Composition for improving athletic performance containing a gypenosidic compound as an active ingredient

Abstract

The present invention relates to a composition for improving athletic performance, or for preventing, improving or treating muscle diseases, which contains a gypenosidic compound as an active ingredient. The composition of the present invention shows excellent effects in improving athletic performance and promoting physical strength by containing a gypenosidic compound as an active ingredient, and is therefore expected to be very useful in the fields of medicine and functional foods.

Description

[Technical field]

[0001] The present invention relates to a composition for improving athletic performance, which contains a gypenosidic compound as an active ingredient. [Background technology]

[0002] In today's complex society, people are exposed to a worsening living environment due to environmental pollution, increased mental stress, lack of physical activity, etc., and are becoming more and more interested in improving their health. Exercise is the most effective and economical way to prevent adult diseases or aging, but modern people who are unable to manage their health due to busy daily lives and fatigue are interested in taking various functional foods as an alternative to exercise. In addition to scientific training and dietary therapy according to the type of exercise, athletes use ergogenic aids to improve the efficiency of their athletic performance. In addition to improving athletic performance, ergogenic aids are also effective in removing fatigue substances that accumulate in the body during physical activity and cause fatigue, and are often used by athletes as well as ordinary people.

[0003] Research into functional supplements for improving athletic performance is being actively conducted in both the East and the West. Supplements containing compounds such as steroids, caffeine, sodium bicarbonate, or sodium citrate can increase athletic performance and add vitality to daily life, but this is only a temporary phenomenon and can be accompanied by potentially devastating side effects on health.

[0004] As a result, there has been a growing need recently for the development of functional adjuvants using natural products or compounds derived from natural products, such as plant extracts, whose safety has been guaranteed.

[0005] Therefore, in the course of researching compounds derived from natural products that have the effect of improving athletic performance and improving or treating muscle diseases, the inventors confirmed that gypenoside compounds significantly enhance the effect of improving athletic performance, and thus completed the present invention. Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a food composition for improving athletic performance, which contains a gypenosidic compound as an active ingredient.

[0007] Another object of the present invention is to provide a pharmaceutical composition for improving athletic performance, which contains a gypenosidic compound as an active ingredient. [Means for solving the problem]

[0008] In order to achieve the above-mentioned object, the present invention provides a health functional food composition for improving athletic performance, which contains a gypenosidic compound represented by the following Chemical Formula 1, its stereoisomer, or a nutrient-based acceptable salt thereof as an active ingredient.

[0009] [ka]

[0010] According to one embodiment of the present invention, the active ingredient may be gypenoside L or gypenoside LI.

[0011] According to one embodiment of the present invention, the active ingredient may be gypenoside L and gypenoside LI.

[0012] According to one embodiment of the present invention, the active ingredients may be gypenoside L and gypenoside LI in a weight ratio of 100:20-80.

[0013] According to one embodiment of the present invention, the active ingredients may be gypenoside L and gypenoside LI in a weight ratio of 100:30-70.

[0014] According to one embodiment of the present invention, the administration dose of the active ingredient may be 0.01 to 200 mg / kg / day.

[0015] The present invention also provides a pharmaceutical composition for improving athletic performance, comprising the gypenosidic compound represented by Chemical Formula 1 above, its stereoisomer, or its pharma- ceutical acceptable salt as an active ingredient.

[0016] The present invention also provides a health functional food composition for preventing or improving muscle diseases, which contains the gypenosidic compound represented by the above Chemical Formula 1, its stereoisomer, or a nutrient-based acceptable salt thereof as an active ingredient. Effect of the Invention

[0017] The composition of the present invention contains a gypenoside compound as an active ingredient and exhibits excellent effects in improving athletic performance and physical strength, and is therefore expected to be very useful in the fields of medicine and functional foods.

[0018] In addition, the composition of the present invention contains a gypenosidic compound as an active ingredient, and thus has excellent effects in reducing ROS generation and activating PGC-1α and AMPK, which are involved in mitochondrial function in muscles. It also activates Nrf2, which protects mitochondria from oxidative stress and regulates the expression of antioxidant genes that can suppress muscle damage. It can also increase the expression of TFAM, CPT-1β, and mtDNA, which are involved in the replication of mitochondria in muscles, and improve the expression of GSY, SIRT1, and PPARγ, which are involved in the change of muscle type and the generation of energy, so it can be usefully used to improve exercise performance. In addition, the effect of improving muscle fatigue, the increase in the exercise time and exercise amount until the time of weakness, and the increase in intramuscular glycogen content can significantly improve exercise performance.

[0019] Since the present invention uses a compound derived from a natural product as an active ingredient, it can be used safely without side effects and can be usefully used in medicines, foods, etc. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0020] The present invention will be described in detail below.

[0021] The term "motor performance ability" or "motor ability" in this specification refers to the degree to which physical movements seen in daily life and sports can be performed quickly, strongly, accurately, long, and well when the movements are externally classified into running, jumping, throwing, swimming, etc., and motor performance ability is defined as factors such as muscle strength, sense of balance, motor coordination, agility, and endurance. The term "improving motor performance ability" refers to improving or enhancing motor performance ability, and specifically means improving or enhancing endurance, sense of balance, or muscle strength.

[0022] As used herein, the term "pharmaceutical acceptable salts" refers to a form of a compound that does not induce significant irritation to an organism to which the compound is administered and does not impair the biological activity and physical properties of the compound.

[0023] The "pharmaceutically acceptable salt" includes, for example, acid addition salts formed by addition of inorganic acids such as hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, hydrobromic acid, hydroiodic acid, etc., or organic acids such as tartaric acid, formic acid, citric acid, acetic acid, trichloroacetic acid, fluoroacetic acid, gluconic acid, benzoic acid, lactic acid, fumaric acid, maleic acid, salicylic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, etc. Examples of pharmaceutically acceptable carboxylic acid salts when a carboxylic acid group is present in the compound of formula 1 above include metal salts or alkaline earth metal salts formed by lithium, sodium, potassium, calcium, magnesium, etc., amino acid salts such as lysine, arginine, guanidine, etc., organic salts such as dicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine, diethanolamine, choline, and triethylamine, etc. The compound of formula 1 according to the present invention can be converted into its salt by a conventional method.

[0024] The term "stereoisomer" as used herein refers to an isomer that has the same chemical or molecular formula but is generated by changing the spatial arrangement of atoms in a molecule, and is classified as an "enantiomer" or a "partial stereoisomer". The "enantiomer" refers to an isomer that does not overlap with its mirror image like the relationship between a right hand and a left hand, and the "partial stereoisomer" refers to an isomer among stereoisomers that does not undergo optical inversion. Partial stereoisomers include geometric isomers that have a non-rotatable bond such as a double bond, shape isomers that have a different temporary arrangement due to the rotation of a single bond, or general partial stereoisomers that have multiple stereocenters but cannot form a mirror image. All of these isomers and mixtures thereof are also included within the scope of the present invention.

[0025] As used herein, the term "active ingredient" refers to an ingredient that exhibits a desired activity alone or that may exhibit activity together with a carrier that is inactive by itself.

[0026] The present invention provides a food composition for improving athletic performance, which comprises a gypenosidic compound, a stereoisomer thereof, or a food-logically acceptable salt thereof as an active ingredient.

[0027] The gypenosidic compound may be a gypenosidic compound L (gypenosidic 50) represented by the following formula 2:

[0028] [ka]

[0029] In the present invention, "gypenoside L" is used interchangeably with "Gypenoside L", "Gyp L", "Gyp 50", "G 50", "gypenoside 50", and "gypenoside 50", and means a type of gypenoside (gypenoside; Gyp).

[0030] The gypenoside L can be chemically synthesized or isolated from natural substances, and when the gypenoside L of the present invention is isolated from natural substances, the concept may include either an extract of a natural product or a fraction thereof, so long as it contains the gypenoside L.

[0031] Additionally, the compounds of the present invention may have asymmetric carbon centers and therefore may exist as R or S isomers, racemates, mixtures of partial stereoisomers, and individual partial stereoisomers, all of which forms and mixtures are within the scope of the present invention.

[0032] The term "gypenoside (Gyp)" as used herein means triterpenoid saponin. Known types of gypenoside include Gyp L and Gyp LI, as well as Gyp LXXV, Gyp XVII, Gyp XLIX, Gyp XXIV, and Gyp XLV.

[0033] The gypenoside L of the present invention can be obtained by hydroxylation from ginsenoside Rg3 by known methods, or can be isolated from a plant extract and used, or can be a commercially available compound.

[0034] As used herein, the term "hydroxylation" refers to a reaction that introduces a hydroxyl group (OH) into an organic compound, either by directly introducing a hydroxy group or by replacing an existing substituent with a hydroxy group.

[0035] Furthermore, the gypenosidic compound may be a stereoisomer of the compound represented by Chemical Formula 2 above, preferably a partial stereoisomer, and more preferably a gypenosidic compound LI (gypenosidic 51) represented by Chemical Formula 3 below.

[0036] [ka]

[0037] The "gypenoside LI" of the present invention is used interchangeably with "Gypenoside LI", "Gyp LI", "Gyp 51", "G 51", "gypenoside 51", and "gypenoside 51", and is a type of gypenoside (Gyp) and is a partial stereoisomer of the gypenoside L compound represented by Chemical Formula 2.

[0038] In one embodiment, the compound represented by Chemical Formula 2 or the compound represented by Chemical Formula 3 may be isolated from a Jiaogulan leaf extract. For example, the Jiaogulan leaf extract may be an ethanol extract, a hot water extract, a hexane extract, an ethyl acetate extract, or an ultra-high pressure extract of Jiaogulan leaves.

[0039] In one embodiment, the Jiaogulan leaf extract may be obtained by extracting Jiaogulan leaves with one or more solvents selected from the group consisting of water, an organic solvent having 1 to 6 carbon atoms, a subcritical fluid, and a supercritical fluid. For example, the Jiaogulan leaves may be extracted under ultra-high pressure conditions of 100 MPa or more. If necessary, the Jiaogulan leaf extract may be prepared by further performing filtration and concentration steps according to methods known in the art. In one embodiment, the organic solvent having 1 to 6 carbon atoms may be one or more selected from alcohol having 1 to 6 carbon atoms, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane, cyclohexane, and petroleum ether.

[0040] The active ingredient contained in the composition of the present invention may preferably be gypenoside L, gypenoside LI, or a mixture thereof.

[0041] The weight ratio of gypenoside L and gypenoside LI may be 100:20-80, preferably 100:30-70, more preferably 100:50-70.

[0042] When the weight ratio of gypenosid LI to gypenosid L is within the above range, the effect of improving athletic performance can be maximized. When the weight ratio of gypenosid LI to gypenosid L is less than the lower limit, the effect of improving athletic performance of the composition is small, and when it exceeds the upper limit, the effect of improving athletic performance of the composition is rather reduced.

[0043] The health functional food composition of the present invention can provide a favorable effect of improving exercise performance when it contains the gypenosidic compound represented by the above formula 2, its stereoisomer, or its food-based acceptable salt in an effective dose. In this specification, the term "effective dose" refers to an amount that shows a higher reaction than the negative control group, and preferably refers to an amount sufficient to improve exercise performance. The health functional food composition of the present invention may contain the gypenosidic compound represented by the above formula 2, its stereoisomer, or its food-based acceptable salt in an amount of 0.001 to 99.99% by weight, preferably 0.05 to 50% by weight, and the remaining amount may be occupied by a food-based acceptable carrier. The effective dose of the active ingredient contained in the health functional food composition of the present invention will vary depending on the form in which the composition is commercialized. For the purpose of achieving favorable effects, the dosage of the gypenosidic compound represented by the above formula 2 of the present invention, its stereoisomer, or its food-based acceptable salt is 0.001-400mg / kg, preferably 0.01-200mg / kg, more preferably 0.01-100mg / kg, even more preferably 0.1-50mg / kg, particularly preferably 1-20mg / kg, and particularly preferably 5-20mg / kg, and may be administered 1-3 times a day. The dosage does not limit the scope of the present invention in any aspect.

[0044] The term "health functional food" as used in the present invention means food manufactured and processed using raw materials or ingredients that have functional properties useful to the human body according to the Health Functional Food Act No. 6727, and "functional" means that it is consumed for the purpose of obtaining beneficial effects for health purposes, such as regulating nutrients for the structure and function of the human body or physiological actions.

[0045] The health functional food composition may contain one or more of a carrier, a diluent, an excipient, and an additive, and may be formulated into any one selected from the group consisting of a tablet, a pill, a powder, a granule, a powder, a capsule, and a liquid dosage form.

[0046] In addition, the health functional food composition can be prepared in the form of a composition by mixing the gypenosidic compound represented by Chemical Formula 2, its stereoisomer, or its food-scientifically acceptable salt with a known substance or active ingredient known to have the effect of improving athletic performance. For example, the health functional food composition of the present invention may further contain, in addition to the gypenosidic compound, trace amounts of minerals, vitamins, sugars, and known ingredients having the effect of improving athletic performance.

[0047] The present invention also provides a pharmaceutical composition for improving athletic performance, comprising the gypenosidic compound represented by the above Chemical Formula 2, its stereoisomer, or its pharma- ceutical acceptable salt as an active ingredient.

[0048] The pharmaceutical composition for improving athletic performance of the present invention can be used to prevent or treat diseases caused by degeneration of athletic performance. Examples of related diseases include degenerative diseases, mitochondrial disorders, decreased stamina, decreased explosiveness, asthenia, muscle wasting, and depression. The composition of the present invention has an effect of improving athletic performance, and does not limit the type and type of exercise. The composition of the present invention has an effect of improving athletic performance, and does not limit the type and type of exercise.

[0049] The pharmaceutical composition may further include a pharma- ceutically acceptable carrier. The pharma-ceutically acceptable carrier is one that is commonly used in formulation, and includes, but is not limited to, saline, sterile water, Ringer's solution, buffered saline, cyclodextrin, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, etc., and may further include other common additives such as antioxidants and buffers, if necessary. In addition, diluents, dispersants, surfactants, binders, lubricants, etc. may be further added to the pharmaceutical composition to be suitably formulated into an aqueous solution, suspension, fluid, and formulation according to each component. The pharmaceutical composition of the present invention is not particularly limited in dosage form, and may be formulated into an injection, oral administration, skin topical agent, etc.

[0050] The pharmaceutical composition can be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method, and the dosage varies depending on the condition and weight of the patient, the degree of the disease, the drug form, the administration route, and the time, but may be appropriately selected by those skilled in the art.

[0051] Also, the dosage level selected from the composition will depend on the activity of the compound, the route of administration, the severity of the condition being treated, and the condition and previous medical history of the patient being treated. However, it is within the knowledge of the art to start with a dose of the compound lower than that required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved, and the preferred dosage may be determined by age, sex, body type, and weight. The composition may be further processed, preferably milled or ground into smaller particles, before being formulated into a pharmaceutically acceptable pharmaceutical preparation. Also, the composition may vary depending on the condition and the patient being treated, which may be determined non-inventively. For a favorable effect, the dosage of the gypenosidic compound represented by the above formula 2 of the present invention, its stereoisomer, or its pharmaceutically acceptable salt may be 0.001-400 mg / kg, preferably 0.01-200 mg / kg, more preferably 0.01-100 mg / kg, and may be administered 1-3 times a day. The above dosage amounts are not intended to limit the scope of the invention in any way.

[0052] The pharmaceutical composition of the present invention may be prepared in the form of a unit dose or in a multi-dose container by formulating it with pharma- ceutically acceptable carriers and / or excipients in a manner that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. In this case, the dosage form may be any form suitable for pharmaceutical preparations, including oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, external preparations such as ointments and creams, suppositories, and sterile injection solutions, and may further include a dispersant or stabilizer.

[0053] The composition containing the gypenosidic compound represented by Chemical Formula 2 according to the present invention, its stereoisomer, or its food-acceptable salt as an active ingredient will be described in detail below with reference to examples. EXAMPLES

[0054] Material preparation .

[0055] Gypenoside L and gypenoside LI were purchased from Embo Co., Ltd., and ginsenoside Rg3 was purchased from Sigma-Aldrich.

[0056] Working Example .

[0057] Compositions of Examples 1 to 4 were prepared according to the formulations in Table 1 below.

[0058] [Table 1]

[0059] In vitro testing.

[0060] Materials and Methods.

[0061] 1. Test Substances .

[0062] Creatine monohydrate (Cr) was used as a positive control substance, and all test substances were provided by BTC Corporation.

[0063] 2. Cell Culture and Differentiation Induction .

[0064] C2C12 cells, which are myoblasts derived from mouse skeletal muscle, were purchased from ATCC (American Type Culture Collection). C2C12 cells were cultured in a humidified CO2 incubator (5% CO2 / 95% air) at 37°C using cell culture medium consisting of Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 100 units / mL penicillin, and 100 μg / mL streptomycin. When the cells filled approximately 80% of the culture dish, the cell monolayer was washed with phosphate-buffered saline (PBS, pH 7.4), and then trypsin-2.65 mM EDTA was added to detach the cells and subcultured. The medium was changed every 2 days.

[0065] When C2C12 cells filled about 90% of the culture dish, the medium was replaced with myocyte differentiation medium (DMEM medium supplemented with 2% horse serum (Gibco-Thermo Fisher Scientific)) to induce differentiation into muscle cells. The myocyte differentiation medium was replaced every 2 days.

[0066] 3. Muscle cell differentiation induction and test substance treatment .

[0067] 2 × 10 C2C12 cells 5 The cells were dispensed into a 6-well plate at 100 cells / well and allowed to stabilize for 24 hours. To examine the effect of each test substance on muscle cell differentiation, each test substance was added to the muscle cell differentiation culture medium and treated with the cells. The muscle cell differentiation culture medium treated with the test substance as shown in Table 2 below was replaced with the cell culture medium, and the cells were cultured for 4 days (mRNA analysis) or 7 days (protein analysis).

[0068] [Table 2]

[0069] 4. Reactive oxygen species (ROS) measurement .

[0070] 1 × 10 C2C12 cells 4 The cells were then dispensed into a 96-well plate at 1000 cells / well and cultured for 24 hours. The medium was then replaced with a muscle cell differentiation medium containing each test substance, and the cells were cultured for 1 hour. After 1 hour, the cells were washed with PBS and further cultured for 3 hours in a medium containing 50 μM TBHP (tert-butyl hydrogen peroxide), after which the intracellular ROS level was measured using a DCF-DA assay kit (Abcam) according to the method suggested by the manufacturer.

[0071] 5. Total cell lysate preparation and protein expression analysis (Western blot analysis) .

[0072] 2 × 10 C2C12 cells 5The cells were dispensed into 6-well plates at 100 cells / well and allowed to stabilize for 24 hours. The cell culture medium was replaced with myocyte differentiation medium containing the test substance and cultured for 7 days. The cells were induced to differentiate for 7 days, and lysis buffer (20 mmol / L HEPES, pH 7.5, 150 mmol / L NaCl, 1% Triton X-100, 1 mmol / L EDTA, 1 mmol / L EGTA, 100 mmol / L NaF, 10 mmol / L sodium pyrophosphate, 1 mmol / L Na3VO4, 20 μg / mL aprotinin, 10 μg / mL antipain, 10 μg / mL leupeptin, 80 μg / mL benzamidine HCl, 0.2 mmol / L PMSF) was added, followed by homogenization and centrifugation to obtain whole cell lysates. The protein content of the whole cell lysates was measured using a BCA protein assay kit (Thermo Scientific). Total cell lysates (50 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was blocked with 5% skim milk-TBST (20 mmol / L Tris.HCl, pH 7.5, 150 mmol / L NaCl, 0.1% Tween 20) for 1 hour, and then antibodies to be measured were added and stirred at 4°C for 16 hours or at room temperature for 1 hour. The antibody information used is shown in Table 3 below. Then, horseradish peroxidase (HRP)-conjugated anti-rabbit IgG or HRP-conjugated anti-mouse IgG was added and stirred for 1 hour. The detected protein bands were analyzed by Luminata TM Protein expression levels were visualized by enhanced chemiluminescence using Forte Western HRP Substrate (Millipore). TMQuantification was performed using LAS 500 imaging systems (GE Healthcare Bio-Sciences AB).

[0073] [Table 3]

[0074] 6. mRNA expression survey (Real-time RT-PCR) .

[0075] 2 × 10 C2C12 cells 5 The cells were dispensed into a 6-well plate at 100 cells / well and allowed to stabilize for 24 hours. The cell culture medium was replaced with myocyte differentiation medium containing the test substance and cultured for 2 or 4 days. After collecting the cells, total RNA was isolated using an RNeasy Plus Mini kit (QIAGEN) and quantified using a micro-volume spectrophotometer (BioSpec-nano, SHIMADZU). RNA with an OD260 / 280 value of 1.8 or higher was used for the experiment.

[0076] cDNA was obtained from total RNA (2 μg) using HyperScriptTM RT master mix kit (GeneAll Biotechnology), and real-time PCR was performed using Rotor-Gene 300 PCR (Corbett Research) and Rotor-GeneTM SYBR Green kit (QIAGEN). The primer information used in the experiment is shown in Table 4 below. Quantitative analysis of gene expression was performed using the Rotor-Gene 6000 Series System Software program (Corbett Research).

[0077] [Table 4]

[0078] 7.Statistical Processing .

[0079] All analytical values ​​were expressed as mean ± SEM. The collected results were analyzed using the GraphPad Prism 5.0 (GraphPad software) program. Student's t-test and one-way analysis of variance (ANOVA) were used to compare the differences between the test substance-treated group and the control group. P<0.05 or more was considered statistically significant.

[0080] <Test example>.

[0081] Test Example 1: Effect on intracellular ROS production in C2C12 cells .

[0082] ROS are mitochondrial by-products generated during normal cellular respiration, and oxidative stress results from an imbalance between ROS production and antioxidant defense. Abnormally increased ROS induces muscle cell dysfunction and damages intracellular macromolecules such as muscle cell proteins, lipids, and nucleic acids, acting as a causative factor in cell death.

[0083] To examine the effect of each composition according to Examples 1 to 4 on intracellular ROS generation in C2C12 cells, ROS generation was measured using 2',7'-dichlorofluorescin diacetate (DCF-DA) and the results are shown in Table 5. DCF-DA is measured based on the principle that it is oxidized by ROS in cells and converted to DCF, which exhibits fluorescence.

[0084] [Table 5]

[0085] *, **, and *** indicate significant differences at p<0.05, p<0.01, and p<0.001, respectively, compared with the G0 group (G1).

[0086] #, ##, and ### mean that there is a significant difference at p<0.05, p<0.01, and p<0.001, respectively, compared to the G1 group (G2, G3, G4, G5, G6).

[0087] As seen in Table 5 above, when oxidative stress was induced in C2C12 cells by treatment with TBHP (G1), the ROS generation was 37.97±2.19, which was significantly increased compared to 16.32±0.66 in the normal control group (G0). The ROS generation increased by oxidative stress was significantly reduced by treatment with the compositions of Examples 1 to 5. The ROS generation increased by STO treatment was 33.97±2.22, which was somewhat reduced compared to the oxidative stress induction group (G1). When the positive control substance Cr was treated, the ROS generation was 21.13±2.74, which was significantly reduced compared to the oxidative stress induction control group (G1). This indicates that the gypenosidic compound effectively inhibits ROS generation due to oxidative stress induced by TBHP in C2C12 cells.

[0088] Test Example 2: Effect on changes in PGC-1α (peroxisome proliferator-activated receptor-gamma coactivator-1 alpha)-related proteins in C2C12 cells .

[0089] Mitochondria are the powerhouses of muscle that oxidize energy sources to produce ATP, and their number and quantity increase when exercise load is sustained to oxidize more energy. PGC-1α plays a role in regulating mitochondrial function, biogenesis, and transcription of cellular energy metabolism. Activation of PGC-1α is induced by AMPK (AMP-activated protein kinase) and Sirt1 (Silent mating-type information regulation 2 homolog 1), and it has been reported that its activation increases with endurance exercise. AMPK is an enzyme that senses the intracellular energy status and is activated when there is a shortage of intracellular energy, i.e., when AMP increases compared to ATP, and regulates various metabolic pathways to restore normal energy balance. Sirt1 is recognized as an important regulator of mitochondrial biogenesis in skeletal muscle due to exercise, as its activity increases due to changes in NAD+ caused by muscle contraction and regulates the activity of PGC-1α. p38 MAPK (mitogen-activated protein kinase) is an enzyme that is activated by various extracellular stimuli and is known to be involved in cell growth and differentiation, cell cycle regulation, etc. Exercise and skeletal muscle contraction increase p38 MAPK activity, and in recent years it has been reported that p38 MAPK activates PGC-1α.

[0090] In this study, to examine the effect of the test substance on protein changes associated with PGC-1α activation, the results of Western blotting using total cell lysates prepared by treating the test substance are shown in Table 6 below. Table 6 below shows the protein expression levels associated with PGC-1α activation in each test group compared with the relative band density (% control group).

[0091] [Table 6]

[0092] Based on the results of the Western blot analysis, the activity of each protein was evaluated based on the ratio of activated protein to each protein, and is shown in Table 7 below.

[0093] [Table 7]

[0094] *, **, and *** indicate significant differences at p<0.05, p<0.01, and p<0.001, respectively, compared to the G1 group (G2 to G6).

[0095] As seen in Tables 6 and 7 above, the expression of p-AMPK was increased by treatment with the composition of the examples (G2-G5) compared to the control group (G1). The expression of p-Sirt1 was significantly increased by treatment with the composition of the examples (G2-G5) compared to the control group (G1). The expression of p-p38 MAPK was significantly increased by treatment with the composition of the examples (G2-G5) compared to the control group (G1). The ratio of p-p38 / p38 was significantly increased by treatment with the examples.

[0096] Activated PGC-1α induces the activity of various other transcription factors, particularly the activation of Nrf2 (nuclear factor erythroid-2 related factor 2), a leucine zipper transcription factor, to regulate the expression of antioxidant genes. When oxidative stress is induced, Nrf2 moves from the cytoplasm to the nucleus and binds to the promoter portion of antioxidant genes, inducing the expression of various antioxidant genes. The expression of p-Nrf2 was significantly increased by the treatment of the example. The ratio of p-Nrf2 / Nrf2 was also increased by the treatment of the example.

[0097] As a result, the treatment with the composition according to the embodiment significantly increased the activation of Sirt1, p38 MAPK, and Nrf2 in C2C12 cells. In contrast, the treatment with STO, an inhibitor of CaMKK, significantly suppressed the activation of AMPK, p38 MAPK, and Nrf2, which were found to be activated in response to changes in Ca2+ levels. The activation of suppressed AMPK, p38 MAPK, and Nrf2 tended to be restored by the composition according to the embodiment, but there was no significant difference.

[0098] Test Example 3: Effect on mitochondrial replication-related mRNA expression in C2C12 cells .

[0099] Mitochondria are essential organelles for life, as they are the central organelles for various cellular processes such as ATP production, cell death, fatty acid beta-oxidation, iron-sulfur conjugate synthesis, etc. Mitochondria are unique in that they possess their own genome in the form of mitochondrial DNA (mtDNA), separate from the chromosomal DNA present in the nucleus.

[0100] Tfam (Mitochondrial transcription factor A) modulates mtDNA stability and mtDNA transcription by transforming the mitochondrial nuclear structure and protecting DNA from ROS attack. CPT-1 (Carnitine palmitoyl transferase-1) is a genetic trait related to ADP phosphorylation in mitochondria that is related to fat oxidation, and is an enzyme involved in allowing fatty acids (long-chain fatty acyl-CoA) that have passed through the outer membrane of mitochondria to pass through the intracellular membrane and enter the mitochondrial matrix.

[0101] The effects of treatment of C2C12 cells with a test substance on the expression of mtDNA, Tfam, and CPT-1β mRNA associated with mitochondrial replication were examined, and the results are shown in Table 8 below.

[0102] [Table 8]

[0103] *, **, and *** indicate significant differences at p<0.05, p<0.01, and p<0.001, respectively, compared to the G1 group (G2 to G6).

[0104] As shown in Table 8, Tfam mRNA expression did not show significant differences in all groups. CPT1-β mRNA expression showed a tendency to increase in the example treatment groups (G2 to G5). mtDNA mRNA expression was significantly decreased in the Cr treatment group (G6) compared to the control group (G1).

[0105] Test Example 4: Effect on muscle type change-related mRNA expression in C2C12 cells .

[0106] The effects of each of the compositions according to Examples 1 to 4 on the expression of MHC1, MHC7, MHC2A, and MHC2B mRNA, which represent muscle strength types, were investigated and the results are shown in Table 9 below.

[0107] [Table 9]

[0108] *, **, and *** indicate significant differences at p<0.05, p<0.01, and p<0.001, respectively, compared to the G1 group (G2 to G6).

[0109] Myosin is the most abundant protein in skeletal muscle and is involved in muscle contraction. Myosin is composed of myosin heavy chain (MHC) and myosin light chain (MLC), and MHC is an important factor that determines muscle contraction properties. Among the well-known subtypes in skeletal muscle, MHC2A and MHC2B subtypes are known to be mainly involved in fast muscle contraction speed, and MHC1 and MHC7 subtypes are known to be involved in slow muscle contraction speed. MHC1 mRNA expression was significantly increased in the example treatment groups (G2 to G5) compared to the control group (G1). MHC2A mRNA expression showed a tendency to increase in the example treatment groups (G2 to G5), but there was no significant difference. In other words, it is considered that treatment with the composition of the example increases the expression of MHC1 and MHC7 mRNA and induces differentiation into a slow muscle type involved in slow muscle contraction speed.

[0110] The effects of each of the compositions according to Examples 1 to 4 on the expression of PGC-1α, PKB, and FNDC5 mRNA, which are representative of muscle strength types, were investigated and are shown in Table 10 below.

[0111] [Table 10]

[0112] *, **, and *** indicate significant differences at p<0.05, p<0.01, and p<0.001, respectively, compared to the G1 group (G2 to G6).

[0113] PGC-1α is known to be a transcriptional coactivator that plays a central role in gene regulation for exercise-induced skeletal muscle adaptation, such as mitochondrial biogenesis in skeletal muscle and muscle fiber morphology specialization (fast-to-slow fiber type switching). PGC-1α mRNA expression was significantly increased in the experimental treatment groups (G2-G5) compared to the control group (G1). PKB (Protein kinase B), also known as Akt, plays an important role in glucose metabolism and various cellular metabolic processes. PKB, an upper signal transmitter of GLUT4, transmits signals to GLUT4 and enables the movement of glucose into muscles. PKB mRNA expression was significantly increased in the experimental treatment groups (G2-G5) compared to the control group (G1). PGC-1α activated in skeletal muscle in response to exercise is not only involved in the oxidative stress regulation mechanism together with FNDC5 (fibronectin type III domain-containing protein 5), a skeletal muscle membrane protein, but also improves insulin sensitivity by activating the insulin signal transduction pathway. FNDC5 mRNA expression was significantly increased in the example treatment groups (G2 to G5) compared to the control group (G1), and none of the other groups showed significant differences.

[0114] Test Example 5: Effect on energy-related mRNA expression in C2C12 cells .

[0115] The effects of each of the compositions according to Examples 1 to 4 on the expression of GSY, SIRT1, and PPARγ mRNA involved in the energy metabolism of muscle cells were examined, and the results are shown in Table 11.

[0116] [Table 11]

[0117] *, **, and *** indicate significant differences at p<0.05, p<0.01, and p<0.001, respectively, compared to the G1 group (G2 to G6).

[0118] GSY (Glycogen Synthase) is a core enzyme in glycogenogenesis, and is a metabolic enzyme involved in the synthesis and storage of glycogen by insulin. Glycogen in skeletal muscles is the main energy source during exercise, and the higher the exercise intensity, the higher the energy dependency on glycogen. GSY mRNA expression was significantly increased in the example treatment groups (G2-G5) compared to the control group (G1), and was also significantly increased in the positive control Cr treatment group (G6). During exercise, the ATP stored in skeletal muscles decreases and the energy requirement increases rapidly, and the associated increase in intracellular NAD+ activates Sirt1, and the activated Sirt1 expresses cytoplasmic and nuclear transcription factors associated with energy generation in order to generate the energy required for exercise. Sirt1 mRNA expression showed a tendency to increase in the example treatment groups (G2-G5) compared to the control group (G1), but there was no significant difference. PPAR-γ (Peroxisome proliferator-activated receptor gamma) is a transcription factor belonging to the nuclear receptors. It is activated by ligands to regulate genes related to lipid and glucose metabolism and energy homeostasis, and plays a role in regulating cell proliferation and differentiation.

[0119] In addition, PPAR-γ regulates the ability to oxidize fatty acids in skeletal muscles through interaction with PGC-1α. PPAR-γ mRNA expression tended to increase in the treatment groups (G2 to G5) compared to the control group (G1), but did not show significant differences.

[0120] From the above results, it was observed that the gypenosidic compound, which is the main component of the composition according to the embodiment, suppresses oxidative stress and increases the expression of various genes related to mitochondrial biosynthesis in muscle cells. This indicates that the composition according to the embodiment can be used as a functional material for suppressing exercise-induced fatigue and improving exercise performance.

[0121] In vivo testing .

[0122] Materials and Methods .

[0123] 1. Test Substances .

[0124] Creatine monohydrate (Cr) was used as a positive control.

[0125] 2.Animal testing approval .

[0126] All animal experiments in this study were performed in accordance with the animal experiment regulations under the approval of the Animal Experiment Ethics Committee of Halim University (Hallym 2019-28).

[0127] 3. Evaluation of the efficacy of gypenosides in improving muscle fatigue in an in vivo system - taking only the sample without exercise .

[0128] (1) Laboratory animals .

[0129] Five-week-old male ICR mice free of specific pathogens were purchased from Doyeol Biotech Co., Ltd. and used. After one week of quarantine and adaptation, healthy animals with no weight loss were selected for use in the experiment. The experimental animals were kept in a breeding environment set at 23±3°C, 50±10% relative humidity, 10-15 ventilation times / hour, 12-hour lighting period (08:00-20:00), and 150-300 Lux illuminance. During the pre-test period, the experimental animals were provided with solid feed for experimental animals (Cargill Agri Purina Co., Ltd.) and drinking water ad libitum.

[0130] [Table 12]

[0131] (2) Test group, test substance administration .

[0132] After one week of adaptation, healthy animals were selected and divided into six groups of 10 mice per group according to the randomized block design. The general control group was orally administered 5% Tween 80-saline, the positive control group was orally administered 75 mg / kg BW (body weight) of Cr (Creatine monohydrate), and the test groups (G2 to G5) were orally administered 7 mg / kg BW (body weight) of the compositions according to Examples 1 to 4 dissolved in drinking water at a fixed time every day for 17 days. During the pre-test period, the experimental animals were provided with solid feed for experimental animals (Cargill Agri Purina Co., Ltd.) and were allowed to consume the food and drinking water ad libitum.

[0133] (3) Weight measurement.

[0134] The experimental animals were weighed at regular times each week during the study period.

[0135] (4) Forced swimming test and blood lactate content measurement .

[0136] The weight-loaded forced swimming test for experimental animals was carried out as follows: A plastic tank (90 x 45 x 45 cm) was filled with water to a depth of 35 cm, and the water temperature was maintained at 25 ± 1°C. A weight equivalent to 5% of the experimental animal's body weight was attached to the base of the tail, and the animal was allowed to swim in the tank. The animal was judged to have lost all of its strength when it was unable to rise to the surface for 7 seconds.

[0137] On the 10th and 12th days after administration of the test substance, the rats were subjected to a total of two 15-minute swimming adaptation exercises. Two days after the final swimming adaptation exercise (the 14th day after administration of the test substance), the rats were fasted for 16 hours and then subjected to a forced swimming test, and the swimming time until fatigue was measured.

[0138] The blood lactate content of the experimental animals was measured using a lactate meter (Lactate Pro2, Arkray) by taking blood samples from the tails of the experimental animals before the forced swimming test, immediately after swimming, 10 minutes after swimming, and 30 minutes after swimming.

[0139] (5) Blood collection .

[0140] On the 17th day after administration of the test substance, the animals were made to swim for a certain period of time (60 min) without weight bearing, and then the animals were anesthetized with an anesthetic made by diluting tribromoethanol with tertiary amyl alcohol, and blood was collected from the orbit. The blood was placed in a serum separate tube (Becton Dickinson) and left at room temperature for 30 min, then centrifuged at 3,000 rpm for 20 min to separate the serum, which was then stored at -70°C until analysis.

[0141] (6) Serum analysis .

[0142] Serum BUN (blood urea nitrogen) and CREA (creatinine) contents, and ALT (alanine aminotransferase), AST (aspartate aminotransferase), CK (creatine kinase), and LDH (lactate dehydrogenase) activities were measured using a blood biochemistry analyzer (KoneLab20XT, Thermo Fisher Scientific). Serum lactate content was measured using a lactate measurement kit (Abcam) according to the method recommended by the manufacturer.

[0143] 4. Evaluation of the efficacy of gypenosides in enhancing exercise performance in an in vivo system - Sample intake in parallel with exercise .

[0144] (1) Laboratory animals .

[0145] Five-week-old male ICR mice free of specific pathogens were purchased from Doyeol Biotech Co., Ltd. and used. After one week of quarantine and adaptation, healthy animals with no weight loss were selected for use in the experiment. The experimental animals were kept in a breeding environment set at 23±3°C, 50±10% relative humidity, 10-15 ventilation times / hour, 12-hour lighting period (08:00-20:00), and 150-300 Lux illuminance. During the pre-test period, the experimental animals were given free access to solid feed for experimental animals (Cargill Agri Purina Co., Ltd.) and drinking water.

[0146] (2) Test Groups and Test Substance Administration .

[0147] After a one-week adaptation period, healthy animals were selected and divided into six groups based on the randomized block design: (G1) non-exercise control group, (G2) non-exercise + 7 mg / kg BW (body weight) Example 1 administration group, (G3) non-exercise + 7 mg / kg BW Example 2 administration group, (G4) non-exercise + 7 mg / kg BW Example 3 administration group, (G5) non-exercise + 7 mg / kg BW Example 4 administration group, (G6) exercise control group, (G7) exercise + 7 mg / kg BW Example 1 administration group, (G8) exercise + 7 mg / kg BW Example 2 administration group, (G9) exercise + 7 mg / kg BW Example 3 administration group, (G10) exercise + 7 mg / kg BW Example 4 administration group, and (G11) exercise + 75 mg / kg BW Cr administration group. Ten experimental animals were used for each test group.

[0148] The test substance was dissolved in drinking water and orally administered at a fixed time every day (2 hours before exercise) for 6 weeks. During the pre-test period, the experimental animals were fed solid feed for experimental animals (Cargill Agri Purina Co., Ltd.) and had free access to food and drinking water.

[0149] [Table 13]

[0150] (3) Body weight and food intake measurement .

[0151] During the test period, the body weight of the experimental animals was measured at a fixed time every week. The food intake of the experimental animals was measured during the test period, and the total food intake and daily food intake were calculated.

[0152] (4) Treadmill testing and endurance exercise .

[0153] The experimental animals were trained for 6 weeks on a small animal treadmill (Exer3 / 6-treadmill, Columbus Instruments). The animals were trained for 15 minutes in the first week, 20 minutes in the second week, 25 minutes in the third week, 30 minutes in the fourth week, 35 minutes in the fifth week, and 40 minutes in the sixth week, with an incline of 10° and a speed of 10 m / min.

[0154] To evaluate endurance exercise performance after 6 weeks of exercise training, the animals were trained for 5 minutes at a 10° gradient and a speed of 10 m / min, and the exercise intensity was increased by increasing the speed by 1 m / min every minute, and the duration of exercise until exhaustion was measured at a maximum speed of 25 m / min. The exhaustion state was determined as the point at which the experimental animals could not run for more than 10 seconds after dropping to the rear part of the treadmill while running. The amount of exercise performed by the experimental animals was calculated using the following formula.

[0155] Exercise capacity =body weight(kg)×speed(m / s)×time(s)×grade×9.8m / s 2 .

[0156] (5) Lean body rate and body fat rate measurement .

[0157] One day before the end of the study, the experimental animals were anesthetized and their body composition was measured using dual-energy X-ray absorptiometry (DEXA, PIXImusTM, GE Lunar) to evaluate the lean body mass and body fat mass.

[0158] (6) Blood collection and tissue extraction .

[0159] Before sacrificing the animals, they were anesthetized with an anesthetic made by diluting tribromoethanol with tertiary amyl alcohol, and blood was collected from the orbit. The blood was placed in a serum separator tube (Becton Dickinson) and left at room temperature for 30 minutes, then centrifuged at 5,000 rpm for 10 minutes to separate the serum, which was then stored at -70°C until analysis. After blood collection, the animals were sacrificed, and the liver and skeletal muscles [quadriceps femoris muscle (QF), gastrocnemius muscle (GA), soleus muscle (SOL), and extensor digitorum longus muscle (EDL)] were removed, washed with cold saline, and excess water was removed using filter paper, and then weighed. A portion of the soleus muscle (SOL) was fixed in 4% PFA (paraformaldehyde) and embedded in paraffin for histoimmunostaining, and another portion was used for total RNA isolation and real-time RT-PCR. A portion of the gastrocnemius muscle (GA) was used for protein isolation and Western blotting. The remaining tissues were stored at -70°C until analysis.

[0160] (7)Blood biochemical analysis .

[0161] Serum glucose, triglycerides, total cholesterol, LDL-cholesterol, and HDL-cholesterol contents, as well as BUN (blood urea nitrogen), CREA (creatinine), and CK (creatine kinase), LDH (lactate dehydrogenase), ALT (alanine aminotransferase), AST (aspartate aminotransferase), and ALP (alkaline phosphatase) activities were measured using a blood biochemistry analyzer (KoneLab 20 XT, Thermo Fisher Scientific). Serum lactate content was measured using a lactate measurement kit (Abcam) according to the manufacturer's instructions.

[0162] (8) Liver tissue fluid production .

[0163] A liver tissue fluid was prepared to measure the glycogen content in liver tissue. 100 mg of liver tissue was added to 1 mL of PBS and homogenized using a homogenizer. The homogenized solution was centrifuged at 5,000 rpm for 10 minutes, and the supernatant was collected and used as liver tissue fluid.

[0164] (9) Muscle tissue fluid production .

[0165] Muscle tissue fluid was prepared to measure glycogen content and enzyme activity in muscle tissue. The isolated skeletal muscles, i.e., quadriceps femoris (QF), gastrocnemius (GA), soleus (SOL), and extensor digitorum longus (EDL), were homogenized with a homogenizer after adding 1 mL of PBS. The homogenized solution was centrifuged at 5,000 rpm for 10 minutes, and the supernatant was collected and used as muscle tissue fluid. The protein content of the muscle tissue fluid was measured using a BCA protein assay kit (Thermo Scientific).

[0166] (10) Measurement of glycogen content in liver and skeletal muscle .

[0167] Glycogen content in the liver and skeletal muscle (GA) was measured using a glycogen measurement kit according to the method provided by the manufacturer (Abcam).

[0168] (11) Enzyme activity measurement in skeletal muscle .

[0169] The activities of citrate synthase (BioVision), beta-hydroxyacyl CoA-dehydrogenase (MyBioSource), and lactate dehydrogenase (Abcam) in skeletal muscles (QF, SOL, and EDL) were measured using the respective kits according to the methods recommended by the manufacturers.

[0170] (12) Histomorphological observation of skeletal muscle (hematocylin & eosin staining) .

[0171] The soleus muscle fixed with 4% PFA was embedded in paraffin, and 5 μm tissue sections were prepared from the embedded tissue. After removing the paraffin, the tissue was hydrated by gradually decreasing the percentage of alcohol, starting from 100% alcohol to 0% alcohol (H2O). For histomorphological observation of the soleus muscle (SOL), the tissue was stained with Accustain® hematoxylin and eosin stain (Sigma-Aldrich Co.) according to the method suggested by the manufacturer. Histological changes were then observed using an optical microscope (Carl Zeiss).

[0172] (13) Protein expression in muscle tissue (Western blot analysis) .

[0173] To investigate protein expression in muscle tissue (gastrocnemius, GA), lysis buffer (20 mmol / L Hepes, pH 7.5, 150 mmol / L NaCl, 1% Triton X-100, 1 mmol / L EDTA, 1 mmol / L EGTA, 100 mmol / L NaF, 10 mmol / L sodium pyrophosphate, 1 mmol / L Na3VO4, 20 μg / mL aprotinin, 10 μg / mL antipain, 10 μg / mL leupeptin, 80 μg / mL benzamidine HCl, 0.2 mmol / L PMSF) was added, followed by homogenization with a homogenizer. The homogenized solution was centrifuged at 12,000 rpm for 10 minutes, and the supernatant was removed to obtain muscle tissue lysate. The amount of protein in the muscle tissue lysate was measured using a BCA protein assay kit (Thermo Scientific).

[0174] Proteins (50 μg) were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride membrane (Millipore). The membrane was blocked with 5% skim milk-TBST (20 mmol / L Tris·HCl, pH 7.5, 150 mmol / L NaCl, 0.1% Tween 20) for 1 hour, and then the antibodies to be measured were added and stirred at 4°C for 16 hours or at room temperature for 1 hour. The antibody information used in the experiment is shown in Table 14 below. Then, horseradish peroxidase (HRP)-conjugated anti-rabbit IgG or HRP-conjugated anti-mouse IgG was added and stirred for 1 hour, and each protein band was visualized by a highly sensitive chemiluminescence method using LuminataTM Forte Western HRP Substrate (Millipore). Protein expression levels were quantified using ImageQuantTM LAS 500 imaging systems (GE Healthcare Bio-Sciences AB).

[0175] [Table 14]

[0176] (14) Investigation of mRNA expression in muscle tissue (real-time RT-PCR) .

[0177] Total RNA was isolated from muscle tissue (SOL) collected at the end of the study using TRIzol Reagent (Thermo Fisher Scientific) and quantified using a micro-volume spectrophotometer (BioSpec-nano, SHIMADZU). RNA with an OD260 / 280 value of 1.8 or higher was used in the experiment. cDNA was obtained from total RNA (2 μg) using HyperScriptTM RT master mix kit (GeneAll Biotechnology), and real-time PCR was performed using Rotor-Gene 300 PCR (Corbett Research) and Rotor-GeneTM SYBR Green kit (QIAGEN). The primer information used in the experiment is shown in Table 15 below. Quantitative analysis of gene expression was performed using the Rotor-Gene 6000 Series System Software program (Corbett Research).

[0178] [Table 15]

[0179] 5.Statistical Processing .

[0180] All analytical values ​​are shown as mean ± SEM. The collected results were analyzed using SAS statistical software, version 9.4 or GraphPad Prism 5.0 (GraphPad software) program. Student's t-test and one-way analysis of variance (ANOVA) were used to compare the differences between the test substance-administered group and the control group. In addition, to compare the differences between the control group and the test substance-administered group, Duncan's multiple range test was used to verify the significance after analysis with ANOVA. Statistical significance was determined only when p<0.05 or more.

[0181] <Test example>.

[0182] <Evaluation of efficacy in improving muscle fatigue in an in vivo system>-When only the sample was taken without exercise .

[0183] Test Example 6: Effects on body weight of experimental animals .

[0184] The body weights of the experimental animals were measured once a week during the study period and are shown in Table 16. The experimental animals in all test groups continued to gain weight during the study period and showed normal body weight changes. There were no significant differences in body weight between the control group (G1) and any of the test substance administration groups (G2-G5) during the study period.

[0185] [Table 16]

[0186] Test Example 7: Effect on exercise (forced swimming) time .

[0187] In order to evaluate the effect of the test substance in improving exercise fatigue, a weight equivalent to 5% of the experimental animal's body weight was attached, and the swimming time until the animal lost strength was measured. The results are shown in Table 17.

[0188] [Table 17]

[0189] Referring to Table 17, it was found that the time taken to reach weakness was significantly increased in the group administered Example 2 (G3) compared to the control group (G1).

[0190] Test Example 8: Effects on lactate concentration before and after exercise (forced swimming) .

[0191] It is known that the increase in blood lactate levels during exercise is proportional to the intensity of the exercise, and the lactate threshold is used as an indicator of aerobic exercise capacity. The ability to remove lactate from the blood during exercise or recovery shows a positive correlation with exercise performance.

[0192] The effects of administration of the test substance on blood lactate concentrations before and after exercise (forced swimming) were investigated and are shown in Table 18.

[0193] [Table 18]

[0194] As seen in Table 18 above, the blood lactate concentration of all test groups was at its highest immediately after exercise (when relaxed) compared to before exercise, and the blood lactate concentration decreased over time. In particular, the composition according to Example 2 significantly reduced the blood lactate concentration before and after exercise.

[0195] Test Example 9: Effects on serum indices .

[0196] After swimming for a certain period of time (60 minutes) without weight bearing, serum lactate, BUN, and CREA contents, as well as CK, LDH, ALT, and AST activities were collected and the results are shown in Table 19.

[0197] [Table 19]

[0198] Lactate is a substance produced by the reduction of pyruvate during anaerobic glycolysis in tissues. It acidifies the environment within muscle cells, inhibits phosphorylase activity and myosin ATPase activity, and acts as a cause of reduced athletic performance. It is therefore used in many studies as an indicator of exercise-induced fatigue. LDH (Lactate dehydrogense) is an enzyme that catalyzes the formation of lactic acid from pyruvate. During high-intensity exercise, excessive pyruvate is produced, and LDH activity, which catalyzes the process of converting it to lactic acid, increases. Therefore, an increase in serum LDH activity indicates increased strain on skeletal muscles and is an indicator of muscle damage.

[0199] As seen in Table 19 above, serum lactate concentration was significantly decreased in the example administration groups (G2 to G4) compared to the control group (G1), and serum LDH activity was significantly decreased in the example administration group (G2) compared to the control group (G1).

[0200] <Evaluation of the effectiveness in improving exercise performance in an in vivo system> - When the sample was taken in parallel with exercise .

[0201] Test Example 10: Body weight of experimental animals .

[0202] The body weights of the experimental animals were measured every two weeks during the study period and are shown in Table 20 below.

[0203] [Table 20]

[0204] As seen in Table 20 above, all experimental animals in the groups continued to gain weight during the test period, showing normal weight changes. Compared to the non-exercise control group (G1), the body weight of the Example-administered group (G2) decreased significantly from the fourth week to the end of the test, and the body weight of the exercise control group (G6) decreased significantly from the first week to the end of the test, compared to the non-exercise control group (G1). In the exercise test groups, the body weight of the Example-administered groups (G7 to G10) began to decrease significantly from the second week, compared to the exercise control group (G6).

[0205] Test Example 11: Effects on exercise duration and exercise volume until relaxation .

[0206] In order to evaluate the endurance exercise performance of the test substance, the subjects were started to exercise for 5 minutes at an incline of 10° and a speed of 10 m / min, and then the exercise intensity was increased by increasing the speed by 1 m / min every minute, and the exercise duration until exhaustion was measured at a maximum speed of 25 m / min. The results are shown in Table 21.

[0207] [Table 21]

[0208] *, **, and *** indicate significant differences at p<0.05, p<0.01, and p<0.001, respectively, compared to the G1 group (G2 to G5, G6).

[0209] +, ++, and +++ indicate significant differences at p<0.05, p<0.01, and p<0.001, respectively, compared to the G6 group (G7 to G11).

[0210] As seen in Table 21 above, the exercise time until weakness was reached was 1,119 ± 58 seconds for the non-exercise control group (G1), the shortest exercise time compared to the other test groups, and the exercise time until weakness was significantly increased in the non-exercise test groups (G2 to G5) and the exercise control group (G6) compared to the non-exercise control group (G1). The exercise test groups (G7 to G10) and the Cr-administered group (G11) compared to the exercise control group (G6) showed a significant increase in the exercise time until weakness.

[0211] As a result of calculating the amount of locomotion of the experimental animals, the amount of locomotion was significantly increased in the non-exercise test groups (G2 to G5) compared to the non-exercise control group (G1). The amount of locomotion in the exercise control group (G6) was significantly increased compared to the non-exercise control group (G1), and the amount of locomotion in the exercise test groups (G7 to G10) and the Cr administration group (G11) was significantly increased compared to the exercise control group (G6).

[0212] This indicates that administration of the composition according to the embodiment has the effect of improving exercise performance.

[0213] Test Example 12: Liver and muscle weight .

[0214] After the test, the weights of the muscles and liver of the experimental animals were measured and are shown in Table 22.

[0215] [Table 22]

[0216] The body weight of the experimental animals was reduced by exercise and administration of the test substance, and was converted into muscle weights and liver weights per 100 g of body weight, as shown in Table 23.

[0217] [Table 23]

[0218] As seen in Table 23 above, the relative weights of the gastrocnemius (GA), soleus (SOL), and extensor digitorum longus (EDL) were significantly increased in the exercise control group (G6) compared to the non-exercise control group (G1). Also, the relative weights of the soleus (SOL) and extensor digitorum longus (EDL) were significantly increased in the example administration group (G2) compared to the non-exercise control group (G1). Also, in the exercise test group, the relative weights of the quadriceps (QF) and gastrocnemius (GA) were significantly increased in the example administration groups (G7 to G10) compared to the exercise control group (G6).

[0219] In other words, it is evident that administration of the compositions according to the examples of the present invention exhibits a muscle-building effect.

[0220] Test Example 13: Blood biochemistry analysis .

[0221] At the end of the study, the contents of lactate, CREA, and BUN in the collected serum, and the activities of CK, LDH, ALT, AST, and ALP were measured and shown in Table 24.

[0222] [Table 24]

[0223] As seen in Table 24 above, the groups administered the composition according to the present invention (G7 to G10) had significantly reduced CK and LDH activity, and serum lactate and BUN (blood nitrogen) concentrations compared to the exercise control group (G6).

[0224] Therefore, the above results show that administration of the compositions according to the examples of the present invention helps improve muscle fatigue.

[0225] Test Example 14: Glycogen content in liver and muscle (gastrocnemius, GA) .

[0226] Carbohydrates, one of the most important energy sources during exercise, provide energy for muscle contraction through the glycogenolysis process. If blood glucose levels decrease during exercise, glycogen stored in the liver or muscles is immediately used as an energy source, so inhibiting glycogenolysis, i.e., using glycogen sparingly, means that muscle contraction can be maintained for a longer period of time.

[0227] As a result, the glycogen content in the muscle (GA) collected at the end of the study was measured and is shown in Table 25 below.

[0228] [Table 25]

[0229] As seen in Table 25 above, the glycogen content in muscle (GA) tissue was significantly increased in the example administration groups (G7 to G10) in the exercise test groups compared to the exercise control group (G6).

[0230] From the above results, it is concluded that the composition according to the embodiment of the present invention increases the glycogen content in muscle (GA) tissue and serves as an energy source that helps reduce muscle fatigue and improve muscle endurance.

[0231] Test Example 15: Changes in protein expression in muscle (gastrocnemius, GA) .

[0232] In this study, to examine the effect of administration of the test substance on the changes in proteins related to the activation of PGC-1α, Western blot was performed using muscle (gastrocnemius, GA) tissue lysates. Based on the results of the Western blot analysis, the activity of each protein was evaluated according to the ratio of activated protein to each protein, and the results are shown in Table 26 below.

[0233] [Table 26]

[0234] As can be seen from Table 26 above, it was confirmed that the activity of proteins associated with the activation of PGC-1α was increased in the example administration groups, and in particular, it was confirmed that in the exercise test group, the example administration groups (G7 to G10) showed significantly increased activity of PGC1α, AMPK, and p-38MAPK compared to the exercise control group (G6).

[0235] From these results, it can be inferred that administration of the composition of the present invention together with regular exercise training significantly increases the activation of AMPK and p38 MAPK, which are proteins associated with the activation of PGC-1α, and regulates the expression of genes involved in mitochondrial biogenesis and glucose metabolism, thereby improving exercise performance.

[0236] Test Example 16: Changes in mRNA expression in muscle (soleus, SOL) .

[0237] Muscles can be classified into slow and fast muscles according to their physiological contraction speed, and slow muscles in particular have a large number of mitochondria and are highly active, which makes them resistant to fatigue and allows for long-term exercise. Therefore, in this study, we investigated the effects of endurance exercise training and administration of test substances on the expression of antioxidant-related genes (SOD2, GPx1, UCP2, UCP3), LDH-related genes (ERRα, LDH B, MCT1), mitochondrial synthesis-related genes (Tfam, CPT-1β, mtDNA, NRF1), and energy metabolism-related genes (PGC-1α, GYS, PPARγ, PPARδ) in the soleus muscle (SOL), which belongs to the slow muscle, and the results are shown in Tables 27 to 30 below.

[0238] [Table 27]

[0239] As seen in Table 27 above, it was confirmed that the activity of proteins related to antioxidants was increased in the groups administered the Example, and in particular, in the exercise test group, it was confirmed that the activity of SOD2, GPx1 and UCP2 was significantly increased in the groups administered the Example (G7 to G10) compared to the exercise control group (G6).

[0240] From these results, it can be inferred that administration of the composition of the present invention together with regular exercise training can protect mitochondria from oxidative stress and suppress muscle cell damage by increasing SOD2, GPx1, and UCP2 mRNA expression.

[0241] [Table 28]

[0242] As seen in Table 28 above, among the genes related to LDH, ERRα and LDH B mRNA expression increased in the group administered the example, and MCT1 mRNA expression increased significantly.

[0243] [Table 29]

[0244] As can be seen from Table 29 above, a tendency for the expression of genes related to mitochondrial synthesis to increase was observed in the groups administered the example, and in particular, in the exercise test group, the groups administered the example (G7 to G10) showed significantly increased expression of mtDNA, Tfam CPT-1β, and NRF1 mRNA compared to the exercise control group (G6).

[0245] From these results, it can be inferred that administration of the composition of the present invention together with regular exercise training can increase mitochondrial biogenesis by increasing the expression of mtDNA, Tfam CPT-1β and NRF1 mRNA, thereby improving exercise performance.

[0246] [Table 30]

[0247] As can be seen from Table 30 above, a tendency for the expression of genes related to energy metabolism to increase was observed in the groups administered the example, and in particular, in the exercise test group, it was confirmed that the expression of PPARγ and PPARδ was significantly increased in the groups administered the example (G7 to G10) compared to the exercise control group (G6).

[0248] From these results, it can be inferred that administration of the composition of the present invention in conjunction with regular exercise training can increase energy metabolism by increasing the expression of PGC1α, PPARγ and PPARδ mRNA, thereby improving exercise performance.

[0249] In conclusion, administration of the composition of the embodiment together with endurance exercise training shows muscle-building effects, reduces the lactate concentration that increases during exercise, thereby helping to improve muscle fatigue, and regulates the expression of genes involved in mitochondrial biogenesis and glucose metabolism, thereby improving exercise performance. This suggests that the composition of the embodiment can be developed into a functional material that improves exercise performance during endurance exercise.

[0250] <Manufacturing example>

[0251] Manufacturing Example 1: Manufacturing of tablets .

[0252] 10 mg of any one of compositions selected from Examples 1 to 4, 9 mg of vitamin E, 9 mg of vitamin C, 200 mg of galactooligosaccharide, 60 mg of lactose, and 140 mg of maltose are mixed and granulated using a fluidized bed dryer, and then 6 mg of sugar ester is added. 500 mg of these compositions are compressed into tablets by a conventional method to produce tablets.

[0253] Manufacturing Example 2: Capsule Manufacturing .

[0254] Using a conventional method for producing soft capsules, 10 mg of any one of the compositions selected from Examples 1 to 4, 9 mg of vitamin C, 2 mg of palm oil, 8 mg of hardened vegetable oil, 4 mg of yellow lead, and 9 mg of lecithin were mixed and filled into a gelatin capsule to produce a soft capsule.

[0255] Manufacturing Example 3: Manufacturing of pills .

[0256] 5 mg of any one of the compositions selected from Examples 1 to 4 was appropriately mixed with honey, dextrin, starch, microcrystalline cellulose, CMC calcium, etc. to prepare pills.

[0257] Production Example 4: Production of energy drinks .

[0258] 20 mg of any one of the compositions selected from Examples 1 to 4, 9 mg of vitamin E, 9 mg of vitamin C, 10 g of glucose, 0.6 g of citric acid, and 25 g of liquid oligosaccharides were mixed, and then 300 ml of purified water was added and the mixture was filled into each bottle to a total volume of 200 ml. After filling into the bottles, the mixture was sterilized at 130°C for 4 to 5 seconds to produce a drink.

[0259] Production Example 5: Production of granules .

[0260] 5 mg of any one of the compositions selected from Examples 1 to 4, 9 mg of vitamin E, 9 mg of vitamin C, 250 mg of anhydrous crystalline glucose, and 550 mg of starch were mixed and formed into granules using a fluidized bed granulator, and then filled into capsules to produce granules.

[0261] From the above description, a person skilled in the art to which the present invention pertains can understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the above-described embodiments should be understood as illustrative and not restrictive in any respect. The scope of the present invention should be interpreted as including any modified or changed forms derived from the meaning and scope of the appended claims and their equivalent concepts rather than the above detailed description.

Claims

1. A functional health food composition for improving athletic performance, comprising as an active ingredient a gypenosidic compound represented by the following Chemical Formula 1, its stereoisomer, or a food-based acceptable salt thereof. 【Chemistry 1】

2. The health functional food composition according to claim 1, characterized in that the active ingredient is gypenoside L or gypenoside LI.

3. The health functional food composition according to claim 1, characterized in that the active ingredients are gypenoside L and gypenoside LI.

4. The health functional food composition according to claim 3, wherein the weight ratio of gypenosid L and gypenosid LI is 100:20-80.

5. The health functional food composition according to claim 1, wherein the dosage of the active ingredient is 0.01 to 200 mg / kg / day.

6. A pharmaceutical composition for improving athletic performance, comprising as an active ingredient a gypenosidic compound represented by the following Chemical Formula 2, a stereoisomer thereof, or a pharma- ceutical acceptable salt thereof. 【Chemistry 2】

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