KR102686892B1 - Composition for preventing, improving or treating of muscular disease comprising Gardenia jasminoides extract - Google Patents

Abstract

The present invention provides a composition for preventing, improving or treating muscle disease comprising gardenia extract as an active ingredient; Composition for muscle strengthening; Composition for promoting muscle differentiation; Or, it relates to a composition for enhancing exercise capacity, wherein the composition promotes muscle differentiation, suppresses muscle atrophy factors and muscle differentiation inhibitors, and increases muscle differentiation regulators, making it useful for preventing, improving or treating muscle diseases. You can use it.

Description

Composition for preventing, improving or treating muscular disease comprising Gardenia jasminoides extract}

The present invention provides a composition for preventing, improving or treating muscle disease comprising gardenia extract as an active ingredient; Composition for muscle strengthening; Composition for promoting muscle differentiation; Or it relates to a food composition for enhancing athletic ability.

Muscle is the most abundant tissue in the human body, and securing an appropriate muscle mass is essential to maintain the functional capacity of the human body and prevent metabolic diseases. Muscle size is regulated by intracellular signaling pathways that induce anabolism or catabolism within the muscle. If more signaling reactions that induce synthesis rather than decomposition of muscle protein occur, muscle protein synthesis increases, resulting in muscle hypertrophy or an increase in the number of muscle fibers (hyperplasia), which increases the size of the muscle (The Korea Journal of Sports Science, 20(3): 1551-1561, 2011).

As the body ages, redistribution of body fat and body protein occurs due to changes in composition, and when the body reaches the age of about 50, the rate of protein synthesis within muscle cells becomes slower than the rate of decomposition, causing muscles to begin to deteriorate rapidly and being exposed to muscle loss diseases. You can.

Sarcopenia, one of the muscle loss diseases, refers to a condition in which approximately 13-24% of one's body mass is reduced, and shows a decrease in protein content, fiber diameter, muscle strength production, and fatigue resistance. Sarcopenia is caused by a variety of causes, including sepsis, cancer, renal failure, excess glucocorticoids, denervation, muscle underuse, and the aging process. Mainly, the causes include a gradual decrease in the quantity and quality of skeletal muscles that occurs as aging progresses and weight loss including fat and body fat components due to inadequate dietary energy intake. It is often due to aging and is highly correlated with age. .

Sarcopenia results from an imbalance between protein synthesis and breakdown. If you have sarcopenia, the amount of movement is significantly reduced, which not only harms your mental health but also reduces your satisfaction with life, and you can easily get injured even in easy everyday life, sometimes resulting in serious injuries.

Additionally, excessive exercise causes muscle fatigue and damage and reduces exercise ability. Muscle injuries include bruises, lacerations, ischemia, strains, and serious injuries to skeletal muscles. Not only do these injuries cause tremendous pain, but they can also be incapacitating, preventing the injured person from continuing to exercise, work, or even perform normal daily activities. When there is significant damage to skeletal muscles, strains are most common, and muscle strain injuries are characterized by destruction of the muscle-tendon unit. This destruction of the muscle-tendon unit can occur anywhere in the muscle. Contusions account for more than 30% of all injuries treated occupationally or by sports medicine professionals.

Treatments that can reduce muscle damage or accelerate muscle tissue recovery have the potential to accelerate recovery of muscle strength after exercise. It may also aid muscle recovery after illness.

However, with the recent increase in interest in appearance and diet, rapid weight loss can cause sarcopenia regardless of age, and muscles can be damaged by strenuous exercise. Accordingly, research and efforts are being focused on treating muscle loss due to general muscle loss diseases or increasing muscles, and research on treatment of muscle loss diseases and muscle strengthening is still required.

The purpose of the present invention is to provide a composition for preventing, improving or treating muscle diseases containing gardenia extract as an active ingredient.

Another object of the present invention is to provide a composition for muscle strengthening containing gardenia extract as an active ingredient.

Another object of the present invention is to provide a composition for promoting muscle differentiation containing gardenia extract as an active ingredient.

Another object of the present invention is to provide a composition for improving athletic performance containing gardenia extract as an active ingredient.

In order to achieve the above object, the present invention provides a composition for preventing, improving or treating muscle diseases containing gardenia extract as an active ingredient.

Additionally, the present invention provides a composition for muscle strengthening containing gardenia extract as an active ingredient.

Additionally, the present invention provides a composition for promoting muscle differentiation containing gardenia extract as an active ingredient.

Additionally, the present invention provides a food composition for improving athletic performance containing gardenia extract as an active ingredient.

The composition according to the present invention has the effect of promoting muscle differentiation, suppressing muscle atrophy factors and muscle differentiation inhibitory factors, increasing muscle differentiation regulators, and promoting or improving exercise performance, thereby preventing and improving muscle diseases. Alternatively, it can be useful for treatment.

Figure 1 shows the results of measuring cell viability after treating mouse-derived myoblasts with gardenia extract at given concentrations (0.5, 1, 2.5, 10, 25, and 50 μg/mL) (C: control; and GJ: gardenia) extract).
Figure 2 shows the results of observing the degree of differentiation into myotube cells through myosin heavy chain (MHC) immunostaining after treating mouse-derived myoblasts with gardenia extract (1 or 2.5 μg/mL) for 24 hours and myotubes. This is a result showing the degree of formation and fusion as a fusion index (%) (C: control; and GJ: gardenia extract).
Figure 3 shows the mRNA of MyoG and MyoD, which are muscle differentiation regulators, through reverse transcription-polymerase chain reaction (RT-PCR) after treatment with gardenia extract (1 or 2.5 μg/mL) during the differentiation of mouse-derived myoblasts into myotubes. This is the result of measuring the expression level (C: control; and GJ: gardenia extract).
Figure 4 shows mouse-derived myoblasts being differentiated into myotube cells, treated with 5 μM dexamethasone to induce muscle atrophy, and gardenia extract (1 or 2.5 μg/mL) treated simultaneously with 5 μM dexamethasone, followed by differentiation into myotube cells. The extent of this was observed through myosin heavy chain (MHC) immunostaining; And the root canal formation and degree of fusion are expressed as fusion index (%) (NC: control group; DEX: dexamethasone treated group; DEX+GJ 1: 1 μg/mL gardenia extract treated group; and DEX+GJ 2.5 : Group treated with 2.5 μg/mL gardenia extract).
Figure 5 shows that mouse-derived myoblasts were differentiated into myotube cells and then treated with 5 μM dexamethasone to induce muscle atrophy, and gardenia extract (1 or 2.5 μg/mL) was simultaneously treated with 5 μM dexamethasone, followed by reverse transcription polymerase chain reaction. mRNA expression levels of muscle atrophy regulators Atrogin-1, MurF-1, and Myostatin through reaction (RT-PCR); and the results of measuring the mRNA expression levels of MyoG and MyoD, which are muscle differentiation regulators (DEX: dexamethasone-treated group; GJ: gardenia extract-treated group; and NC: control group).
Figure 6 shows the results of measuring bone density and body composition of a mouse animal model after feeding gardenia extract to mice (red: body fat); and the results of measuring fat in tissue (%) and lean body mass (%, real body weight) (CTL: control group; DEX: mice administered dexamethasone; GJL: 0.05% mice fed with gardenia extract; and GJH: mice fed with 0.1% gardenia extract).
Figure 7 shows the results of measuring grip strength, hanging test, running time, and running distance after feeding gardenia extract to mice (CTL: control group; DEX: Mice administered dexamethasone; GJL: mice fed 0.05% gardenia extract and GJH: mice fed 0.1% gardenia extract).
Figure 8 shows the muscle mass of the quadriceps, gastrocnemius, triceps, tibialis anterior, extensor digitorum longus, and soleus after feeding gardenia extract to mice. These are the measured results (CTL: control group; DEX: mice administered dexamethasone; GJL: mice fed 0.05% gardenia extract; and GJH: mice fed 0.1% gardenia extract).
Figure 9 shows the results of measuring the cross-sectional area (CSA) of muscle fibers and distribution according to muscle fiber size after feeding gardenia extract to mice (CTL: control group; DEX: mice administered dexamethasone; GJL: mice fed 0.05% gardenia extract and GJH: mice fed 0.1% gardenia extract).
Figure 10 shows the results of analyzing the composition ratio (%) of MHC isoforms after feeding gardenia extract to mice (CTL: control group; DEX: mouse administered dexamethasone; GJL: fed 0.05% gardenia extract and GJH: mice fed with 0.1% gardenia extract).

Hereinafter, the present invention will be described in detail.

The present invention provides a composition for preventing, improving, or treating muscle diseases containing Gardenia jasminoides extract as an active ingredient.

The extract according to the present invention can be obtained by extraction and separation from nature using extraction and separation methods known in the art, and “extract” as defined in the present invention is extracted from gardenia using an appropriate solvent. , for example, includes all crude extracts, polar solvent-soluble extracts, or non-polar solvent-soluble extracts of gardenia.

An appropriate solvent for extracting the extract from the gardenia may be any pharmaceutically acceptable organic solvent. Water or an organic solvent may be used, but is not limited thereto, for example, purified water, methanol ( Alcohols with 1 to 4 carbon atoms including methanol, ethanol, propanol, isopropanol, butanol, acetone, ether, benzene, chloroform ( Various solvents such as chloroform, ethyl acetate, methylene chloride, hexane, and cyclohexane can be used individually or in combination.

As an extraction method, any one of the following methods can be selected and used: hot water extraction, cold needle extraction, reflux cooling extraction, solvent extraction, steam distillation, ultrasonic extraction, elution, and compression. In addition, the desired extract may be further subjected to a conventional fractionation process or may be purified using a conventional purification method. There is no limitation to the method for producing the gardenia extract of the present invention, and any known method can be used.

For example, the gardenia extract included in the composition of the present invention can be prepared in powder form by performing additional processes such as reduced pressure distillation and freeze-drying or spray-drying of the primary extract extracted by the hot water extraction or solvent extraction method. In addition, the primary extract was further purified into fractions using various chromatographies such as silica gel column chromatography, thin layer chromatography, high performance liquid chromatography, etc. You can also get

Therefore, in the present invention, gardenia extract is a concept that includes all extracts, fractions, and purified products obtained at each stage of extraction, fractionation, or purification, as well as their dilutions, concentrates, or dried products.

In one embodiment of the present invention, the composition may inhibit the expression of Atrogin-1 and MuRF-1 (Muscle RING-finger protein-1), which cause muscle atrophy, and the expression of Myostatin, which inhibits muscle differentiation. It may inhibit and increase the expression of MyoG (myogenin) and MyoD (myoblast determination protein 1), which promote muscle differentiation.

The above-mentioned muscle disease is a progressive disease characterized by loss of walking ability due to gradual decrease in muscle strength, weakened respiratory muscle, weakened heart function, etc. It is a chronic disease that eventually leads to physical disability and makes one dependent on others for all daily life. It is a human disease. In addition, muscle diseases can be divided into congenital diseases and acquired diseases, but are not limited to atony, muscular atrophy, muscular dystrophy, muscular dystrophy, muscular rigidity, and amyotrophic axonal sclerosis. , myasthenia gravis, cachexia, or sarcopenia.

Additionally, the present invention provides a food composition for preventing or improving muscle disease containing gardenia extract as an active ingredient.

Food compositions using gardenia extract according to the present invention include all forms such as functional foods, nutritional supplements, health foods, and food additives. The above types can be manufactured in various forms according to conventional methods known in the art.

For example, as a health food, the food composition of the present invention itself can be prepared and consumed in the form of tea, juice, and drink, or it can be consumed by granulating, encapsulating, and powdering. Additionally, the food composition of the present invention can be prepared in the form of a composition by mixing it with known substances or active ingredients known to have hair growth promotion and anti-inflammatory effects.

In addition, functional foods include beverages (including alcoholic beverages), fruits and their processed foods (e.g. canned fruits, bottled foods, jams, marmalade, etc.), fish, meat and their processed foods (e.g. ham, sausages, etc.) corned beef, etc.), breads and noodles (e.g. udon, buckwheat noodles, ramen, spaghetti, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, It can be manufactured by adding the food composition of the present invention to margarine, vegetable protein, retort food, frozen food, and various seasonings (e.g., soybean paste, soy sauce, sauce, etc.).

The preferred content of the food composition according to the present invention is not limited thereto, but is preferably 0.01 to 50% by weight of the total weight of the finally manufactured food. In order to use the food composition of the present invention in the form of a food additive, it can be prepared and used in the form of a powder or concentrate.

Additionally, the present invention provides a pharmaceutical composition for preventing or treating muscle diseases containing gardenia extract as an active ingredient.

The pharmaceutical composition according to the present invention may contain the gardenia extract alone or may be formulated in a suitable form together with a pharmaceutically acceptable carrier, and may additionally contain an excipient or diluent. In the above, 'pharmacologically acceptable' refers to a non-toxic composition that is physiologically acceptable and does not usually cause allergic reactions such as gastrointestinal disorders, dizziness, or similar reactions when administered to humans.

Pharmaceutically acceptable carriers may further include, for example, carriers for oral administration or carriers for parenteral administration. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. In addition, it may contain various drug delivery substances used for oral administration of peptide preparations. Additionally, the carrier for parenteral administration may include water, suitable oil, saline solution, aqueous glucose, glycol, etc., and may further include stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite or ascorbic acid. Suitable preservatives include benzalkonium chloride, methyl- or propyl-paraben and chlorobutanol. The pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, etc. in addition to the above components. Other pharmaceutically acceptable carriers and agents may be referred to as described in the following literature (Remington's Pharmaceutical Sciences, 19th ed., Mack Publishing Company, Easton, PA, 1995).

The composition of the present invention can be administered to mammals, including humans, by any method. For example, it can be administered orally or parenterally. Parenteral administration methods include, but are not limited to, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal administration. It can be.

The pharmaceutical composition of the present invention can be formulated as a preparation for oral administration or parenteral administration according to the administration route described above.

In the case of a formulation for oral administration, the composition of the present invention can be formulated into powder, granules, tablets, pills, sugar-coated tablets, capsules, solutions, gels, syrups, slurries, suspensions, etc. using methods known in the art. You can. For example, oral preparations can be prepared by combining the active ingredient with solid excipients, grinding them, adding suitable auxiliaries, and processing them into a granule mixture to obtain tablets or dragees. Examples of suitable excipients include sugars including lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol and maltitol, starches including corn starch, wheat starch, rice starch and potato starch, cellulose, Fillers such as cellulose, including methyl cellulose, sodium carboxymethylcellulose, and hydroxypropylmethyl-cellulose, gelatin, polyvinylpyrrolidone, etc. may be included. Additionally, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate may be added as a disintegrant. Furthermore, the pharmaceutical composition of the present invention may further include anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, and preservatives.

In the case of preparations for parenteral administration, they can be formulated in the form of injections, creams, lotions, external ointments, oils, moisturizers, gels, aerosols, and nasal inhalants by methods known in the art. These formulations are described in Remington's Pharmaceutical Science, 19th ed., Mack Publishing Company, Easton, PA, 1995, a commonly known text in all pharmaceutical chemistry.

The total effective amount of the composition of the present invention can be administered to a patient in a single dose, or can be administered by a fractionated treatment protocol in which multiple doses are administered over a long period of time. The pharmaceutical composition of the present invention may vary the content of the active ingredient depending on the severity of the disease. Preferably, the total preferred dosage of the pharmaceutical composition of the present invention may be about 0.01 μg to 10,000 mg, most preferably 0.1 μg to 500 mg per kg of patient body weight per day. However, the effective dose for the patient is determined by considering various factors such as the formulation method, administration route, and number of treatments as well as the patient's age, weight, health status, gender, severity of the disease, diet, and excretion rate. Therefore, taking this into account, anyone skilled in the art will be able to determine an appropriate effective dosage of the composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited in its formulation, administration route, and administration method as long as it exhibits the effects of the present invention.

Additionally, the present invention provides a composition for muscle strengthening containing gardenia extract as an active ingredient.

The term "muscle strengthening" of the present invention refers to the effect of increasing muscle strength and/or size, and does not limit the type of muscle. Preferably, it has the effect of increasing muscle mass and has the effect of suppressing muscle loss.

The increase in muscle mass improves muscle performance, and muscle mass can be increased through physical exercise and improved endurance, and muscle mass can be increased by administering a substance with a muscle-increasing effect into the body. In addition, the above-mentioned muscle loss is characterized by gradual loss of muscle mass, weakness and degeneration of muscles, especially skeletal or voluntary muscles and cardiac muscles, and can be caused by genetic or acquired factors. Aging may be the cause.

Additionally, the present invention provides a composition for promoting muscle differentiation containing gardenia extract as an active ingredient.

The term "myogenesis" in the present invention refers to the formation of muscle tissue, and the muscle fibers that make up the muscle are formed by differentiating into myotubes through the fusion of myoblasts.

Additionally, the present invention provides a composition for enhancing athletic performance containing gardenia extract as an active ingredient.

The term "athletic ability" of the present invention refers to physical movements that can be seen in daily life or sports, such as running, jumping, throwing, swimming, etc., when performing the movements quickly, strongly, accurately, long, and skillfully. As it indicates the extent to which an exercise can be performed, exercise performance is defined by factors such as strength, agility, and endurance.

As used herein, the term "enhancement of athletic ability" refers to improving or improving athletic ability. Preferably, the food composition has the effect of increasing muscle strength and has the effect of increasing muscle endurance. In addition, but not limited thereto, it may prevent or improve symptoms of degenerative diseases, mitochondrial abnormalities, low endurance, low agility, lethargy, muscle wasting, or depression. Preferably, the degenerative disease may be a degenerative disease that causes a decrease in muscle function, the mitochondrial abnormality disease may be a mitochondrial abnormality disease that causes a decrease in muscle function, and the depression is a depression that causes a decrease in muscle function. It may be.

Hereinafter, the present invention will be described in more detail through examples. These examples are for illustrating the present invention in more detail, and the scope of the present invention is not limited to these examples.

Example 1. Experimental method

1.1. statistical analysis

All research results were performed by one-way ANOVA using GraphPad Prism 7 (GraphPad Software Inc., SanDiego, CA, USA), and statistical significance was verified by Boneferroni multiple comparison post test, *p<0.05, **p Indicated as <0.01, ***p<0.001 and ****p<0.0001.

1.2. Preparation of Gardenia Extract

Dried gardenia was crushed and made into powder. Hydrothermal extraction was performed for 3 hours using a 50% ethanol solvent in a volume 10 times the weight of the powder. The extract was concentrated under reduced pressure, frozen at -80°C, and freeze-dried. The resulting final product was finely ground and used in the experiment.

Example 2. Cytotoxicity test of gardenia extract

The present inventors performed an experiment to determine cytotoxicity after treating mouse-derived myoblasts with gardenia extract. Briefly, the C2C12 cell line (CRL1772 ATCC, USA), a mouse-derived myoblast, was cultured in DMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS (fetal bovine serum) and 1% PS (penicillin-streptomycin). Gardenia extract was treated at given concentrations (0.5, 1, 2.5, 10, 25, and 50 μg/mL), and cell growth rate was analyzed after 24 hours.

As a result, it was confirmed that the gardenia extract did not exhibit cytotoxicity up to a concentration of 50 μg/mL (Figure 1).

Example 3. Effect of gardenia extract on promoting muscle differentiation (myogenesis)

3.1. Increased effect of root canal formation

The present inventors conducted an experiment to determine whether gardenia extract had the effect of promoting muscle differentiation after treating mouse-derived myoblasts. Briefly, the C2C12 cell line (CRL1772 ATCC, USA), a mouse-derived myoblast, was cultured in DMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS (fetal bovine serum) and 1% PS (penicillin-streptomycin). When the cells grow to 100% confluence in the culture vessel, change to DMEM (Dulbecco's Modified Eagle Medium) containing 2% HS (horse serum) and 1% PS (penicillin-streptomycin), and leave for a total of 4 days to form myotube cells. Differentiation took place during the period.

On the 4th day of differentiation, the cells were treated with 1 and 2.5 μg/mL of gardenia extract for 24 hours, and then myosin heavy chain (MHC) immunostaining was performed to determine the degree of differentiation into myotube cells. Differentiated myotube cells were fixed with 4% formaldehyde, permeabilized with 0.05% saponin, and blocked with 1% bovine serum albumin (BSA). Afterwards, the cells were stained with primary and secondary antibodies against myosin heavy chain (MHC), and the nuclei were stained with DAPI. The fusion index (%) was calculated using the following formula: fusion index = (nucleus in myotube cells / nuclei present in all cells) × 100 (%).

As a result, it was confirmed that when treated with gardenia extract, differentiation into myotube cells was enhanced over time (FIG. 2). In addition, it was confirmed that the fusion index, which indicates the degree of root canal formation and fusion, was significantly increased in the group treated with gardenia extract (1 and 2.5 μg/mL) compared to the control group (Figure 2). As a result, it was confirmed that gardenia extract has an effect of promoting muscle differentiation by increasing root canal formation.

3.2. Increased effect of muscle differentiation (myogenesis) regulators

The present inventors conducted an experiment to determine whether gardenia extract affects the expression levels of MyoG (myogenin) and MyoD (myoblast determination protein 1), which are muscle differentiation regulators. Briefly, the mouse-derived myoblast C2C12 cell line (CRL1772 ATCC, USA) was treated with 1 and 2.5 μg/mL of gardenia extract for 24 hours. Afterwards, the cells were washed with PBS, the cells were obtained, and total RNA was extracted using the NucleoSpin RNA Kit (Nacherey-Nagel, Duren, Germany) according to the manufacturer's instructions. Afterwards, cDNA was synthesized using Synthesize cDNA for real-time PCR Kit (ReverTra Ace qPCR RT Master Mix, FSQ-201, TOYOBO), and then PCR was performed using SYBR Green MasterMix (TOYOBO Co. Ltd., Osaka, Japan). The mRNA expression levels of MyoG (myogenin) and MyoD (myoblast determination protein 1) were measured. Primer sequences specific to each gene are shown in Table 1 below.

Base sequence (5' -> 3') sequence number Atrogin Sense GACTGGACTTCTCGACTGCC One Anti-sense TCAGGGATGTGAGCTGTGAC 2 MurF-1 Sense GCTGGTGGAAAACATCATTGACAT 3 Anti-sense CATCGGGTGGCTGCCTTT 4 Myostatin Sense ACGCTACCACGGAAACAATC 5 Anti-sense GGAGTCTTGACGGGTCTGAG 6 MyoG Sense GCAGGCTCAAGAAAGTGAATGA 7 Anti-sense TAGGCCTCAATGTACTGGAT 8 MyoD Sense CGGGACATAGACTTGACAGGC 9 Anti-sense TCGAAACACGGGTCATAGA 10 18s RNA Sense GTAACCCGTTGAACCCCATT 11 Anti-sense CCATCCAATCGGTAGTAGCG 12

As a result, it was confirmed that in the group treated with gardenia extract, the mRNA expression level of MyoG significantly increased compared to the control group, and the mRNA expression level of MyoD tended to increase (Figure 3). As a result, the present inventors confirmed that gardenia extract has the effect of enhancing muscle differentiation by increasing the expression levels of MyoG and MyoD.

Example 4. Effect of gardenia extract on promoting muscle differentiation and suppressing myotube atrophy

4.1. Increased effect of root canal formation

The present inventors conducted an experiment to determine whether gardenia extract had the effect of promoting muscle differentiation and suppressing myotube atrophy. Briefly, the C2C12 cell line (CRL1772 ATCC, USA), a mouse-derived myoblast, was cultured in DMEM (Dulbecco's Modified Eagle Medium) containing 10% FBS (fetal bovine serum), and DMEM containing 2% HS (horse serum). Differentiation induction medium was treated for 4 days. After differentiation induction was completed, the cells were treated with 1 and 2.5 μg/mL of gardenia extract and 5 μM of dexamethasone (DEX) for 24 hours. Afterwards, the degree of differentiation into myotube cells was observed by performing myosin heavy chain (MHC) immunostaining, and the degree of myotube formation and fusion was calculated as a fusion index (%).

As a result, in the group treated with dexamethasone, root canal formation decreased and fusion of root canal cells decreased compared to the control group, while in the group treated with gardenia extract, root canal formation increased compared to the group treated with dexamethasone, and root canal formation and fusion decreased. It was confirmed that the fusion index, which indicates the degree of fusion, significantly increased (Figure 4). As a result, it was confirmed that gardenia extract has the effect of suppressing muscle atrophy induced by dexamethasone and promoting muscle differentiation.

4.2. Inhibition of muscle atrophy regulators and increasing effect of muscle differentiation regulators

Afterwards, the cells were obtained after washing with PBS, and total RNA was extracted using the NucleoSpin RNA Kit (Nacherey-Nagel, Duren, Germany) according to the manufacturer's instructions. Afterwards, cDNA was synthesized using Synthesize cDNA for real-time PCR Kit (ReverTra Ace qPCR RT Master Mix, FSQ-201, TOYOBO), and then PCR was performed using SYBR Green MasterMix (TOYOBO Co. Ltd., Osaka, Japan). The mRNA expression levels of Atrogin-1, MurF-1 (Muscle RING-finger protein-1), and Myostatin were measured. Primer sequences specific to each gene are shown in Table 1 above.

As a result, the mRNA expression levels of Atrogin-1, MurF-1, and Myostatin increased in the group treated with dexamethasone compared to the control group (DMSO), while the mRNA expression levels of Atrogin-1, MurF-1, and Myostatin increased in the group treated with gardenia extract compared to the group treated with dexamethasone. It was confirmed that the mRNA expression levels of -1 and Myostatin were significantly decreased (Figure 5). In addition, in the group treated with dexamethasone, the mRNA expression levels of MyoG and MyoD decreased compared to the control group, whereas in the group treated with gardenia extract, the mRNA expression levels of MyoG and MyoD were significantly increased compared to the group treated with dexamethasone ( Figure 5).

Atrogin-1 and MurF-1 are genes known to cause muscle atrophy by destroying muscle proteins, and Myostatin is known to be an inhibitor of muscle differentiation. Additionally, MyoG and MyoD are known to be factors that promote muscle differentiation.

From the above results, the present inventors found that gardenia extract suppresses the expression levels of Atrogin-1 and MurF-1, which cause muscle atrophy, suppresses the expression levels of Myostatin, which suppresses muscle differentiation, and MyoG and MyoD, which promote muscle differentiation. It was confirmed that it has the effect of increasing the expression level.

Example 5. Effect of improving exercise performance and increasing muscle mass in a mouse animal model

5.1. Body fat reduction effect

The present inventors conducted an experiment to determine whether gardenia extract had an effect in reducing body fat in a mouse animal model. Mice were 8-week-old C57BL/6 male mice, and were fed a diet prepared by adding 0.05 or 0.1% (w/v) gardenia extract to the AIN-93M Adult maintenance Rodent Diet (Research diet, D10012M) base for 8 weeks. It was paid. The body weight of the tested mice was measured starting before the experiment was conducted and until the mouse was sacrificed.

Mice were intraperitoneally administered dexamethasone (Handong Pharmaceutical Dexasone) at 5 mg/kg starting 19 days before sacrifice. On the 15th day of intraperitoneal administration, the percentage of fat in tissue (%, fat in tissue) and percentage of body weight excluding fat (%, lean body mass, real body weight) of mice were measured using a bone density meter (InAlyzer, Medicors).

As a result of measuring the body composition of mice using a bone density meter (Inalyzer), mice administered dexamethasone (DEX) had a higher percentage of body fat shown in red compared to the control group, while mice fed gardenia extract (GJL, GJH) had a higher percentage of body fat. It was confirmed that it decreased to a level similar to that of the normal control group (FIG. 6).

In particular, in mice fed dexamethasone (DEX), tissue fat ratio (%) increased compared to the control group, while in mice fed gardenia extract (GJL, GJH), tissue fat ratio (%) increased compared to mice fed dexamethasone. ) was confirmed to be significantly reduced in a concentration-dependent manner (Figure 6). In addition, in mice fed dexamethasone (DEX), the percentage of body weight excluding fat (%) decreased compared to the control group, while in mice fed gardenia extract (GJL, GJH), the percentage of body weight excluding fat (%) recovered similar to that of the control group. It was confirmed that there was an effect (Figure 6). As a result, it was confirmed that gardenia extract is effective in reducing body fat increased by dexamethasone.

5.2. Effect of improving exercise performance

Afterwards, the present inventors conducted an experiment to determine whether gardenia extract had the effect of improving exercise performance in a mouse animal model. First, in the grip strength and hanging test, on the 18th day of intraperitoneal administration, the average value was calculated after having the mouse perform grip strength and hanging with the forelimbs 5 times repeatedly, and the skeletal muscle strength was expressed in g. Additionally, the running time and running distance of the mouse were measured using a treadmill (Ugo Basile). At this time, the movement distance of the mouse was measured at an incline of 10 degrees and 10 m/min from 0 to 20 minutes, and increased by 2 m/min every 2 minutes after 20 minutes.

As a result, mice fed dexamethasone (DEX) showed significantly reduced exercise performance in grip strength and hanging tests compared to the control group, while mice fed gardenia extract (GJL, GJH) showed significantly reduced exercise performance compared to the control group. It was confirmed that compared to mice fed dexamethasone, there was an effect of recovering or improving motor performance in grip strength and hanging tests (FIG. 7). In addition, in mice fed demetasone (DEX), running time and running distance decreased compared to the control group, while in mice fed gardenia extract (GJL, GJH), the running time and running distance decreased to a level similar to that of the control group. It was confirmed that there was an effect of recovering exercise time and exercise distance (Figure 7). As a result, it was confirmed that gardenia extract has the effect of recovering or improving exercise performance reduced by dexamethasone.

5.3. Effect of increasing muscle mass

Afterwards, the present inventors sacrificed the mice the next day after the end of 18 days of intraperitoneal administration, and analyzed the quadriceps, gastrocnemius, triceps, tibialis anterior, and extensor digitorum longus muscles. And muscle mass (muscle weight, expressed in mg per mouse body weight, mg/g body weight) was measured in 6 muscle parts of the soleus muscle.

As a result, mice fed dexamethasone (DEX) had a decrease in body muscle mass such as quadriceps and gastrocnemius compared to the control group, while mice fed gardenia extract (GJL, GJH) had a decrease in body muscle mass compared to the control group. In comparison, it was confirmed that the body muscle mass of the quadriceps and gastrocnemius muscles was significantly recovered in a concentration-dependent manner (Figure 8). As a result, it was confirmed that gardenia extract has the effect of restoring muscle mass reduced by dexamethasone.

5.4. Effect of increasing muscle fiber size

Laminin is a protein that exists in the extracellular matrix and is known to be an essential protein for the formation of cell structure. To determine whether gardenia extract affects the size of muscle fibers, the present inventors observed the cross sectional area of muscle fibers through laminin staining. Briefly, the gastrocnemius muscle of the mouse was fixed at -20°C with OCT compound, then sectioned to a thickness of 7 μm and attached to a slide. Afterwards, the sectioned muscle tissue was fixed with 20% acetone, blocked with 10% FBS, and stained with alexa fluor 488-conjugated laminin at 4°C for 16 hours. The stained tissue was washed with PBS, covered with a cover slide, and observed through a confocal microscope. In immunohistochemical staining, laminin appears green.

As a result, it was confirmed that in mice fed dexamethasone, the cross-sectional area of muscle fibers was smaller than that in the control group, whereas in mice fed gardenia extract, the cross-sectional area of muscle fibers significantly increased in a concentration-dependent manner (FIG. 9). In addition, from the distribution results according to muscle fiber size, it was confirmed that in mice fed dexamethasone, muscle fibers of 1,000 μm ² or less were about 45%, and muscle fibers of 1,500 μm ² or less were about 50%, whereas in mice fed gardenia extract, the muscle fibers were 1,500 μm 2 or less. It was confirmed that compared to mice fed dexamethasone, the number of muscle fibers smaller than 1,000 μm ² decreased, and the number of muscle fibers smaller than 2,000 μm ² increased in a concentration-dependent manner (FIG. 9). As a result, it was confirmed that gardenia extract has the effect of restoring the size of muscle fibers reduced by dexamethasone.

5.5. Effect of restoring MHC (myosin heavy chain) isoform composition ratio

MHC (myosin heavy chain) present in skeletal muscle has isoforms such as MHC1, MHC2A, and MHC2B, and each isoform is known to have different characteristics. In particular, MHC1 is a slow type and MHC2B is a fast type of muscle, and as aging progresses, fast-to-slow type conversion occurs. In order to determine whether gardenia extract affects the composition ratio of MHC subtypes, the present inventors performed an experiment to perform immunohistochemical staining of MHC types in muscle and analyze the composition ratio of MHC subtypes.

Briefly, the gastrocnemius muscle of the mouse was fixed at -20°C with OCT compound, then sectioned to a thickness of 7 μm and attached to a slide. Afterwards, the sectioned muscle tissue was fixed with 20% acetone, blocked with 10% FBS, and stained with MHC1, MHC2A, and MHC2B primary antibodies at 4°C for 16 hours. Afterwards, it was washed with PBS and stained with secondary antibody for 30 minutes at room temperature. The stained tissue was washed with PBS, covered with a cover slide, and observed through a confocal microscope. In immunohistochemical staining, MHC1 appears blue, MHC2A appears green, and MHC2B appears red.

As a result, in mice fed dexamethasone, slow type MHC1 increased and fast type MHC2B decreased compared to the control group, while in mice fed gardenia extract, MHC1 was at a similar level to the control group compared to mice fed dexamethasone. It was confirmed that MHC2B decreased significantly and MHC2B increased significantly (FIG. 10). As a result, it was confirmed that gardenia extract has the effect of reducing slow type MHC1 increased by dexamethasone and increasing fast type MHC2B decreased by dexamethasone.

<110> KOREA FOOD RESEARCH INSTITUTE <120> Composition for preventing, improving or treating muscular disease comprising Gardenia jasminoides extract <130> 1070467 <150> KR 10-2021-0002232 <151> 2021-01-07 <160> 12 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Atrogin_sense <400> 1 gactggactt ctcgactgcc 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Atrogin_anti sense <400> 2 tcagggatgt gagctgtgac 20 <210> 3 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> MurF-1_sense <400> 3 gctggtggaa aacatcattg acat 24 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> MurF-1_anti sense <400> 4 catcgggtgg ctgccttt 18 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Myostatin_sense <400> 5 acgctaccac ggaaacaatc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Myostatin_anti sense <400> 6 ggggtcttga cgggtctgag 20 <210> 7 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> MyoG_sense <400> 7 gcaggctcaa gaaagtgaat ga 22 <210> 8 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> MyoG_anti sense <400> 8 taggcgctca atgtactgga t 21 <210> 9 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> MyoD_sense <400> 9 cgggacatag acttgacagg c 21 <210> 10 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> MyoD_anti sense <400> 10 tcgaaacacg ggtcatcata ga 22 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 18s RNA_sense <400> 11 gtaacccgtt gaaccccatt 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> 18s RNA_anti sense <400> 12 ccatccaatc ggtagtagcg 20

Claims (12)

A food composition for preventing or improving muscle disease containing Gardenia jasminoides extract as an active ingredient,
The muscle diseases include atony, muscular atrophy, muscular dystrophy, muscle degeneration, muscle rigidity, amyotrophic axonal sclerosis, myasthenia gravis, cachexia, sarcopenia, and combinations thereof. It is selected from a group consisting of
A food composition wherein the gardenia extract is extracted using an ethanol solvent.
delete delete According to claim 1,
The composition inhibits the expression of Atrogin-1 and MuRF-1 (Muscle RING-finger protein-1), which cause muscle atrophy.
According to claim 1,
The composition inhibits the expression of Myostatin, which inhibits muscle differentiation.
According to claim 1,
The composition increases the expression of MyoG (myogenin) and MyoD (myoblast determination protein 1), which promote muscle differentiation.
delete A pharmaceutical composition for preventing or treating muscle diseases containing gardenia extract as an active ingredient,
The muscle diseases include atony, muscular atrophy, muscular dystrophy, muscle degeneration, muscle rigidity, amyotrophic axonal sclerosis, myasthenia gravis, cachexia, sarcopenia, and combinations thereof. It is selected from a group consisting of
A pharmaceutical composition, wherein the gardenia extract is extracted using an ethanol solvent.
delete delete A composition for improving athletic performance containing gardenia extract as an active ingredient,
The composition suppresses the expression of Atrogin-1 and MuRF-1 (Muscle RING-finger protein-1), which cause muscle atrophy, and suppresses the expression of Myostatin, which suppresses muscle differentiation,
The exercise capacity enhancement prevents or improves one or more symptoms selected from the group consisting of degenerative diseases that cause decreased muscle function, mitochondrial abnormalities, lethargy, and muscle wasting,
A composition wherein the gardenia extract is extracted using an ethanol solvent. delete