COMPOSITION FOR IMPROVING PHYSICAL STRENGTH OR ATHLETIC ABILITY, CONTAINING NUTMEG EXTRACT AS ACTIVE INGREDIENT

Abstract

The present invention relates to a composition for improving physical strength or athletic ability, containing a nutmeg extract as an active ingredient and, more specifically, to a composition for improving physical strength or athletic ability, for treating sarcopenia, for improving muscle function, and for anti-fatigue, containing a nutmeg extract as an active ingredient.

The nutmeg extract of the present invention contains a high quantity of ligand-based compounds and exhibits, when taken, very excellent effects of improving athletic ability, improving endurance and improving muscular strength, and thus is effective for treating sarcopenia and can be effectively used in medical supplies, food and the like for improving physical strength or athletic ability.

Claims

1-12. (canceled)

13. A method for improving physical strength or athletic ability or improving muscle function or increasing the amount of muscle in a subject, the method comprising administering an effective amount of a composition comprising a nutmeg extract as an active ingredient to the subject in need thereof.

14. (canceled)

15. A method for treating or improving sarcopenia in a subject, the method comprising administering an effective amount of a composition comprising a nutmeg extract as an active ingredient to the subject in need thereof.

16. (canceled)

17. (canceled)

18. (canceled)

19. An anti-fatigue method, the method comprising administering an effective amount of a composition comprising a nutmeg extract as an active ingredient to a subject in need thereof.

20. The method of claim 13, wherein the nutmeg extract is an extract obtained with one or more solvents selected from the group consisting of water, alcohol having 1 to 6 carbon atoms, acetone, ether, benzene, chloroform, ethyl acetate, methylene chloride, hexane, cyclohexane, petroleum ether, subcritical fluid and supercritical fluid.

21. The method of claim 13, wherein the improvement in athletic ability is to increase muscle endurance or strengthen muscle strength.

22. The method of claim 13, wherein the nutmeg extract further comprises a property that increases the level of glycogen at a cellular level.

23. The method of claim 13, wherein the composition is a pharmaceutical composition, a food composition, or a feed additive.

24. The method of claim 13, wherein the method is for a middle-aged or older individual.

25. The method of claim 15, wherein the composition is a pharmaceutical composition or a food composition.

26. The method of claim 19, wherein the composition is a pharmaceutical composition or a food composition.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0132] FIG. 1 shows the content of glycogen that appears as a result of treating the nutmeg extract of the present invention to muscle cells.

[0133] FIG. 2 shows the weight change of the mice to which the nutmeg extract of the present invention was administered.

[0134] FIG. 3 is a graph showing the results of the rotarod test in order to confirm the effect of nutmeg extract on athletic ability and physical strength.

[0135] FIG. 4 is a graph showing the results of the rotarod test to confirm the effect of nutmeg extract on athletic ability and physical strength, and more specifically, the results of confirming the athletic ability and endurance of mice in a rod rotating at a low speed to determine the athletic ability according to muscle strengthening.

[0136] FIG. 5 is a graph showing the results of the rotarod test in order to confirm the effect of nutmeg extract on athletic ability and physical strength, and more specifically, the results of confirming the athletic ability and endurance of mice in a rod rotating at high speed.

[0137] FIG. 6 shows the results of the Wire hang test confirming the effect of improving the athletic ability of muscle strengthening of the old mouse according to the administration of nutmeg extract.

[0138] FIG. 7 is a graph measuring the swimming speed by Morris Water Maze test of mice according to the nutmeg extract administration.

[0139] FIG. 8 is a graph showing the results of measuring the swimming speed by Morris Water Maze test of the Old mouse group.

[0140] FIG. 9 is a result graph showing the maximum swimming duration of a mouse as a result of the forced swim endurance test.

[0141] FIG. 10 is a photograph showing changes in the volume of the femur of a mouse as a result of administering a nutmeg extract to a young mouse.

[0142] FIG. 11 is a photograph showing changes in the volume of the femur of a mouse as a result of administering a nutmeg extract to an old mouse.

[0143] FIG. 12A is a graph showing the results of rotarod testing on mice to which a nutmeg extract was administered to mice, and is a result of confirming the athletic ability and endurance of mice on rods rotating at low speeds.

[0144] FIG. 12B is a photograph observed by extracting the liver of a normal mouse and a mouse to which creatine has been administered for a long time.

[0145] In the figure, *: p<0.05; **: p<0.01 and ***: p<0.001 indicate a significant difference, respectively.

[0146] Abbreviations in the drawings mean:

[0147] Y: Young, O: old, V: vehicle; Y-V: Y-vehicle, Y-10: Y-10 mg/kg, Y-30: Y-30 mg/kg, Y-100: Y-100 mg/kg. O-V: O-vehicle, O-10: O-10 mg/kg, O-30: O-30 mg/kg, O-100: O-100 mg/kg.

MODE FOR CARRYING OUT INVENTION

[0148] Hereinafter, the present invention will be described in detail.

[0149] However, the following examples are illustrative of the present invention, and the present invention is not limited to the following examples.

Preparation for the Experiment

[0150] 1. Plant Materials

[0151] Dried Myristica fragrans Houtt was collected in Jakarta, Indonesia, and was identified by Dr. Nam-In Paik, Department of Herbal Medicine Ingredients and Manufacturing, Kyunghee University (Yongin, Korea). Evidence samples were deposited with the Department of Biotechnology, Yonsei University (Seoul, Korea).

[0152] 2. Test Substances and Test Systems

[0153] C57BL/6 male mice were prepared with 12 mice of 3 months (Young) and 16 mice of 20 months (Old) according to age, and the doses were set to 10, 30, and 100 mg/kg, bw/day. It was approved by JC Pharma Laboratory Animal Ethics Committee in accordance with the ethical regulations of laboratory animals (deliberation number: 17-M001).

[0154] The route of administration applies the clinically planned route, and the number and duration of administration were administered once/day, 7 days/week, until the end of the test.

[0155] Table 1 describes the test group configuration for in vivo experiments of the present invention.

TABLE-US-00001 TABLE 1 Dosage Dosage Days Routes Month Test Numbers Animal amounts amounts of of age group Gender of animals number (ml/kg/day) (mg/kg/day) administration administration 3 Y1 M 3 1~3 4 0 22 Oral Y2 M 3 4~6 4 10 22 Oral Y3 M 3 7~9 4 30 22 Oral Y4 M 3 10~12 4 100 22 Oral 20 O1 M 4 13~16 4 0 22 Oral O2 M 4 17~20 4 10 22 Oral O3 M 4 21~24 4 30 22 Oral O4 M 4 25~28 4 100 22 Oral Y1 and O1: negative control (with excipients) Y2~Y4 and O2~O4: Group administrating Test substance

[0156] As a test substance, a 30% ethanol extract of nutmeg (Basic Disaster Team, Korea Basic Science Institute) was used, and Polyethylenglycol 400 (PEG 400) (Samchun Pure Chemical Co., Ltd. P0638) was used as an excipient (negative control group).

[0157] 3. Statistical Analysis

[0158] For statistical analysis, GraphPad Prism 7 was used and the significance level was set to p<0.05. The measurement values of each test group classified according to the administration concentration were converted into percentages compared to the control group, and an average value (M) and a standard error average (SEM) were used as test results. The difference between each group was statistically analyzed by One-Way or Two-Way ANOVA, and Neuman Keuls Test or Tukey Test was performed by post-test analysis. The difference between each group was analyzed by statistical analysis by One-Way or Two-Way ANOVA, and if significant results were seen, a post-test analysis was performed to evaluate the significance between each group.

Example 1: Preparation of Nutmeg Extract

1-1. Preparation of Nutmeg Extract

[0159] 100 g of pulverized nutmeg was added to 500 ml of each solvent (see Table 2) and extracts were prepared for each solvent condition using an ultrasonic extractor three times over two hours. The separated extract was prepared and used at the same concentration, and the myristicin standard was purchased from Sigma (product number: M9237). Each nutmeg solvent extract and myristicin standard were analyzed using HPLC [Optima Pak C18 column 4.6250 mm, 5 m particle size, flow rate 1 ml/min, UV detection: 260 nm] under MeOHH2O (0-32 min: 63% MeOH, 32-37 min: 63.fwdarw.100% MeOH) conditions, and the myristicin content of each nutmeg extract is shown in Table 1.

TABLE-US-00002 TABLE 2 The content of The content of Solvent extraction of myristicin in the nectarine B in the alcohol extract (%) extract (%) Water extract 0.19 0.62 10% aqueous ethanol 0.31 0.94 extract 20% aqueous ethanol 0.33 2.98 extract 30% aqueous ethanol 0.45 7.48 extract 40% aqueous ethanol 1.34 1.95 extract 50% aqueous extract 1.48 2.77 using ethanol 75% aqueous ethanol 1.27 2.95 extract 75% aqueous 1.85 7.47 methanol extract

[0160] As shown in Table 2, it was confirmed that the myristicin content of the 30% aqueous ethanol extract is extracted on average about three times less than that of the other conditions. In addition, as a result of analyzing the content of the nectarine B using the HPLC [OptimaPak_C18 column 4.6250 mm, 5 m particle size, flow rate 1 ml/min, UV detection: 205, 280nm] of MeOHH2O (0-35 min: 60% MeOH, 35-60 min: 60.fwdarw.100% MeOH) for the nutmeg solvent extract under each condition, it was found that the nectarine B was most extracted from the 30% aqueous ethanol extract. Therefore, it was confirmed that the condition for minimizing the content of toxic substances while maximizing the amount of the active substance is extracting nutmeg with an aqueous ethanol solution of 30% or less.

1-2. Identification of Compounds Extracted from Nutmeg

[0161] 2,5-bis-aryl-3,4-dimethyltetrahydro furan lignan-based compound was isolated using HPLC in the nutmeg extract of a 30% aqueous ethanol solution of the Example 1.

[0162] As a result, tetrahydrofuroguaiacin, Saucernetindiol, Verrucosin, Nectandrin B, Nectandrin A, and Fragransin C1, a total of 6 compounds were isolated.

Example 2: Measurement of Weight Change in Mice

[0163] Body weights were measured before administration (when taken and separated) and from the start of administration to the end of the experiment. As a positive control, creatine (120 mg/kg) was administered.

[0164] As shown in FIG. 2 the weight change of the group to which the nutmeg extract was administered was similar to that of the excipient control group, and the weight change was the same in the tested Young (Y) and Old (O) age groups.

[0165] A temporary weight loss was observed in all test groups on the 20th day of administration, which was judged to be due to the result of discontinuing the dietary supply one day prior to performing the neurological deficit test by administration to the nutmeg extract. It showed that the weight had been recovered by supply.

Example 3: Measurement of Glycogen Content in Muscle Cells (In Vitro)

[0166] C2C12 Cell was resuspended in a 25 cm2 flask using Growth Medium containing 10% fetal bovine serum, Penicillin-Streptomycin-Glutamine, and 50 l Sodium Pyruvate added to Dulbecco's Modified Eagle's Medium. The growth medium was changed after 6 hours. After maintaining the 50-70% confluency, the cells were cultured and subcultured with 10020 mm dish. At that time, the cell concentration was 1:13-15 dilutions. The cells were seedling at a concentration of 2.5105/6 well in a 6 well plate. When it was 90% confluency, the medium was changed to a differentiation medium containing 2% Horse Serum and Penicillin-Streptomycin-Glutamine in Dulbecco's Modified Eagle's Medium. The medium was changed to a differentiation medium every 24 hours (for 5 days). The differentiation under a microscope (make sure to differentiate and form a myotube) was confirmed. After the differentiation was completed, the extract or indicator was treated.

[0167] The glycogen content of the cells was measured using a Glycogen assay kit (Biovision). Specifically, the C2C12 muscle cells from which the culture solution was removed were washed twice with cold PBS, and then the plate was placed on ice, 200 l of distilled water per well was added, scraped, placed in an ep-tube, and then lysed with an ultrasonicator. Using 96-well plates, 50 l and 2 l hydrolysis enzyme were respectively added to the nutmeg extract sample (10 mg/kg/day, 20 mg/kg/day) and the standard solution in a well, and reacted at room temperature for 30 minutes. After completion of the reaction for 30 minutes, 50 l of the reaction mixture was dispensed, and then reacted for 30 minutes at room temperature at a shade, and absorbance was measured at 570 nm. The results were shown in FIG. 1.

[0168] As a result, it was confirmed that the content of glycogen in the differentiated muscle cells was significantly increased according to the nutmeg extract treatment as shown in FIG. 1. This can be said that the nutmeg extract of the present invention is a direct basis for indicating endurance exercise capacity.

Example 4: Rotarod Test

[0169] The equipment used for the test was a rotarod from SB Technology, and was tested in a fixed mode and an acceleration mode rotating at a speed of 4 or 4-40 rpm with a rotarod equipped with a rod for a mouse. The mice to be tested 1 day before the start of the test, 7 days after administration, were adapted in the laboratory for 1 hour, and then pre-training was performed for 1 minute at a fixed 4 rpm condition. At this time, the mouse that fell within 1 minute was put on the rod again, and trained on the rod for a total time of 1 minute. Before starting the test, the test was conducted after being adapted to the test environment for at least 30 minutes, and the test was conducted for 5 minutes in an accelerated mode rotating at a speed of 4-40 rpm per mouse individual. And the time of falling moment on the rod was recorded.

[0170] As a result, as shown in FIGS. 3 to 5, it was confirmed that the athletic ability and endurance of the mouse significantly increased since the nutmeg methanol extract has been treated.

[0171] As shown in FIG. 3, Young and Old mice not administered with nutmeg extract showed significantly lower the athletic ability and endurance compared to the Young and the Old mice administered with nutmeg extract in rotarod rotating at 4 rpm.

[0172] In other words, Young and Old mice administered with nutmeg extract showed superior athletic ability and endurance compared to mice without nutmeg extract, despite no concentration-dependent experience in rotarod.

[0173] As shown in FIG. 4, in order to determine the athletic ability according to muscle strengthening by the administration of nutmeg extract, the criterion showing that both Young and Old mice showed similar athletic ability regardless of whether or not the test substance was administered in a rod rotating at a low speed was found in training continuously at a low speed of 4 rpm.

[0174] It can be confirmed from FIG. 4 that after 3 days of training, the conditions for the movement and endurance of all mice were secured (F(7,20)=0.8985, p=0.5265).

[0175] As shown in FIG. 5, as a result of testing in the accelerated mode of rotarod, the athletic ability and endurance were improved in a concentration-dependent manner in both Young and Old mice by administration of nutmeg extract (F(7,19)=4.3180, p=0.0052). In Young mice, the improvement of athletic ability and the increase of endurance with concentration of nutmeg extract showed concentration-dependent effects, but there was no statistical significance (F(3,8)=0.5841, p=0.6421). In the case of Old mice, the concentration-dependent effect of nutmeg extract showed statistical significance (F(3,11)=10.79, p=0.0013).

[0176] In conclusion, it was shown that the old mice dosed with 30 and 100 mg/kg, bw showed statistically significant improvement in athletic ability and endurance of 2.3 times and 2.6 times, respectively, compared to old mice without nutmeg extract (p<0.01). Old mice dosed with 10 mg/kg, bw showed an increase in athletic ability and endurance of 1.4 times. Particularly, in the case of the old mouse to which 30 and 100 mg/kg, bw of the nutmeg extract were administered, it showed similar athletic ability and endurance as the Young mouse, and thus it was confirmed that the nutmeg extract of the present invention is effective in improving exercise capacity and endurance.

[0177] The statistically significant improvement in concentration-dependent athletic ability in the old mouse according to nutmeg extract means that it has the same effect as that in the Young mouse by restoring the athletic ability of the old mouse with reduced exercise ability due to muscle weakness. In addition, nutmeg extract suggests that the results of improving the motor skills of Young mice may strengthen the normal muscle strength.

Example 5: Wire Hang Test

[0178] The test was performing using a wire having a diameter of 2 mm installed at a height of 25 cm. Each mouse was placed on the wire using only the front legs, and the time of hanging without falling was measured. The time when the mouse was suspended and then dropped was measured and the test was conducted for a maximum of 120 seconds. In this way, each mouse was repeated 3 times at approximately 20 minute intervals. The results of each mouse were analyzed for the average value of the first and longest hanging time in three trials. All Young mice in the nutmeg extract-administered group and the excipient control group were tested only in the Old mice because they were not suitable for evaluating the exercise ability of the pure forelimbs only because they showed the behavior of pulling the wire by the muscles of the strong forelimbs and pulling up the wire with the hind limbs. In addition, creatine 120 mg/kg (creatine-120) was administered to mice as a positive control, and the test was conducted under the same conditions.

[0179] As shown in FIG. 6, as a result, it was found that the improvement of athletic ability and endurance according to muscle strengthening was statistically significantly increased depending on the concentration of nutmeg extract administration (F(3,11)=4.555, p=0.0262). The effect of enhancing athletic ability by muscle strengthening by administration of nutmeg extract was increased by 25%, 58% and 97% at 10, 30 and 100 mg/kg, bw, respectively and the administration group of 100 mg/kg, bw showed a statistically significant increase effect (p<0.05).

[0180] In addition, when compared with the results of the positive control group (creatine-120), it was confirmed that it was more effective than the aging mouse group, but when the nutmeg extract 10mg/kg was administered, the effect of improving motor function was similar. On the other hand, when 30 mg/kg and 100 mg/kg of nutmeg extract respectively were administered, it was confirmed that they showed a remarkable effect on improving motor function compared to the positive control group.

[0181] Through these results, it means that nutmeg extract can strengthen the muscles of the fore limbs of aged or degenerated Old mice, which can improve athletic ability and endurance, suggesting that these effects are non-specifically involved in exercise-related muscles.

Example 6: Morris Water Maze Test

[0182] Morris water maze placed mice swimming in a circular pool with a featureless interior surface in a room with walls with various visible external markers attached. The water temperature in the water labyrinth was adjusted to room temperature or 21 C., and skim milk powder was mixed to make the water labyrinth surface opaque, and the only platform that could escape the water in the labyrinth was placed in one of the four quadrants of the round labyrinth. The water labyrinth test was performed with a visible version where the pretraining and platform can be seen from the surface. Pretraining allowed all mice to swim freely in a water labyrinth without platforms for 60 seconds, and the swimming speed of each mouse was used as an indicator of athletic ability.

[0183] As shown in FIG. 7, in the Young mice, swimming speed was consistent in all groups regardless of the nutmeg extract administration. In the Old mouse, it showed a tendency to improve swimming speed by nutmeg extract administration statistically significantly (F(7,20)=6.647, p=0.0004).

[0184] As shown in FIG. 8, the same tendency was observed when only the Young and Old mice, which were not treated with nutmeg extract, were analyzed statistically (F(4,14)=10.23, p=0.0004).

Example 7: Neurological Deficit Test

[0185] To determine the presence or absence of neurological deficit according to the administration of a nutmeg extract, rotarod fixed at 6 rpm was used.

[0186] In order to exclude interactions with ingested food, all experimental animals were fasted for 18 hours, and nutmeg extract was administered at 100, 300, and 1,000 mg/kg, and then the number of experimental animals falling from the rotarod for 1 minute at 0.5, 1 and 2 hours was recorded.

[0187] As a result, as shown in Table 3, 1,000 mg/kg, bw of nutmeg extract was administered to each of Young and Old mice and tested by time period. As a result, all mice tested did not drop successfully from the rotarod.

[0188] In the Old mice, at 0.5 and 1 hour after administration of the test substance, all mice did not fall under the tested conditions, but at 2 hours, only one was not satisfied with the conditions.

TABLE-US-00003 TABLE 3 Test time (number of mice fallen/number of mice tested) 0.5 hr 1 hr 2 hr Young-control 3/3 2/3 3/3 Young-1000 mg/kg, bw 3/3 3/3 3/3 Old-control 2/4 4/4 2/4 Old-1000 mg/kg, bw |4/4 4/4 3/4

[0189] In addition, the test result of Neurologic deficit means that there is no effect on the nervous system according to the administration of nutmeg extract. That is, the nutmeg extract is estimated to have a TD50 value of 1,000 mg/kg, bw or more, with 50% toxic dose that shows neurological deficit, and at the same time, based on the results of the rotarod test, when predicted to be at least 10 to 30 mg/kg at a concentration of 50% of nutmeg to show the athletic ability of the Young mice, the protective index (PI) value is judged to be at least 34, suggesting that it is a very safe material.

Example 8: Forced Swim Endurance Test

[0190] The forced swim endurance test was conducted by modifying Jacob and Michud's method (Jacob J, Michud G. Action of various pharmacological agents on the exhaustion time and behavior of mice swimming at 20 degree C. I. Description of the technic. Actions of amphetamine, cocaine, caffeine, hexobarbital and merprobamate. Arch. Int. Pharmacodyn. Ther. (1961) 133: 101.). A circular water tank with a diameter of 120 cm was filled with water to a depth of 30 cm and the water temperature was maintained at 211 C. A weight about 5% of the weight of each mouse was hung on the tail, and 3-4 mice from each group are put into the tank at the same time. The time until swimming and exhaustion was measured. The test was performed within a limited time of 5 minutes.

[0191] As a result, as shown in FIG. 9, both Young and Old mice administered with the nutmeg extract showed a tendency to increase swimming endurance in a concentration-dependent manner. During the forced swim endurance test, the Young mice showed more activity than the Old mice, and few inappropriate floating behaviors were observed compared to the Old mice.

Example 9: The Effect of Increasing the Muscle Mass of Nutmeg Extract

[0192] Three and 10-month C57BL/6 mice were randomly divided into 8 groups of 3 animals, respectively, according to Table 1, and used in the experiment.

[0193] In the experimental group, 30% ethanol extract of nutmeg was administered at a constant time for a total of 22 days once a day at three concentrations of 10 mg/kg, 30 mg/kg, and 100 mg/kg. At this time, as a control, polyethylene glycol as an excipient was used. After the end of the test, the rat's thigh was separated and the volume was measured.

[0194] As a result, as shown in FIGS. 10 and 11, compared to the control group, the muscle mass and volume increased overall in the group administered with the nutmeg extract regardless of the age of the rat, especially in the Old mouse. As a result, it was confirmed that the nutmeg extract of the present invention increased the muscle mass, and thus, the effects of the athletic ability and muscle strength improvement were very excellent.

Example 10: Rotarod Test and Liver Toxicity Confirmation

[0195] The rotarod test was performed under the same conditions and methods as in Example 4. At this time, as a positive control, mice administered with creatine 120 mg/kg (creatine-120) were used. Specifically, the mice to be tested 1 day before the start of the test, 7 days after administration, were adapted in the laboratory for 1 hour, and then pre-training was performed for 1 minute at a fixed 4 rpm condition. At this time, the mouse that fell within 1 minute was put on the rod again, and trained on the rod for a total time of 1 minute. Before starting the test, the test was performed after being adapted to the test environment for at least 30 minutes, and the test was conducted in an accelerated mode rotating at a speed of 4-40 rpm per mouse. And the time of falling moment on the rod was recorded.

[0196] As a result, as shown in FIG. 12A, the effect of exercise improvement was similar when creatine and 10 mg/kg of nutmeg extract was administered. On the other hand, when the nutmeg extract was administered at a concentration of 30 mg/kg, and 100 mg/kg, it was confirmed that the effect of exercise improvement was remarkably higher than when the creatine was administered.

[0197] And then, experiments were performed as follows to confirm the effect of nutmeg extract on the liver.

[0198] After the acclimatization of the three-month-old mouse, each group was divided into a group that was orally administered with creatine 120 mg/kg for 22 days, a group that was orally administered with nutmeg extract 100 mg/kg for 22 days, and a group that had only normal diet for 22 days. Then, the liver was removed and observed at the expense of each mouse.

[0199] As a result, as shown in FIG. 12B, when creatine was administered, it was confirmed that the liver lobe was enlarged, and it was confirmed that liver toxicity occurred during long-term use. On the other hand, when the nutmeg extract was administered, the liver lobe was not enlarged, and the shape of the liver was found to be the same as that of a normal mouse (data not shown).

[0200] Therefore, it was confirmed that nutmeg extract did not show liver toxicity even after long-term use.

INDUSTRIAL APPLICABILITY

[0201] As discussed above, the nutmeg extract of the present invention contains a high quantity of ligand-based compounds and exhibits, when taken, very excellent effects of improving athletic ability, improving endurance and improving muscular strength, and thus is effective for treating sarcopenia and can be effectively used in medical supplies, food and the like for improving physical strength or athletic ability.