KR101523953B1 - Composition for preventing or treating muscle atrophy comprising loquat leaf extract - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating muscle hypoxia comprising the extract of Beecha leaf as an active ingredient, wherein the extract of Beecha leaf reduces the amount of creatine kinase induced by dexamethasone administration, It was confirmed that the damage was reduced. It was also found that muscle hypoxia was greatly influenced by the degradation of MyHC by MuRF1, which is regulated by FoxO1, and that administration of Becker leaf extract decreases muscle degradation in dexamethasone-induced muscle hypoxia By confirming, it was suggested that the extract of Beech leaf can be used as a therapeutic agent for hypersensitivity.

Description

TECHNICAL FIELD [0001] The present invention relates to a composition for preventing or treating muscular dysgenesis comprising, as an active ingredient,

The present invention relates to a composition for preventing or treating hypocholesterolemia comprising an extract of Beech leaf as an active ingredient.

Muscle hypoxia is caused by a variety of causes, including sepsis, cancer, renal failure, excess glucocorticoids, nerve removal, muscle disuse, and aging processes. Muscle hypoxia refers to a decrease in protein content, fiber diameter, muscle strength and fatigue resistance. Muscle hypoxia results from imbalance between protein synthesis and degradation. Muscle ring finger 1 (MuRF1) and Muscle atrophy F box (MAFbx), two genes specific for myopenia, are upregulated in myopenia. On the other hand, the Akt / mTOR pathway involved in protein synthesis is downregulated in the hypoxic process. In addition, cell death is activated by various atrophic stimuli in the skeletal muscle. These genes are important for protein degradation by ubiquitin as E3 ubiquitin ligase. In particular, MuRF1 is involved in the degradation of myosin heavy chain (MyHC), a major component of muscle. The MuRF1 promoter contains a nearly complete palindromic glucocorticoid response element (GRE), a Forkhead box O (FoxO) binding element (FBE) and a Nuclear Factor kappa B response element. Indicating that the FoxO factor is important in regulating the transcription of MuRF1. Thus, inhibiting the activity of these genes may be an effective strategy for inhibiting myopathy.

Loquat, Eriobotrya japonica ) belongs to Rosaceae . Vipa leaves are widely used as traditional herbal tea. In folk remedies, Vipa leaves have been used for skin diseases, colds, nausea and itching. It contains approximately 1.5 mg of ursolic acid and 0.7 mg of oleanolic acid per gram of loquat leaf.

Therefore, the present inventors confirmed that the extract of Vipara leaf reduced the amount of creatine kinase induced by dexamethasone administration and decreased muscle damage. It was also found that muscle hypoxia was greatly influenced by the degradation of MyHC by MuRF1, which is regulated by FoxO1, and that administration of Becker leaf extract decreases muscle degradation in dexamethasone-induced muscle hypoxia And completed the present invention.

Korean Patent Laid-Open No. 10-2012-0093534 relates to a pharmaceutical composition for the prevention and treatment of rheumatoid arthritis containing non-leaf extract, and more particularly, to a pharmaceutical composition for preventing and treating rheumatoid arthritis containing non-leaf extract, which can be safely used without toxicity and side- Prevention and treatment, but there is no mention of the use of the present invention for the prevention and treatment of hypersensitivity.

It is an object of the present invention to provide a composition for preventing or treating hypocholesterolemia containing an extract of Beech leaf as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating muscle hypoxia, comprising an extract of Beech leaf as an active ingredient.

Specifically, the loquat leaf extract is characterized by being extracted with any one solvent selected from the group consisting of water, C1 to C4 lower alcohols, hexane, methylene chloride, ethyl acetate, and a mixed solvent thereof.

In more detail, the Beech leaf extract is a residue obtained by extracting Beech leaf with ethanol, concentrating it with ethanol, dissolving it in ethanol again, and adding 2 times water to the residue.

In detail, the pea leaf extract is characterized by inhibiting the transcription activity of FoxO1 (Forkhead box O1) and decreasing MuRF1 (Muscle ring finger 1) expression.

In addition, the composition of the present invention may be provided in various forms selected from a pharmaceutical composition or a health food composition.

When the composition of the present invention is a pharmaceutical composition, the pharmaceutical composition may include a pharmaceutically acceptable carrier in addition to the non-peanut leaf extract. Such a pharmaceutically acceptable carrier is commonly used in pharmaceutical preparations, Starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxyacetate, Benzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, but are not limited thereto. In addition, the pharmaceutical composition may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. as an additive.

The pharmaceutical composition may be administered according to the severity of symptoms of muscle hypoxia. The extract of the present invention may be administered to mammals such as rats, mice, livestock, and humans in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections. The dosage of the active ingredient in the pharmaceutical composition may vary depending on the route of administration, the severity of the disease, the age, sex, and weight of the patient, and may be administered once to several times per day. Preferably, the loquat leaf extract may comprise from 0.01 to 50% by weight of the total pharmaceutical composition, but is not limited thereto.

The pharmaceutical composition may be prepared in unit dose form by formulating it with a pharmaceutically acceptable carrier and / or excipient, or may be prepared by inserting it into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions, or may be in the form of elixirs, excipients, powders, granules, tablets, alerts, lotions, ointments and the like.

When the composition of the present invention is a health food composition, there is no particular limitation on its kind, and examples thereof include meat, sausage, bread, chocolates, candies, snacks, confectionery, pizza, ramen noodles, other noodles, gums, Dairy products, various soups, beverages, tea, drinks, alcoholic beverages and vitamin complexes.

In some cases, in addition to the above-described Beech leaf extract, a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and thickening agents (cheese, chocolate etc.), pectic acid and its salts, Alginic acid and its salts, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated drinks and the like. All of these ingredients can be used independently or in combination.

On the other hand, the non-leaf extract used in the composition of the present invention is a natural product and can be safely used for long-term administration for preventive purposes with little toxicity and side effects.

The present invention relates to a composition for preventing or treating muscle hypoxia comprising an extract of Beech leaf, as an active ingredient, wherein the extract of Beech leaf extract reduces creatine kinase induced by dexamethasone administration, . It was also found that muscle hypoxia was greatly influenced by the degradation of MyHC by MuRF1, which is regulated by FoxO1, and that administration of Becker leaf extract decreases muscle degradation in dexamethasone-induced muscle hypoxia By confirming, it was suggested that the extract of Beech leaf can be used as a therapeutic agent for hypersensitivity.

Fig. 1 shows changes in muscle strength of forelegs according to administration of loquat leaf extract.
Figure 2 shows the amount of creatine kinase liberated in the blood.
Figure 3 shows the results of histological analysis through Haematoxylin & eosin (H & E) staining.
Fig. 4 shows the amount of MyHC protein by administration of the peach leaf extract.
FIG. 5 shows inhibition of muscle degradation-related gene expression by administration of non-pea leaf extract.
FIG. 6 shows the effect of inhibiting ubiquitination of MyHC upon administration of Beech leaf extract.
Fig. 7 shows the inhibitory mechanism for the hypoxia of Leek leaf extract.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the present invention is not limited by these examples.

< Example  1> Loquat leaf  Changes in muscle strength of forelegs following administration of extracts

1. Preparation of loquat leaf extract

The non-foliar ethanol was added to 100 g of non-foliage and heated to 70 ° C for 3 hours after adding 99% ethanol so as to contain non-foliage. Then, the extracted non-foliar ethanol was filtered using a filter paper. After concentrating the non-foliar ethanol extract with a concentrator, the completely concentrated non-foliar ethanol extract was dissolved in 150 ml of 99% ethanol and 300 ml of water was added to the non-foliar ethanol extract dissolved in 150 ml of 99% ethanol to obtain a precipitate. After removing the supernatant, the precipitate remaining in the flask was dissolved in 150 ml of 99% ethanol, and 300 ml of water was added thereto. The supernatant was washed twice with washing, and the remaining precipitate was filtered to dry the residue in the desiccator to obtain a pure precipitate.

2. SD rat

Male 6 - week - old SD rats were purchased from Samtako (Osan, Korea) and raised in an animal breeding room of the College of Pharmacy, Pusan National University. Experimental animals were fed three cages per cage, and bottled water and feed (Superfeed Co., Wonju, Korea) were frozen. The animals were stabilized for at least 1 week before starting the experiment. Dexamethasone was administered intraperitoneally daily for 5 days, and ursolic acid and Lipid leaf extract were orally administered daily for 5 days. The animal protocols used in the present invention have been reviewed and approved by the Pusan National University Animal Experimental Ethics Committee.

After the stabilization period, the experimental animals were randomly grouped. The body weight change of the experimental animals was measured and recorded before administration of the active substance daily. Dexamethasone was diluted in physiological saline at a concentration of 600 μg / kg. As a positive control, leucine and ursolic acid were diluted to 600 mg / kg and 100 mg / kg, respectively. The active ingredient, Beech leaf extract, was diluted to 50 mg / kg and 200 mg / kg.

3. Measurement of muscle strength of experimental animals

The forelimb muscle strength of the experimental animals was measured using a T-shaped pull bar (Columbus instrument, OH, USA).

4. Results

Grip strength meter was used to measure the muscle strength of the forelimb of the experimental animals. As a result, dexamethasone-treated control group showed a significant decrease in muscle strength compared to the normal group. However, it was observed that the muscle strength was restored in a dose-dependent manner in the leucine-treated group, the ursolic acid-treated group, and the Beecha leaf extract-treated group (FIG. 1).

< Example  2> Loquat leaf  Inhibitory effect of extracts on muscle damage

1. Measurement of creatine kinase

The creatine kinase and creatine kinase levels in the blood were measured using the creatine kinase assay kit SICDIA CPK (Sinyang Co, Seoul, Korea). Reagents 1 and 2 were prepared by mixing CPK reagent-1B and CPK reagent-1A into CPK reagent-2B and CPK reagent-2A, respectively. Reagent 1 was mixed with 6 μl of sample and reacted at 37 ° C for 5 minutes. The following reagent 2 was added and the sample was measured at a wavelength of 340 nm using a microplate reader (TECAN, Salzburg, Austria).

2. Quantitative analysis of blood creatine kinase (creatine kinase)

Creatine kinase is an enzyme specifically present in the heart and muscle. Creatine kinase is used as an indicator of muscle or heart damage. In the control group treated with dexamethasone, the amount of creatine kinase in the blood was increased compared to the normal group. However, it was confirmed that the free amount of creatine kinase was decreased in a concentration-dependent manner by leucine (Leucine), ursolic acid, and non-leaf extract (Fig. 2).

3. Inhibitory effect of pea leaf extract on muscle damage determined by histological analysis

Haematoxylin & eosin (H & E) staining revealed that the muscle bundles were tightly structured in the normal group. However, in the control group treated with dexamethasone, it was confirmed that the degree of muscle damage indicated by the arrow was frequent. On the other hand, it was confirmed that the frequency of muscle damage was significantly lowered by the administration of leucine and ursolic acid, which were positive control groups, and that the extract of Beecha leaf was inhibited the degree of muscle damage in a concentration-dependent manner 3).

< Example  3> Loquat leaf  Inhibition of Muscle-related Gene Expression by Extract Administration

1. Protein Isolation from Muscle

All reagents, tubes and centrifuges were run at 0-4 ° C. The frozen muscle tissue was resuspended in 2 ml of hypotonic lysis buffer (buffer A: 10 mM KCl, 2 mM MgCl ₂ , 1 mM dithiothreitol (DTT), 0.1 mM EDTA, 0.1 mM PMSF, 1 mM pepstatin, 2 mM leupeptin, glycerophosphate, 20 mM NaF and 2 mM Na ₃ VO ₄ , 10 mM 4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid (HEPES), pH 7.4]. After 15 minutes, 125 μl of 10% Nonidet P-40 (NP-40) was added and mixed for 15 seconds. After centrifugation at 12,000 g for 5 minutes, the supernatant was collected and used as a cytoplasmic fraction. To the remaining pellet, 400 μl of A buffer and 25 μl of 10% NP-40 were added, and the mixture was centrifuged at 12,000 g for 5 minutes. The remaining pellet was added to 50 μl of buffer C [50 mM KCl, 300 mM NaCl, 20 mM β-glycerophosphate, 20 mM NaF, 2 mM Na ₃ VO _4, and 50 mM HEPES, pH 7.8], and the mixture was cryotomized for 30 minutes. After the reaction, centrifugation was performed at 12,000 g for 10 minutes. The supernatant was used as the nuclear fraction. Each sample was stored at -80 ° C. The protein content was determined by bicinchonic acid (BCA) assay and the standard was bovine serum albumin (BSA).

2. Western blotting &lt; RTI ID = 0.0 &gt;

The tissue homogenate containing a certain amount of protein was mixed with gel-loading buffer (0.125M Tris-HCl, pH 6.8, 4% SDS, 10% 2-mercaptoethanol and 0.2% bromophenolblue) and reacted for 5 min. Samples with the same total protein content were electrophoresed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for 1 hr 30 min at 100 V at 10-15%. And transferred to PVDF (polyvinylidene difluoride) membrane using Towbin buffer (25 mM Tris-Cl, 192 mM glycine, 20% MeOH). The membrane was blocked with blocking buffer (10 mM Tris, pH 7.5, 100 mM NaCl, and 0.1% Tween 20) containing 5% non-fat milk to block nonspecific protein binding. And washed three times with washing buffer for 10 minutes each. Each primary antibody was reacted at room temperature for 5 hours. After washing three times for 10 minutes with washing buffer, anti-rabbit antibody (Santa Cruz, 1: 10,000) or an anti-goat antibody (Santa Cruz, Santa Cruz, 1: 10,000) at room temperature for 1 hour. The ECL detection reagent, Westsave ^tm , was used to confirm the signal of the antibody, and the generated chemiluminescence was confirmed by Davinch-chem ™ SP (CoreBio, Seoul, Korea). The molecular weight was confirmed using pre-stained protein markers (Bio-Rad).

3. Changes in the amount of protein in MyHC following administration of Beech leaf extract

MyHC is a gene that is used as an indicator of muscle synthesis and degradation as a constituent of muscle. Observation of the amount of MyHC protein by Western blot revealed that the amount of MyHC protein was significantly decreased in the control group treated with dexamethasone as compared with the normal group. However, it was confirmed that the amount of MyHC protein was maintained at a level similar to that of the normal group in the positive control group and the non-pea leaf extract administration group (FIG. 4).

4. Inhibition of muscle degradation-related gene expression by treatment with Vile leaf extract

MuRF1, an E3 ligase, is known to directly target MyHC and participate in myopenia. As a result of confirming the amount of MuRF1 protein by Western blotting, the amount of protein was significantly increased in the control group treated with dexamethasone as compared with that of MyHC. Similarly, the amount of MuRF1 protein in the control group and the group treated with the Vile leaf extract was decreased compared with the control group. On the other hand, MAFbx indirectly involved in MuRF1 and hypoxia did not show any significant change. In addition, we confirmed that the nuclear level of the transcription factor FoxO1, which regulates MuRF1 transcription, is somewhat similar to that of MuRF1. Based on the above experimental results, it was found that the extract of Beecha leaf inhibits the migration of FoxO1 into the nucleus and inhibits the expression of MuRF1, thereby suppressing the degradation of MyHC, which is effective in suppressing muscle weakness and inhibiting muscle damage ).

< Example  4> Loquat leaf  Depending on the administration of the extract MyHC of Ubiquitination  Inhibitory effect

Since the degradation of MyHC by MuRF1 occurs via ubiquitination, the ubiquitination of MyHC was confirmed by immunoprecipitation. As a result of calculating the amount of ubiquitinated MyHC in the expression amount of MyHC, it was confirmed that the amount of ubiquitinated MyHC was significantly increased only in the control group to which dexamethasone was administered (FIG. 6).

Claims (6)

The peach leaf extract is a residue obtained by dissolving the extract of the peanut leaf with ethanol and then concentrating the extract with ethanol and then adding water to the extract. The extract of Foxa1 (Muscle ring finger 1) expression is suppressed by inhibiting the transcriptional activity of the Forkhead box O1. delete delete delete delete The extract of Beech leaf is extracted with ethanol, and the concentrated extract is dissolved again in ethanol. The residue is precipitated by addition of water. The extract of Foxhe (Forkhead box O1) Wherein the expression of MuRF1 (Muscle ring finger 1) is reduced by inhibiting the activity of the protein.

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