COMPOSITION FOR STRENGTHENING, DEVELOPING, DIFFERENTIATING, OR REGENERATING MUSCLE, OR PREVENTING, AMELIORATING, OR TREATING MUSCLE LOSS OR MUSCLE FATIGUE COMPRISING SCHIZONEPETA TENUIFOLIA EXTRACT AS EFFECTIVE COMPONENT

Abstract

A composition for strengthening, developing, differentiating, or regenerating muscle, or preventing, ameliorating, or treating muscle loss or muscle fatigue includes a Schizonepeta tenuifolia extract as an effective component. The Schizonepeta tenuifolia extract as an effective component has the effects of restoring the viability of muscle cells, increasing the endurance time on the rotarod performance test, i.e., the holding time without falling off from the rod, compared to the control group, reducing the content of LDH, AST, and ALT in blood, increasing the grip strength, enhancing the expression amount of energy metabolism-related genes in muscle tissues, increasing the density of muscle fibers in muscle tissues, inducing a change in the fibroblast arrangement to have a well-organized structure, and reducing the expression of MuRF1 in muscle tissues, as compared to the control group.

Claims

1-6. (canceled)

7. A method for strengthening, developing, differentiating, or regenerating muscle, and/or ameliorating muscle loss or muscle fatigue, the method comprising administering to a subject in need thereof a composition comprising a Schizonepeta tenuifolia extract as an effective component.

8. The method of claim 7, wherein an extraction solvent for extracting the Schizonepeta tenuifolia is any one selected from water, C.sub.1-C.sub.4 lower alcohol, and a mixture thereof.

9. A method for increasing muscle mass or promoting muscle generation, the method comprising administering to a subject in need thereof a composition comprising a Schizonepeta tenuifolia extract as an effective component.

10. A method for treating a muscular disorder, the method comprising administering to a subject in need thereof a composition comprising a Schizonepeta tenuifolia extract as an effective component.

11. The method of claim 10, wherein the muscular disorder is a muscular disorder that is caused by loss of muscle function, muscle atrophy, muscle loss, and/or muscle degeneration.

12. The method of claim 10, wherein the muscular disorder is one more selected from the group consisting of atony, muscular atrophy, muscular dystrophy, muscle weakness, cachexia, rigid spine syndrome, amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, and sarcopenia.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0012] FIG. 1 shows the results showing that the decreased cell viability induced by oxidative stress (i.e., H.sub.2O.sub.2 treatment) is restored by a treatment with Schizonepeta tenuifolia extract. #### indicates that there is a statistically significant decrease in cell viability of the oxidative stress (H.sub.2O.sub.2) group compared to the control group, with a p value of <0.0001. **** indicates that there is a statistically significant increase in cell viability of the group treated with the Schizonepeta tenuifolia extract of the present invention compared to the oxidative stress group, with a p value of <0.0001.

[0013] FIG. 2 shows the results demonstrating that the hanging time on the rotarod bar, which has been reduced by dexamethasone induction, was increased by a treatment with Schizonepeta tenuifolia extract. ## indicates a statistically significant decrease in hanging time in the dexamethasone-induced group compared to the normal group, with a p value of p<0.01. * indicates a statistically significant increase in hanging time on the rotarod bar in the group treated with Schizonepeta tenuifolia extract compared to the dexamethasone-induced group, with a p value of p<0.05. Oxy50 is the positive control group which has been administered with 50 mg/kg of oxymetholone.

[0014] FIG. 3 shows a change in the levels of LDH, AST, and ALT in blood after the administration of Schizonepeta tenuifolia extract. #### indicates a statistically significant increase in AST and ALT levels in the dexamethasone-induced group compared to the normal group (N), with a p value of p<0.0001. * indicates a statistically significant decrease in AST and ALT content in the group treated with 200 mg/kg Schizonepeta tenuifolia extract of the present invention compared to the dexamethasone-induced group, with a p value of p<0.05.

[0015] FIGS. 4A and 4B show the result of determining a change in grip strength after the administration of Schizonepeta tenuifolia extract. Specifically, FIG. 4A shows the grip strength evaluation results at the 3-week time point of Schizonepeta tenuifolia extract administration, while FIG. 4B shows the grip strength evaluation results at the 6-week time point. *, **, ***, and **** indicate a statistically significant increase in grip strength in the group administered with Schizonepeta tenuifolia extract compared to the control group. Specifically, * denotes a p value of p<0.05, ** denotes a p value of p<0.01, *** denotes a p value of p<0.001, and **** denotes a p value of p<0.0001.

[0016] FIGS. 5A to 5C show the result of determining a change in the expression amount of the genes that are related to energy metabolism, specifically PGC-1a (FIG. 5A), FNDC5 (FIG. 5B), and SIRT1 (FIG. 5C), in the muscle tissues of an animal model after the administration of Schizonepeta tenuifolia extract. * and ** indicate a statistically significant increase in the expression amount of PGC-1a, FNDC5, or SIRT1 genes in the group administered with Schizonepeta tenuifolia extract compared to the control group. Specifically, * denotes a p value of p<0.05 and ** denotes a p value of p<0.01.

[0017] FIG. 6 illustrates the results of examining the muscle tissues in an animal model after administration of Schizonepeta tenuifolia extract, in which the examination was made by using H&E staining technique. The areas indicated with oval represent the region where the tissues of the group administered with Schizonepeta tenuifolia extract are compared to the control group. Specifically, those areas clearly demonstrate differences in the density of fibers forming muscles and the organization level of the arrangement of fibroblast cells along the collagen fibers.

[0018] FIG. 7 presents the results of examining the muscle tissue in an animal model after the administration of Schizonepeta tenuifolia extract, in which the examination was made by using the PTAH staining technique. The areas indicated with arrow represent the region where the tissues of the group administered with Schizonepeta tenuifolia extract are compared to the control group. Specifically, those areas clearly demonstrate differences in the density of fibers forming muscles and the organization level of the arrangement of fibroblast cells along the collagen fibers.

[0019] FIG. 8 shows the results of examining the muscle tissues in an animal model after the administration of Schizonepeta tenuifolia extract, in which the examination was made by using immunohistochemical staining. The areas indicated by arrow represent the regions where the tissues of the group administered with Schizonepeta tenuifolia extract are compared to the control group. These areas show a clear difference in the expression of MuRF1.

DETAILED DESCRIPTION

[0020] The present invention relates to a functional health food composition for strengthening, developing, differentiating, or regenerating muscle, or preventing or ameliorating muscle loss or muscle fatigue comprising Schizonepeta tenuifolia extract as an effective component.

[0021] The Schizonepeta tenuifolia extract can be produced by a method including the following steps: [0022] (1) carrying out extraction by adding an extraction solvent to dried Schizonepeta tenuifolia; [0023] (2) centrifuging the extract of the step (1); and [0024] (3) drying the centrifuged extract of the step (2) to produce extract, but the method is not limited thereto.

[0025] The extraction solvent of the above step (1) is preferably selected from water, C.sub.1-C.sub.4 lower alcohol, and a mixture thereof. It is more preferably ethanol, but not limited thereto. With regard to the production method, any kind of common methods that are generally known as extraction method in the pertinent art, e.g., filtration, hot water extraction, impregnation extraction, extraction by reflux condensation, and ultrasonic extraction, can be used. It is preferable that the extraction is carried out by adding an extraction solvent in an amount of 1 to 20 times the weight of Schizonepeta tenuifolia. The extraction temperature is preferably between 60? C. and 100? C., but it is not limited thereto. Furthermore, the extraction time is preferably between 1 hour and 6 hours, but it is not limited thereto. It is preferable that the drying of the step (3) in the above method is preferably carried out by drying under reduced pressure, drying under vacuum, drying under boiling, spray drying, or freeze-drying, and it is more preferably freeze-drying, but it is not limited thereto.

[0026] The present invention further relates to a functional health food composition for increasing muscle mass or promoting muscle generation comprising Schizonepeta tenuifolia extract as an effective component.

[0027] The functional health food composition can be preferably prepared in any formulation selected from a powder, a granule, a pill, a tablet, a capsule, a candy, a syrup, and a drink, but it is not limited thereto. The functional health food composition of the present invention may be prepared by adding the effective component by itself or mixing it with other food product or other food component. The composition can be suitably prepared by following a common method. Examples of the food product to which the Schizonepeta tenuifolia extract of the present invention can be added include caramel, meat, sausage, bread, chocolate, candies, snacks, biscuits, pizza, ramen, other noodles, gums, dairy products including ice cream, various kinds of soup, beverage, tea, drink, alcohol beverage, and vitamin complex, and all functional health food products in general sense are included therein. Namely, the type of the food product is not particularly limited. The functional health food composition may further comprise various nutritional supplements, a vitamin, a mineral (i.e., electrolyte), a natural or synthetic flavor, a coloring agent, an enhancing agent (e.g., cheese and chocolate), pectinic acid and a salt thereof, alginic acid and a salt thereof, an organic acid, a protective colloidal thickening agent, a pH adjusting agent, a stabilizer, a preservative, glycerin, alcohol, and a carbonating agent used for carbonated drink. Other than those, fruit juice or fruit pulp for producing vegetable drink may be additionally comprised. Those ingredients may be used either singly or in combination thereof. Furthermore, the functional health food composition of the present invention may comprise various flavoring agents and natural carbohydrates as an additional component. Examples of the natural carbohydrates include monosaccharides like glucose and fructose, disaccharides like maltose and sucrose, polysaccharides like dextrin and cyclodextrin, and sugar alcohols like xylitol, sorbitol, and erythritol. Ratio of the natural carbohydrates is, although not critically important, preferably 0.01 g to 0.04 g relative to 100 g of the composition of the present invention. More preferably, it is comprised in an amount of 0.02 g to 0.03 g, but not limited thereto. As a sweetening agent, a natural sweetening agent like thaumatin and stevia extract and a synthetic sweetening agent like saccharine and aspartame can be used.

[0028] The present invention still further relates to a pharmaceutical composition for preventing or treating muscular disorders comprising Schizonepeta tenuifolia extract as an effective component.

[0029] The aforementioned muscular disorder is a muscular disorder that is caused by muscle function loss, muscle atrophy, muscle loss, or muscle degeneration, and it is more preferably any one selected from the group consisting of atony, muscular atrophy, muscular dystrophy, muscle weakness, cachexia, rigid spine syndrome, amyotrophic lateral sclerosis, Charcot-Marie-Tooth disease, and sarcopenia, but it is not limited thereto.

[0030] The pharmaceutically acceptable carrier to be contained in the pharmaceutical composition of the present invention is a carrier that is commonly used for preparing a formulation, and example thereof include saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, lactose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium carbonate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil, but it is not limited thereto. In addition to the mentioned ingredients, the pharmaceutical composition of the present invention may also contain an antioxidant, a buffer solution, an antimicrobial agent, a diluent, a surfactant, a binding agent, a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, or the like. The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the method of preparation, route of administration, patient's age, weight, gender, pathological condition, food, administration time, administration route, discharge rate, sensitivity to treatment, or the like. The route of administration for the pharmaceutical composition for preventing or treating muscle disorders of the present invention can generally be any route that allows it to reach the target tissue. Although not limited thereto, the pharmaceutical composition of the present invention can be administered via routes such as intramuscular administration, intraocular administration, intraperitoneal administration, intravenous administration, subcutaneous administration, intradermal patch administration, oral administration, intranasal administration, pulmonary administration, rectal administration, or the like, depending on the intended purpose. Specifically, the pharmaceutical composition can be administered via the intramuscular route.

[0031] Hereinbelow, the present invention is explained in greater detail in view of Examples. However, the following Examples are given only for specific explanation of the present invention and it is evident to a person who has common general knowledge in the pertinent art that the scope of the present invention is not limited by them.

EXAMPLES

Example 1. Preparation of Schizonepeta tenuifolia Extract

[0032] Schizonepeta tenuifolia was extracted by adding 15 liters of 70% ethanol to 1 kg of Schizonepeta tenuifolia and refluxing the mixture at 85? C. for 3 hours. The extract was then filtered, and the filtrate was concentrated and dried to give ethanol extract of Schizonepeta tenuifolia.

Example 2. Evaluation of Viability Recovery of Muscle Cells

[0033] Mouse myoblast cell line (i.e., C2C12 cells) was purchased from ATCC (Manassas, VA, USA) and cultured at conditions of 37? C. and 5% CO.sub.2 in DMEM medium containing 10% fetal bovine serum. Once the cultured cells reached 80% confluency, the medium was replaced with other DMEM medium (Gibco) containing 2% horse serum, 100 units/ml of penicillin, and 100 ?g/ml of streptomycin to induce cell differentiation. The cells were cultured for 5 days after replacing the medium to induce cell differentiation. The culture medium was replaced with fresh medium every 2 days during the differentiation process. To induce muscular atrophy, the cells were treated with H.sub.2O.sub.2 at a final concentration of 250 ?M. Concurrently, the cells were treated with Schizonepeta tenuifolia extract obtained from Example 1 at a concentration of 100 ?g/ml. After treating the cells with Schizonepeta tenuifolia extract and culturing them for 24 hours, the medium was replaced with fresh medium containing 10% CCK8 (Dojindo) solution. After incubating for 2 hours, the absorbance was measured at 450 nm to evaluate cell viability. Each sample was measured three times, and the mean value was calculated.

[0034] The results show that, as illustrated in FIG. 1, the muscle cell proliferation rate was found to be higher in the group treated with the Schizonepeta tenuifolia compared to the group treated with H.sub.2O.sub.2.

Example 3. Efficacy Evaluation Using Animal Model of Fatigue

[0035] A 6-week-old male BALB/c mice was purchased from Orient Bio Inc. (Seongnam, South Korea) and used them for the test. The test animals were acclimated for one week in a breeding environment with a temperature of 25?2? C., humidity of 50?5%, and a 12-hour light-dark cycle, while being provided with ad libitum access to a standard diet (AIN-76A diet) and water. After the acclimation period, the animals were used for the test. The animal test of the present invention was carried out after obtaining an approval of Korea Institute of Oriental Medicine Institutional Animal Care and Use Committee.

[0036] The acclimated mice were randomly assigned to different groups: the normal group (Normal), the group administered with dexamethasone group (25 mg/kg), the group administered with Schizonepeta tenuifolia extract (HG, 200 mg/kg), and the positive control group administered with oxymetholone (50 mg/kg). Each group consisted of 6 mice.

[0037] The dosage of Schizonepeta tenuifolia extract was set by using the formula that is based on the daily human intake adjusted according to the body surface area [Animal dose (mg/kg)=Human dose (mg/kg)?conversion factor]. The extract prepared as a dry powder was dissolved in physiological saline and used for the administration. After two weeks of oral administration of the test sample, the evaluation was made by Rotarod test and also in terms of a change in fatigue-related factors in the blood.

(1) Rotarod Test

[0038] Mouse was orally administered for 2 weeks with each test material, and then placed on the rod of a Rotarod apparatus and rotated at a speed of 20 rpm. The time until the mouse fell off the rod was then measured. Six animals were used in each group, and each animal was subjected to the test three times. The mean value of the three measurements for each animal was taken as the test result value, and the mean and standard deviation were calculated for each group of the six animals.

[0039] According to the results described in FIG. 2, the group administered with dexamethasone (DEX) showed significantly decreased hanging time on the Rotarod compared to the normal group (N). On the contrary, the group administered with Schizonepeta tenuifolia extract (HG) of the present invention and the positive control group administered with oxymetholone showed a statistically significant increase in the time spent hanging on the Rotarod.

(2) Effect Exhibited on Fatigue-Related Factors in the Blood

[0040] At the end of the 1-hour time point after the oral administration of each test material for 2 weeks, the Rotarod test was carried out, and blood samples were collected immediately from the animal heart. The collected blood was centrifuged at 3,000 rpm, 4? C. for 15 minutes to separate the blood serum, which was then stored at ?70? C. until further analysis. Thereafter, concentrations of lactate dehydrogenase (LDH), aspartate aminotransferase (AST), and alanine aminotransferase (ALT) in the blood serum were measured by using a biochemical analyzer (AU480 chemistry analyzer, Beckman Coulter Inc., Brea, CA, USA).

[0041] According to the results illustrated in FIG. 3, the group administered with Schizonepeta tenuifolia extract showed a tendency of reducing the elevated levels of LDH, ALT, and AST which had been induced by the administration of dexamethasone.

Example 4. Evaluation of Muscle Strength Using Animal Model

[0042] Nine week-old male mice and twelve month-old male mice were purchased from Orient Bio Inc. and acclimated for one week before the test. They were provided with ad libitum access to food and water. Schizonepeta tenuifolia extract was prepared at the following concentrations and administered orally every day. The 9 week-old mice were divided into the following three groups: (1) Control (untreated group), (2) group administered with Schizonepeta tenuifolia extract at a dose of 100 mg/kg, and (3) group administered with Schizonepeta tenuifolia extract at a dose of 200 mg/kg. The 12 month-old mice were divided into the following three groups: (4) Control (untreated group), (5) group administered with Schizonepeta tenuifolia extract at a dose of 100 mg/kg, and (6) group administered with Schizonepeta tenuifolia extract at a dose of 200 mg/kg. Each group consisted of 7 to 8 mice, and the drugs were administered orally for a period of 6 weeks. Grip strength test was conducted at Week 3 and Week 6 to evaluate muscle strength.

[0043] The grip force test involved measuring the hanging time on a crossed wire mesh to evaluate grip strength. To ensure reliability, three separate measurements were taken in total, and the highest value recorded was used for analysis. A full 20-minute rest period was provided between each measurement. The measurement procedure involved gently holding the body of the mouse while grasping its tail and guiding it to hold onto a bar positioned 10 cm above the floor on a grip strength meter. The tail was then pulled backward at a constant speed until both forelimbs of the mouse are released from the bar. The maximum grip strength (g) recorded on the grip strength meter was recorded, and the strongest force among the three measurements was considered as the grip strength.

[0044] The results showed that, at (A) the time point of Week 3, grip strength was significantly improved in both the young mouse group (i.e., only few month-old) and the adult mouse group (i.e., older than few month-old), which had been administered with 200 mg/kg of Schizonepeta tenuifolia extract, compared to the control group (untreated group). At (B) the time point of Week 6, grip strength in the young mouse group was significantly improved in the group administered with 200 mg/kg Schizonepeta tenuifolia extract compared to the control group. In the adult mouse group, grip strength was significantly higher in both groups which have been administered with either 100 mg/kg or 200 mg/kg Schizonepeta tenuifolia extract compared to the control group (FIGS. 4A and 4B).

Example 5. Analysis of Expression of Energy Metabolism-Related Genes Isolated from Muscle Tissues of Animal Model

[0045] PGC-1? (peroxisome proliferator-activated receptor-gamma coactivator-1 alpha) is a transcription factor known to be involved in muscle metabolism, specifically in mitochondria responsible for cellular energy metabolism, where it promotes the oxidation of fatty acids, leading to ATP production. FNDC5 (fibronectin type III domain containing 5) is known as a protein activated by PGC1-? and serves as a precursor protein of irisin. SIRT1 (silent mating type information regulation 2 homolog; sirtuin) is known to mediate dietary restriction as a strategy for regulating aging. It is also known as a transcription factor which is, upon the activation, involved in mitochondrial biosynthesis in muscle cells.

[0046] Following the muscle strength evaluation in Example 4 described above, muscles of both the adult and young mice were separated, and real-time polymerase chain reaction (PCR) was performed to examine a change in mRNA expression amount of PGC-1?, FNDC5, and SIRT1.

[0047] As a result, it was found that there is no significant change observed in the young mouse group. However, in comparison to the young mouse group, the expression amount of PGC-1a, FNDC5, and SIRT1 was higher in the adult mouse group.

[0048] Furthermore, in the adult mouse group, the administration of 200 mg/kg of Schizonepeta tenuifolia extract significantly increased the expression amount of PGC-1?, FNDC5, and SIRT1 compared to the control group (untreated group) (FIGS. 5A to 5C)

Example 6. Histological Analysis of Muscles of Animal Model

(1) Hematoxylin and Eosin (H&E) Staining

[0049] After Example 4 described above, the gastrocnemius muscles were excised during autopsy, and their weights were measured. The muscles were then separated for RNA analysis and protein analysis, and stored at ?80? C. until further analysis.

[0050] For H&E staining, the gastrocnemius muscle tissues excised from both the adult mouse group and young mouse group were fixed using 10% neutral-buffered formalin. Subsequently, the tissues were embedded in paraffin. The peeled sections were immersed in hematoxylin solution for 5 minutes, distilled water for 5 minutes, and eosin solution for 30 seconds. After that, the sections were fixed by drying.

[0051] According to the H&E staining results, it was observed that, compared to the control group (untreated group), both the young mouse group and adult mouse group administered with 200 mg/kg of Schizonepeta tenuifolia extract exhibited densely packed muscle fibers for forming muscles and orderly arrangement of fibroblasts along the collagen fibers (FIG. 6).

(2) PTAH (Phosphotungstic Acid Hematoxylin) Staining

[0052] The paraffin-fixed tissues were thinly sliced into 4 ?m sections and stained using the PTAH staining method. The peeled sections were treated with a 1% potassium permanganate solution for 5 minutes and then washed three times with PBS. After the washing, the sections were treated with PTAH solution for 24 hours, followed by clear sealing and examination under an optical microscope.

[0053] As a result, it was found that there was a loss of muscle fibers and a decrease in fiber density in the adult mouse group compared to the young mouse group. A reduction in the number of fibroblasts and loss of muscle fiber striations were also observed. The arrangement of fibroblasts appeared irregular and unrelated compared to the control group (untreated group), with some clusters observed. In the adult mouse group, it was also found that many fibroblasts with a small and stick-like shape were aligned somewhat in rows. (FIG. 7).

(3) Immunohistochemical Staining

[0054] MuRF1 (muscle RING-finger protein-1) is an E3 ubiquitin ligase expressed in skeletal muscle and it is known as one of the factors involved in muscle degradation. Based on immunohistochemical staining using MuRF1, protein expression of MuRF1 was analyzed.

[0055] Specifically, the fixed gastrocnemius muscles were embedded in paraffin and attached to a slide after being sliced into 5 ?m-thick section. For immunohistochemical staining, the sections were deparaffinized, and then subjected to a treatment with citrate buffer (i.e., citrate buffer antigen, epitope). After removing the epitope, the sections were incubated overnight at 4? C. with MuRF1 antibody which has been diluted at 1:500 dilution. After rinsing with PBS for 1 hour, the LSAB (labeled streptavidin-biotin) kit was applied, and the sections were stained with DAB (3,3-diaminobenzidine) for visualization and then examined under an optical microscope.

[0056] As a result, it was found that, in the case of the adult mice, there is an increase in the expression of MuRF1 in the muscle of the control group (untreated group), but the expression was reduced by the administration of 200 mg/kg Schizonepeta tenuifolia extract of the present invention (FIG. 8).

[Statistical Processing]

[0057] In the Examples of the present invention, the obtained data were analyzed by using one-way analysis of variance (ANOVA) to determine the statistically significant variance among the groups compared to each measured final value. The statistical significance between each group was assessed by using nonparametric Mann-Whitney test and Dunnett's multiple comparison test (IBM SPSS Statistics version 19.0 Software, Inc., IBM, USA). The given results are described with statistical significance based on the criteria of *p<0.05, **p<0.01, and ***p<0.001.