by centrifugation at 12000rpm after leaving on ice for 10 minutes. The mass of protein in the recovered supernatant was quantified by the BCA method, electrophoresed with SDS-PAGE, then transferred to a nitrocellulose membrane (nitrocellulose membrane) and subjected to immunoblotting (immunoblot) using an antibody to the target protein. Horseradish peroxidase (Horse-radish peroxidase) conjugated secondary antibody was reacted for 2 hours and printed on a film with ECL. The antibodies used were atrogin-1/MAFbx, MuRF1, FOXO3A, FOXO1 and β -actin (. beta. -actin), and the expression level of the target protein was expressed as the ratio (ratio) after β -actin (. beta. -actin) normalization (normalization).
As a result of confirming the effect of the potentilla chinensis extract (example 1) or the polygonum tinctorium extract (example 2) on the protein expression of the muscle atrophy factors (FOXO3A, FOXO1, MuRF1, atrogin-1), as shown in fig. 4, it was confirmed that the potentilla chinensis extract and the polygonum tinctorium extract reduced the protein expression of FOXO3A, FOXO1, atrogin-1, MuRF1 in a concentration-dependent manner. Thus, as shown by the results of the above-mentioned experiments on the mRNA expression of the muscle atrophy factor, it was confirmed that the potentilla extract or the polygonum tinctorium extract is effective for the improvement of muscle atrophy.
4) Evaluation of mRNA expression level and protein expression level of muscle atrophy factor in differentiated muscle cells
Differentiated muscle cells (L6) were treated with potentilla chinensis extract (example 1) or polygonum tinctorium extract (example 2), and mRNA expression and protein expression morphology of FOXO3A, FOXO1, MuRF-1, atrogin-1 as muscle atrophy factors were observed. The same experimental procedure as described above for mRNA expression and protein expression experiments was used, except that dexamethasone was not used for the treatment.
As a result of confirming the effect of the tormentum chinensis extract (example 1) or the polygonum tinctorium extract (example 2) on mRNA expression and protein expression of the muscle atrophy factors (FOXO3A, FOXO1, MuRF1, atrogin-1), as shown in parts (a) to (C) of fig. 5, it could be confirmed that the tormentum chinensis extract and the polygonum tinctorium extract reduced mRNA expression of FOXO3A, MuRF1, atrogin-1 in a concentration-dependent manner in the differentiated muscle cells themselves that were not treated with dexamethasone. Also, as shown in parts (D) to (F) of fig. 5, it was confirmed that cinquefoil extract and polygonum tinctorium extract reduced the protein expression of FOXO3A, FOXO1 in a concentration-dependent manner in the differentiated muscle cells themselves that were not treated with dexamethasone. From this, it was confirmed that the potentilla extract or the polygonum tinctorium extract is a substance effective in improving muscular atrophy even in the differentiated muscle cells themselves.
5) Diameter of observation myotube
Cells induced muscle atrophy by treatment of differentiated muscle cells (L6) with dexamethasone were treated with potentilla extract (example 1) and the diameter of the myotubes was observed under 200-fold microscope. The diameter was randomly determined for each group and observed. For comparison, commercially available drug creatine was used.
As a result, as shown in fig. 6, when differentiated muscle cells were treated with dexamethasone, muscle atrophy was induced and the size of myotubes (Myotube) was reduced. However, it was confirmed that the reduction in the size of myotubes was suppressed by the treatment with Potentilla chinensis extract. Thus, it was confirmed that the potentilla extract is effective for ameliorating muscular atrophy.
6) Study of the binding Capacity of the muscle atrophy factor Glucocorticoid Receptor (GR), FOXO3a, and RNA polymerase II by chromatin immunoprecipitation (ChIP)
The binding capacity of muscle wasting factors was studied in differentiated muscle cells (L6) by Chromatin immunoprecipitation analysis (ChIP assay). Differentiated muscle cells (L6) treated with potentilla extract (example 1) or polygonum tinctorium extract (example 2) were placed in 1% formalin, after sufficient fixation at a temperature of 4 ℃ for 1 hour, Lysis buffer containing a proteolytic enzyme antagonist was added, and sonication was performed to cut nucleosomes (nucleosomes) to a size of 200-1000 bp. 1.0. mu.g of an antibody showing binding ability was added and reacted for 16 hours. Protein G Sepharose (Protein G agarose) was added for reaction, and the supernatant was removed by centrifugation and washed. After adding Elution buffer (Elution buffer) for reaction, 1. mu.L of proteinase K (protease K) was added to the supernatant obtained by centrifugation at 14000rpm, and the reaction was carried out at 45 ℃ for 1 to 2 hours. After DNA was isolated using a DNA purification column (column), the binding capacity of muscle atrophy factor was analyzed by qRT-PCR.
The binding ability of the promoters of FOXO3a, MuRF1 and Atrogin1 to Glucocorticoid receptors (Glucocorticoid receptors, GR), FOXO3a and RNA polymerase ii (Pol ii) was investigated by chromatin immunoprecipitation analysis when differentiated muscle cells (L6) were treated with potentilla extracts (example 1) or polygonum blue extracts (example 2) and treated at cumulative concentrations.
As a result, as shown in fig. 7 (a), cinquefoil extract or polygonum tinctorium extract decreased the ability of the promoter of FOXO3a to bind to Glucocorticoid Receptor (GR) and RNA polymerase ii (Pol ii). Also, as shown in parts (b) and (c) of FIG. 7, the binding ability of the promoters of MuRF1 and Atrogin1 to FOXO3a and RNA polymerase II (PolII) was reduced.
7) Investigating the enzymatic Activity of Histone Deacetylases (HDACs) 6
Histone Deacetylases (HDACs), which are regulators of epigenetics, inhibit the expression of cell proliferation inhibitors by inhibiting acetylation of histones, thereby promoting cell proliferation, and regulating tumorigenicity and differentiation of cells. Therefore, the pathological activity of HDACs and the down-regulation of various diseases such as muscular atrophy (muscular atrophy) are involved. That is, HDACs affect muscle atrophy and muscle dysfunction caused by denervation, muscular dystrophy, costs, and modulate side effects in skeletal muscle. In particular, HDACs were overexpressed in a muscle atrophy model, and HDACs inhibitors reduced muscle atrophy factors. Class I HDACs (class ihdacs) are involved in skeletal muscle metabolism and exhibit myopathies characterized by muscle degeneration, mitochondrial dysfunction, autophagy (autophagy) inhibition, and redox metabolism in mice lacking HDAC 1/2.
HDAC6 is a member of the HDACs family of class IIb and is the only cytoplasmic deacetylase (cytoplasmic deacetylase). It does not directly catalyze histones, but uses alpha tubulin (alpha-tubulin) and heat shock protein (HSP90) as substrates to regulate cellular transport, adhesion and movement. Therefore, the influence on the genetic-related physiological functions is very small, so that the side effects are reduced, and deacetylation is realized. Can inhibit muscle atrophy factor by inhibiting HDAC6 enzyme activity and has muscle protection function. Fig. 8 shows the mechanism of HDAC activity.
The enzymatic activity of Histone Deacetylases (HDACs) 6 was studied by treatment with potentilla extract (example 1) or polygonum tinctorium extract (example 2) and treatment at cumulative concentrations. In the experimental procedure, the degree of acetyl (O ═ C-CH3) removal by HDACs from an acetyl lysine (K) amino acid was determined. That is, an HDAC fluorogenic substrate containing an acetylated lysine (Lys-Ac) side chain is incubated with the purified HDAC enzyme. That is, when HDAC removes acetyl group from lysine (K), the solution releases dye that can emit fluorescence. HDAC activity was analyzed by measuring fluorescence values thereof. For comparison, Tricostatin a was used as an HDAC inhibitor.
As a result, as shown in fig. 9, it was revealed that the potentilla extract or the polygonum tinctorium extract inhibited the enzymatic activity of Histone Deacetylase (HDACs) 6 in a concentration-dependent manner.
Claims (8)
1. A food composition for ameliorating muscular atrophy, characterized by comprising an extract of Potentilla chinensis as an active ingredient.
2. A food composition for improving muscle atrophy, characterized by comprising Polygonum tinctorium extract as an active ingredient.
3. A pharmaceutical composition for preventing or treating muscular atrophy, characterized by comprising an extract of Potentilla tormentilla as an active ingredient.
4. A pharmaceutical composition for preventing or treating muscle atrophy, characterized by comprising an extract of Polygonum tinctorium as an active ingredient.
5. An animal pharmaceutical composition for preventing or treating muscular atrophy, characterized by comprising an extract of Potentilla chinensis as an active ingredient.
6. An animal pharmaceutical composition for preventing or treating muscle atrophy, characterized by comprising Polygonum tinctorium extract as an active ingredient.
7. A feed composition for ameliorating muscular atrophy, characterized by comprising an extract of Potentilla chinensis as an active ingredient.
8. A feed composition for ameliorating muscle atrophy, characterized by comprising Polygonum tinctorium extract as an active ingredient.
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