Method of producing myokine producing promotor

Abstract

To provide a method for producing a myokine producing promotor supported by evidence; and to provide various agents exhibiting excellent effects on skeletal muscles and immunization by promoting production of myokine in a body (skeletal muscles, etc.).SOLUTION: There is provided a method of producing a myokine producing promotor, in which, while a temperature of an object to be separated into solid and liquid in a vessel is maintained at 45°C or lower, a pressure inside the vessel is reduced to separate the object into solid and liquid without using both extraction liquid and extraction steam which are extraction media, and the liquid contained in the object is recovered as an active ingredient, the object being an undried product which is cypress not completely dried in the method of producing a myokine producing promotor. There are provided a myokine producing promotor produced by the method, and an anti-inflammatory agent for skeletal muscles, a skeletal muscle formation promoter, a repair agent for damaged skeletal muscles, an enlargement agent for skeletal muscles, and a post-movement immunosuppressant, which contain the myokine producing promotor.SELECTED DRAWING: Figure 4

Description

[Technical field]

[0001] The present invention relates to a method for producing a myokine production promoter, and the myokine production promoter produced by said method. It also relates to an anti-inflammatory agent for skeletal muscle, an agent for promoting skeletal muscle formation, an agent for repairing skeletal muscle damage, an agent for hypertrophying skeletal muscle, and an agent for suppressing post-exercise immune decline, each containing the myokine production promoter. The present invention further relates to creams, aqueous liquids, lotions, gels, etc. for muscle building or massage, each containing the agent. [Background technology]

[0002] Essential oils extracted from coniferous trees using an extraction device often contain aromatic components such as monoterpenes, and therefore coniferous trees are often used as the subject of extraction. In addition to being used for extraction, trees, vegetables, fruits, etc. are also dried in specific drying devices and used for biomass power generation, waste disposal, etc.

[0003] Patent Document 1 describes a method for extracting high-purity monoterpenes by performing steam distillation under reduced pressure while irradiating with microwaves, thereby reducing the content of unnecessary nitrogen dioxide and formaldehyde.

[0004] Furthermore, Patent Document 2 describes an apparatus for obtaining various extracts by drying wood, vegetables, etc., using a specific wood drying and extraction device.

[0005] However, in Patent Documents 1 and 2, there is no upper limit on the temperature during extraction, so the active ingredients contained in the target substances such as trees, vegetables, and fruits end up being decomposed. In addition, in Patent Document 1, an extraction medium called water vapor is added from the outside, so not only do the active ingredients dissipate when the water is removed, but impurities also enter along with the water vapor, so it is not possible to obtain an extract that is entirely natural.

[0006] As an example of an extraction method and extraction device that specifies an upper limit temperature, Patent Document 3 describes a method for producing aroma components such as essential oil derived from Citrus depressur by reducing pressure at 65°C or lower. However, in Patent Document 3, the subject of extraction is limited to Shikuwasa, and the uses of the obtained aroma components are for aromatherapy, fragrances, cosmetics, etc. In other words, the focus was basically on the smell and aroma of Shikuwasa essential oil.

[0007] As an extraction method and extraction device with a specified upper limit temperature, Patent Document 4 describes a method for obtaining an aqueous extract by performing solid-liquid separation of lavender, and describes that the obtained aqueous extract can be used as an autonomic nervous system regulator. However, in Patent Document 4, the subject of extraction is limited to a specific lavender, and the extract described in Patent Document 4 is taken intranasally to act on a specific part of the cerebrum and obtain the above-mentioned effect. In other words, the focus was on the smell and aroma of the extract from lavender.

[0008] On the other hand, Patent Document 5 describes an agent for promoting the production of IL-6 (a type of cytokine) that contains chamomile essential oil, rosemary essential oil, menthol, limonene, and the like. However, Patent Document 5 does not mention extraction from Hinoki cypress, and only confirmed the production of IL-6, so the invention was limited to IL-6.

[0009] In recent years, it has become clear that myokines, physiologically active substances (cytokines) secreted from skeletal muscles, have various actions and effects on skeletal muscles. However, there has not been much research into what specific substances promote the production of myokines, and it is not clear in detail whether the fluids (compositions) within plant cells have such effects. No investigation has been conducted into whether the substance exists within plants, or what kind of intracellular fluid (composition) in a plant has such an effect (the substance), or, if it does exist, what agents contain the substance.

[0010] In recent years, as sports have become more popular, it has become increasingly important for athletes to maintain their health and increase their muscle strength. In addition, as life expectancy increases, it has also become increasingly important to prevent muscle loss due to aging. Problems have arisen as to how athletes can efficiently (through training, etc.) maintain their health and increase their muscle strength, and how elderly people can be prevented from losing muscle strength.

[0011] However, there are almost no muscle-building agents that are directly supported (with evidence) by medical measurements, analytical measurements, or measurements directly on humans (skeletal muscles) that target skeletal muscles, and in particular, there have been no reports of "extracts from plants (plant-derived substances)" as such agents. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] JP 2013-100520 A [Patent Document 2] Patent No. 7154643 [Patent Document 3] JP 2013-203911 A [Patent Document 4] JP 2020-147547 A [Patent Document 5] JP 2019-147777 A Summary of the Invention [Problem to be solved by the invention]

[0013] The present invention has been made in consideration of the above-mentioned background technology, and its object is to solve the above-mentioned problems, to provide a "manufacturing method for a myokine production promoter" that is supported by scientific measurement results (evidence), and to provide various agents that promote the production of said myokines in the body of the person who takes them (skeletal muscles, etc.) and exert excellent effects on skeletal muscles and the immune system. [Means for solving the problem]

[0014] As a result of extensive research into solving the above problems, the inventors discovered that the myokine production effect is particularly remarkable when cypress in a specific condition is used and the "liquid obtained by solid-liquid separation" is obtained using a specific method, thereby completing the present invention.

[0015] That is, the present invention is a method for producing a myokine production promoter, which comprises maintaining a temperature of a solid-liquid separation target placed in a container at 45°C or lower, reducing the pressure inside the container without using an extraction liquid or extraction steam as an extraction medium, to separate the target into solid and liquid, and recovering the liquid contained in the target as an active ingredient, The present invention provides a method for producing a myokine production promoter, characterized in that the object is a cypress product that is not completely dried but is still undried.

[0016] The present invention also provides a method for producing the myokine production promoter, wherein the myokine is Interleukin-6 (IL-6), Recombinant Murine IP-2 (CXCL2), Recombinant Murine IP-10 (CXCL10), Rps6kb1 (RPS6KB1), or Musclin (OSTN).

[0017] The present invention also provides a myokine production promoter which is produced using the above-mentioned "method for producing a myokine production promoter".

[0018] The present invention also provides an anti-inflammatory agent for skeletal muscle, an agent for promoting skeletal muscle formation, an agent for repairing skeletal muscle damage, an agent for hypertrophying skeletal muscle, and an agent for suppressing post-exercise immune decline, each of which is characterized by containing the above-mentioned myokine production promoter.

[0019] The present invention also provides a muscle spray liquid characterized by containing the above-mentioned myokine production promoter; a cream, aqueous liquid, emulsion, or gel for muscle strengthening; and a cream or gel for muscle massage.

[0020] The present invention also provides a muscle-building cream, aqueous liquid, emulsion, or gel, which contains the above-mentioned skeletal muscle formation promoter. Effect of the Invention

[0021] The present invention solves the above-mentioned problems and issues, and was made by discovering that the liquid phase obtained by solid-liquid separation of undried cypress at 45°C or below has the effect of promoting the production of myokines in the human body. A "myokine" is a physiologically active substance (cytokine) secreted from skeletal muscle (muscle) cells.

[0022] In the present invention, multiple evaluation subjects were added to "C2C12 cells that can be made to produce a state of movement by applying electrical stimulation (EPS)," and the expression of various "myokine genes" was confirmed by RT-PCR. When the object of evaluation was "liquid obtained by solid-liquid separation of undried cypress using a specific method," unexpectedly, the expression of at least five kinds of "myokine genes" was confirmed. As a result of measuring multiple plants using the above method, the above fact was confirmed particularly notably in cypress. Although the number and types of "myokine genes" that were confirmed to be expressed in cedar were smaller than in cypress, the above facts were confirmed when compared to other plants.

[0023] Here, "C2C12 cells" are mouse skeletal muscle cells, and can also be called "exercise-like skeletal muscle cultured cells" that can create an in vitro exercise state by applying electrical stimulation (EPS) to cells grown in vitro (see, for example, Hatakeyama H, Kanzaki M. Mol Biol Cell, 24(6), 809-817, (2013)).

[0024] Hereinafter, the "method of separating the object into solid and liquid by reducing pressure without using the extraction liquid or extraction steam as the extraction medium while maintaining the temperature of the object below 45°C" may be abbreviated to "low-temperature vacuum extraction method." However, since this method does not extract or transfer the active ingredient to the extraction medium, it is not an extraction method in the strict sense, but rather a solid-liquid separation method.

[0025] It has already been shown that various myokines are secreted from the skeletal muscles of people who exercise (see, for example, Pedersen BK, J Appl Physiol, 103(3), 1093-8, 2007, etc.). Furthermore, these myokines are believed to have various physiological actions and have a significant impact on promoting health, and should be particularly effective on human skeletal muscles (after exercise).

[0026] In light of recent trends in exercise physiology, we investigated training (exercise) and post-exercise care of skeletal muscles to achieve more efficient and effective muscle building and strength building. In the present invention, it has been discovered that in order to achieve and realize the above, it is necessary to solve the problem of "how to increase the amount of effective myokines secreted from skeletal muscles" (the problem has been found).

[0027] The multiple myokines found to be effective by this invention (multiple myokines produced in C2C12 cells) are known to have anti-inflammatory effects, skeletal muscle formation promotion effects, skeletal muscle damage repair effects, skeletal muscle hypertrophy effects, or immune suppression effects, respectively (see literature listed in the examples). After the liquid of the present invention is administered via the skin, etc., the "multiple myokines" produced in the body each have one of the above-mentioned effects, and therefore, by administering the liquid (liquid-solid separated from cypress by low-temperature vacuum extraction), all of the above-mentioned effects will be achieved.

[0028] In the present invention, it was revealed that at least five kinds of myokines are produced in skeletal muscles by the liquid derived from Hinoki cypress. Therefore, even if some myokines are not produced, the present invention can be generalized to a "method for producing a myokine production promoter."

[0029] The myokine production promoter of the present invention, and the skeletal muscle anti-inflammatory agent, skeletal muscle formation promoter, skeletal muscle damage repair agent, skeletal muscle hypertrophy agent, and post-exercise immune suppressant containing the myokine production promoter, exert their effects even when administered (preferably to the skin above the muscle) when the general public is in a normal state. Therefore, by incorporating the agent, it can be applied to health-related products, cosmetics-related products, etc.

[0030] However, the above-mentioned effects are more pronounced when applied to the muscles of athletes, people who have exercised, or people who will exercise before, during, or after exercise. Furthermore, the above-mentioned effects are particularly pronounced for athletes after exercise.

[0031] When the myokine production promoter is contained in a muscle spray liquid, or a muscle massage liquid, cream, gel, etc., the manner of use particularly enhances the above-mentioned effects. [Brief description of the drawings]

[0032] [Figure 1] FIG. 1 is a schematic diagram showing one embodiment of an apparatus used in the extraction step of the low-temperature vacuum extraction method of the present invention. [Diagram 2] FIG. 2 is a schematic diagram showing another embodiment of an apparatus used in the extraction step of the low-temperature vacuum extraction method of the present invention. [Diagram 3] Schematic diagram of the device used to evaluate and confirm myokine production in the present invention. (a) Schematic diagram of the evaluation device that applies electrical stimulation (EPS) to C2C12 cells to create a state of movement in vitro. (b) Microscopic magnified image of C2C12 cells. [Figure 4]This is a bar graph showing the level of IL-6 gene expression immediately, 60 minutes, and 120 minutes after C2C12 cells were given a liquid separated from solid-liquid cedar or cypress and then subjected to electrical stimulation (EPS). [Diagram 5] This is a bar graph showing the level of CXCL2 gene expression immediately, 60 minutes, and 120 minutes after C2C12 cells were given a liquid separated from solid-liquid cedar or cypress and then subjected to electrical stimulation (EPS). [Figure 6] This is a bar graph showing the gene expression level of CXCL10 immediately, 60 minutes, and 120 minutes after applying an electrical stimulus (EPS) to C2C12 cells after applying a liquid separated from solid-liquid separation of cedar or cypress. [Figure 7] This is a bar graph showing the expression level of the RPS6KB1 gene immediately, 60 minutes, and 120 minutes after C2C12 cells were given a liquid separated from solid-liquid separation of cedar or cypress and an electrical stimulus (EPS). [Figure 8] This is a bar graph showing gene expression levels immediately, 60 minutes, and 120 minutes after C2C12 cells were given a liquid separated from solid-liquid separation of cedar or cypress and then subjected to electrical stimulation (EPS). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0033] The present invention will be described below, however, the present invention is not limited to the specific embodiments below and can be modified as desired within the scope of the technical concept of the present invention.

[0034] The method for producing the myokine production promoter of the present invention is a method in which the target substance is separated into solid and liquid by the "low-temperature vacuum extraction method" defined above, and the liquid contained therein is recovered as an active ingredient, and is characterized by the use of "undried cypress wood that is not completely dried" as the target substance.

[0035] <The subject is a cypress tree> Cypress is a plant belonging to the class Pinaceae, order Pinales, family Cupressaceae, and genus Chamaecyparis. In the present invention, the age of the cypress tree, place of origin (including trademarks indicating various places of origin), etc. are not limited. Furthermore, the parts of the cypress are not particularly limited, and examples thereof include the trunk, bark, branches, leaves, flowers, and fruits. Preferably, the bark, branches, or leaves, and also the above parts contained (mixed) in the bark or branches and leaves. Particularly preferably, the object 41 of the present invention is the bark of the cypress, or the branches and leaves of the cypress.

[0036] In addition, when classified based on their origin, the object 41 of the present invention is preferably, but not limited to, thinning materials of cypress trees that have been cut down in places as whole trees in order to obtain excellent building materials, etc.; branches and leaves of cypress trees that have been cut off (pruned) in order to obtain building materials, etc.; driftwood of cypress trees that are retained or dammed downstream of rivers, dams, ponds, lakes, etc.; bark of cypress trees that has been peeled off to obtain building materials, etc.; etc. These can be obtained in large quantities as unwanted materials and can also be used for waste disposal, which reduces the cost of the present invention.

[0037] In the present invention, it is essential to use cypress that is not completely dried as the object of solid-liquid separation 41. If a dried product is used, it is not possible to perform solid-liquid separation and recover the liquid 42. Usually, if the cypress used is of the above-mentioned origin and is not dried deliberately, it falls into the category of "undried products that are not completely dried."

[0038] In the present invention, it is preferable to cut the cypress, which is the object 41 of solid-liquid separation, before placing it in the container. The cypress may be placed without cutting, but solid-liquid separation may not proceed well. There are no particular limitations on the degree of cutting, but the number-average length of the longest point of each piece is preferably 0.1 cm to 50 cm, more preferably 0.3 cm to 40 cm, even more preferably 1.0 cm to 30 cm, and particularly preferably 3 cm to 20 cm. If the time is too short, drying will occur easily, the yield of the liquid 42 contained in the cypress will decrease, and active ingredients with low boiling points may dissipate. If the time is too long, the efficiency of mixing and solid-liquid separation may decrease, and the equipment may be overloaded.

[0039] <Solid-liquid separation method (low-temperature vacuum extraction method)> In the manufacturing method of the present invention, while maintaining the temperature of the cypress placed in a container at 45°C or lower, the container is depressurized without using an extraction liquid or extraction steam as an extraction medium, and the target 41 is subjected to solid-liquid separation, and the liquid contained in the target 41 is recovered as an active ingredient. In other words, the present invention uses cypress as the target 41, performs solid-liquid separation by low-temperature vacuum extraction, and recovers the liquid side.

[0040] The extraction medium not used includes water, organic solvents such as alcohols, supercritical fluids and subcritical fluids such as carbon dioxide, etc. The above water vapor refers to the water vapor used in steam distillation. Here, "not used" means that substantially no use is made, and "substantially no use" means that only 3% by mass or less of the target is used, preferably 2% by mass or less, more preferably 1% by mass or less, and particularly preferably not used at all.

[0041] The low-temperature vacuum extraction method is preferable in that it produces an extract containing only natural ingredients because no extraction medium remains, it leaves no odor of the extraction medium, it does not require the removal of the extraction medium, the ingredients do not thermally decompose, the ingredients do not dissipate, liquid 42 is obtained with almost no dissipation of water within the cypress, it actually produces the above-mentioned effects of the present invention more effectively than extracts produced by other extraction methods, and the solid side (residue 44) after solid-liquid separation can also be effectively utilized.

[0042] When cypress is subjected to solid-liquid separation by low-temperature vacuum extraction, mainly an aqueous extract is obtained. The "aqueous extract" includes "a case in which the oily component, also known as essential oil, is dissolved and is uniform as an aqueous solution" and "a case in which a small amount of oily component separates to the upper layer and disperses uniformly when stirred." When the oily component (essential oil) separates to the upper layer, it can be used as a uniform liquid 42 by stirring or the like, or it can be used as the "liquid 42" in the present invention without the oily component.

[0043] The liquid 42 obtained by solid-liquid separation in the present invention, i.e. the myokine production promoter, contains water (intracellular fluid) within the cells of the cypress, and therefore the liquid 42 (myokine production promoter) is entirely derived from the natural product (plant) of the cypress. Furthermore, since no deterioration due to heat occurs when extracted using low-temperature vacuum extraction, the liquid remains completely natural (plant).

[0044] <<Solid-liquid separation equipment and conditions>> 1 and 2 are schematic diagrams showing an example of an apparatus used for solid-liquid separation of cypress, but the present invention is not limited to solid-liquid separation using the apparatus actually shown in FIGS. 1 and 2 as long as it is within the scope of the present invention.

[0045] The vessel 21 holds the raw material 41, cypress, and stirs it with stirring blades 23 while reducing the pressure inside the vessel to separate the solid and liquid, while maintaining a predetermined temperature range (this temperature refers to the temperature of the raw material, cypress). The cooler 22 is a device that cools the gas (steam) coming out of the vessel 21.

[0046] Around the container 21, there is a heating device 24 that applies heat to the inside of the container 21 and to the target object (raw material) 41. A cooling device (not shown) may be provided in the container 21. At the top of the container 21, there is an inlet for the target object 41, cypress, and a lid that covers the inlet, and at the center of the bottom of the container 21, there is a discharge outlet 26 for taking out the solid residue 44 of cypress after solid-liquid separation. The vessel 21 is provided with an exhaust port 25 for the sucked steam, and this exhaust port 25 is connected to a pipe 27 leading to the cooler 22 .

[0047] The container 21 is equipped with measuring instruments such as a vacuum gauge 32 for measuring the degree of vacuum within the container, and a thermometer 33. These are installed to measure the pressure (degree of reduced pressure) and temperature within the container during extraction, and to indirectly measure the temperature of the cypress, which is the subject of extraction (raw material) 41, and to determine the start and end of extraction.

[0048] The solid-liquid separation is carried out, for example, as follows. At the start of the operation, the cooling unit is filled with cooling water. The target object (raw material) 41, cypress, is put into the container through the inlet and the lid is closed. Stirring is started, and heat is applied from the outside by the heating device 24. The heat applied to the container 21 is transferred to the cypress, which is the target (raw material) 41, and the cypress is stirred, promoting solid-liquid separation. At that time, the heating device 24 and cooling device are adjusted to bring the temperature of the cypress into the preferred range described below.

[0049] By sucking with the pressure reducing device 35, the gas in the container, i.e., the liquid vapor and air, is sucked through the piping 27, and the liquid 42 contained in the cypress in the container, i.e., the components that are liquid at room temperature, begins to evaporate. At this time, the amount of suction and the suction power of the pressure reducing device 35 are adjusted to keep the pressure (degree of reduced pressure) within a preferred range described below. Also, the temperature range of the object 41 is maintained within a preferred range described below. From the viewpoint of an industrially feasible (realistic) "relationship between exhaust volume and degree of vacuum," the pressure reducing device 35 is preferably an ejector having a large exhaust volume.

[0050] The vapor of the active ingredients, such as “vapor of the aqueous components contained in the cypress” in the container, is sucked through piping 27 and introduced into the cooler 22, where it is cooled and liquefied, becoming a recovered liquid that accumulates in the recovery tank 34. When a predetermined amount of the aqueous liquid 42 has accumulated in the recovery tank 34, suction by the pressure reducing device 35 is stopped, and the aqueous liquid 42 is recovered by appropriately opening and closing the valves. In the case of liquid 42 from cypress, most of it is aqueous. However, if an oil layer (essential oil) has been separated, it is possible to leave it to stand and separate the liquids to remove the oil layer (essential oil) and recover only the aqueous liquid 42, or it is possible to disperse the oil layer (essential oil) evenly in the aqueous layer and recover all of it as aqueous liquid 42.

[0051] In the present invention, it is particularly preferable to use aqueous liquid 42, which is the liquid side obtained after solid-liquid separation, because it can be suitably used as a cream, aqueous liquid, emulsion, gel, etc. for application to muscles for massage, etc.; and it is particularly effective as a myokine production promoter for various agents described below. In addition to the main effect of the aqueous liquid 42 being superior to oil-based extracts (essential oils, etc.) as an agent of the present invention, the aqueous liquid 42 is superior to oil-based extracts (essential oils, etc.) in that a larger amount can be recovered in a single solid-liquid separation; the odor is weaker than that of oil-based extracts (essential oils, etc.), and is relatively pleasant.

[0052] The temperature (range) of the target material (raw material) 41 during solid-liquid separation must be maintained at 45°C or less, preferably 10°C or more and 43°C or less, more preferably 15°C or more and 40°C or less, and particularly preferably 20°C or more and 38°C or less. If the temperature is too high, the active ingredient may be thermally decomposed or deteriorated, the active ingredient may be dispersed, ingredients other than natural products may be produced, etc. On the other hand, if the temperature is too low, solid-liquid separation may not proceed properly, etc.

[0053] The degree of reduced pressure during solid-liquid separation is not particularly limited, but at least during the main solid-liquid separation, the pressure inside the vessel is preferably maintained at 20 kPa or less, more preferably at 15 kPa or less, and particularly preferably at 1 kPa or more and 10 kPa or less.

[0054] When the upper limit of the pressure is within the above range, it is possible to extract the active ingredient at a low temperature taking into consideration the boiling point of water, so that the active ingredient can be prevented from being denatured or decomposed by heat, and the active ingredient can be sufficiently extracted. In addition, the extraction time can be shortened, so that the decomposition or loss of the active ingredient is suppressed, and there is no unnecessary time loss, making it economical. For industrial implementation, a large exhaust capacity is required to maintain the temperature range within the above range, etc. From the practical "relationship between exhaust capacity and degree of vacuum", that is, from the viewpoint that the pressure reducing device 35 must actually exist, etc., the lower limit of the pressure is preferably within the above range.

[0055] <Cedar as the object> The manufacturing method for the myokine production promoter of the present invention is characterized in that the subject 41 is "a wet cypress product that is not completely dried." However, when the subject 41 is "a wet cedar product that is not completely dried," the production of several myokines was confirmed (expression of myokine genes was confirmed), although not to the same extent as with cypress (see the examples).

[0056] Therefore, it is also preferable to mix the "myokine production promoter obtained by solid-liquid separation of Japanese cypress" of the present invention with the "liquid obtained by solid-liquid separation of Japanese cedar." The present invention also relates to a method for producing the myokine production promoter, in which the object 41 is made of undried cedar that has not been completely dried.

[0057] The definitions and preferred aspects of "cedar", including the part (such as branches and leaves), origin (such as thinned wood), cutting, etc., are the same as those of "cypress" described above. In addition, the method and conditions for solid-liquid separation are also the same as those of "cypress" described above.

[0058] The cypress and cedar may be subjected to solid-liquid separation separately and the resulting liquid 42 may be mixed to produce the myokine production promoter, or the cypress and cedar may be placed simultaneously in container 21 and subjected to solid-liquid separation to produce the myokine production promoter. The mixing ratio of the cypress-derived liquid and the cedar-derived liquid (the ratio of cypress to cedar) is not particularly limited, but taking into consideration the myokine production effect and the type and strength of the odor, a cypress (derived liquid) / cedar (derived liquid) ratio of 100 / 0 to 60 / 40 is preferable, 95 / 5 to 65 / 35 is more preferable, and 90 / 10 to 70 / 30 is particularly preferable.

[0059] <<Comparison with other methods (extraction methods)>> Currently, the most commonly used methods for extracting functional ingredients from plants are solvent extraction and steam distillation. Among these, the solvent extraction method leaves behind residual solvent, which has a detrimental effect on the myokine production promoter. Note that the solvent also contains water.

[0060] In addition, steam distillation is a method of extracting essential oils and other components from plants using steam, and is a traditional technique with a high level of safety, but the components originally contained in the plant are decomposed due to exposure to high temperatures of around 100°C. In addition, the distilled water containing a large amount of aroma extracted together with the essential oil has its components altered due to exposure to high temperatures, and therefore cannot be used as the myokine production promoter of the present invention, or as "various agents containing myokine production promoters" described below.

[0061] In addition, in the solvent extraction method and steam distillation method, the residue after component extraction has no particular use and is discarded as industrial waste. However, in the present invention, not only the liquid 42 obtained by solid-liquid separation but also the solid residue 44 can be effectively utilized with great potential.

[0062] The manufacturing method of the present invention, which uses cypress to separate into solid and liquid form to obtain liquid 42, solves the problems associated with other manufacturing (extraction) methods and provides the various effects described above, but also has the following advantages (including overlapping ones): (1) Because solid-liquid separation is carried out at low temperatures below 45°C, heat-sensitive components such as enzymes can be extracted in a form close to their natural state. (2) Since no solvents or steam are used, it is possible to obtain natural active ingredients that are 100% derived from cypress. (3) There is no need to worry about residual organic solvents, making it highly reliable and safe. (4) The concentration of ingredients is higher than with other extraction methods.

[0063] <Myokine> When the liquid 42 obtained by solid-liquid separation of cypress using the above-mentioned method was added to the aforementioned "C2C12 cells that can be made to move by applying electrical stimulation (EPS)," the expression of various "myokine genes" was confirmed (see Examples).

[0064] The myokines (proteins) for which expression of the corresponding genes was confirmed were Interleukin-6 (IL-6), Recombinant Murine IP-2 (CXCL2), Recombinant Murine IP-10 (CXCL10), Rps6kb1 (RPS6KB1), and Musclin (OSTN).

[0065] In this embodiment, the production of at least the five types of myokines mentioned above was confirmed, and because this finding is a new discovery, it is natural that there are some myokines that are not produced. However, the myokines in the "manufacturing method for myokine production promoter of the present invention" are not particularly limited, but cover the entire higher-level concept of myokines.

[0066] However, a preferred embodiment of the present invention is the above-mentioned "method for producing a myokine production promoter" in which the myokine produced is Interleukin-6 (IL-6), Recombinant Murine IP-2 (CXCL2), Recombinant Murine IP-10 (CXCL10), Rps6kb1 (RPS6KB1), or Musclin (OSTN).

[0067] Here, the official names of the above five myokines (proteins) and the official names of the genes of the proteins are as follows, in the order of the above myokines (proteins). The top row shows the myokine name (protein name) and the bottom row shows the gene name. In addition, for both myokine names (protein names) and gene names, the text following the colon (:) is the respective abbreviation.

[0068] Protein name: abbreviation (Gene name: abbreviation) Interleukin 6: IL6 (Interleukin 6: Il6) ·Recombinant Murine IP-2, CXC motif chemokine ligand 2: MIP-2, CXCL2 (CXC motif chemokine ligand 2: Cxcl2) ·Recombinant Murine IP-10, CXC motif chemokine ligand 10: MIP-10,CXCL10 (CX-Cmotif chemokine ligand 10: Cxcl10) ·Ribosomal protein S6 kinase, polypeptide 1: RPS6KB1 (Ribosomal protein S6 kinase, polypeptide 1: Rps6kb1) Muscle, Osteocron: OSTN (Osteocrin: Ostn)

[0069] The above myokines (proteins) are expressed as described above (as claimed) in the present specification, including the examples.

[0070] <Myokine production promoter> The present invention also relates to a myokine production promoter which is produced using the above-mentioned "method for producing a myokine production promoter".

[0071] The myokine production promoter produced by the manufacturing method of the present invention contains a very large number of active ingredients, and it is impossible or extremely impractical (there are ``impossible or impractical circumstances'') to identify these ingredients or to identify from these ingredients the active ingredient that exhibits the effects of the present invention. Therefore, there is no way to specify the "liquid 42 obtained by low-temperature vacuum extraction from Japanese cypress" in the present invention other than by the production method (solid-liquid separation method).

[0072] <<Anti-inflammatory agent for skeletal muscles>> The present invention also relates to an anti-inflammatory agent for skeletal muscle, which comprises the above-mentioned myokine production promoter.

[0073] The anti-inflammatory effect of Interleukin-6 (IL-6) in skeletal muscle is shown in, for example, the Examples.<Interleukin-6(IL-6)> This is already known from the literature etc. described in the section.

[0074] Therefore, it is clear that the "liquid 42 obtained by low-temperature vacuum extraction from cypress" in the present invention is an anti-inflammatory agent for skeletal muscles, as the liquid 42 produced Interleukin-6 (IL-6) in this embodiment (see Examples).

[0075] <<Skeletal muscle formation promoter>> The present invention also relates to a skeletal muscle formation promoter comprising the above-mentioned myokine production promoter.

[0076] The function of Interleukin-6 (IL-6) in promoting skeletal muscle formation is shown in, for example, the Example.<Interleukin-6(IL-6)> This is already known from the literature etc. described in the section. In addition, the function of Rps6kb1 (RPS6KB1) in promoting skeletal muscle formation is shown in, for example,<Rps6kb1(RPS6KB1)> This is already known from the literature etc. described in the section. In addition, the function of Musclin (OSTN) in promoting skeletal muscle formation is shown in, for example, the Example.<Musclin(OSTN)> This is already known from the literature etc. described in the section.

[0077] Therefore, it is clear from the fact that "liquid 42 obtained by low-temperature vacuum extraction from Japanese cypress" in the present invention is a promoter of skeletal muscle formation in this embodiment, since said liquid 42 produced Interleukin-6 (IL-6), Rps6kb1 (RPS6KB1), and Musclin (OSTN) (see Examples).

[0078] <<Skeletal muscle damage repair agent>> The present invention also relates to an agent for repairing damaged skeletal muscle, which comprises the above-mentioned myokine production promoter.

[0079] The function of Interleukin-6 (IL-6) in repairing skeletal muscle damage is described in, for example, the Examples.<Interleukin-6(IL-6)> This is already known from the literature etc. described in the section. In addition, the function of recombinant Murine IP-10 (CXCL10) in repairing skeletal muscle damage is shown in, for example, the Example.<Recombinant Murine IP-10(CXCL10)> This is already known from the literature etc. described in the section.

[0080] Therefore, it is clear from the fact that "liquid 42 obtained by low-temperature vacuum extraction from Japanese cypress" in the present invention can be said to be an agent for repairing damaged skeletal muscles in this embodiment, since said liquid 42 produced Interleukin-6 (IL-6) and Recombinant Murine IP-10 (CXCL10) (see Examples).

[0081] <<Skeletal muscle enlargement agent>> The present invention also relates to a skeletal muscle hypertrophy agent comprising the above-mentioned myokine production promoter.

[0082] Rps6kb1 (RPS6KB1) has a skeletal muscle hypertrophy effect, as described in, for example, the Examples.<Rps6kb1(RPS6KB1)> This is already known from the literature etc. described in the section.

[0083] Therefore, it is clear from the fact that in this embodiment, the liquid 42 produced Rps6kb1 (RPS6KB1) that the "liquid 42 obtained by low-temperature vacuum extraction from cypress" in the present invention can be said to be a skeletal muscle hypertrophy agent (see Examples).

[0084] <<Post-exercise immune suppressant>> The present invention also relates to an agent for suppressing post-exercise immunosuppression, characterized by containing the above-mentioned myokine production promoter.

[0085] Recombinant Murine IP-2 (CXCL2) has an effect of suppressing immune decline after exercise, as described in, for example, the Examples.<Recombinant Murine IP-2 (CXCL2)> This is already known from the literature etc. described in the section. In addition, Recombinant Murine IP-10 (CXCL10) has an effect of suppressing post-exercise immune decline, as shown in, for example, the Examples.<Recombinant Murine IP-10(CXCL10)> This is already known from the literature etc. described in the section.

[0086] Therefore, it is clear that the "liquid 42 obtained by low-temperature vacuum extraction from Japanese cypress" in the present invention can be said to be an agent for suppressing post-exercise immune decline, as shown in this embodiment in which the liquid 42 produced Recombinant Murine IP-2 (CXCL2) and Recombinant Murine IP-10 (CXCL10) (see Examples).

[0087] <Specific uses (products) containing the myokine production promoter of the present invention> There are no particular limitations on the type of product (form) that may be made containing the myokine production promoter of the present invention, but it is preferable for the product to be applied to the skin above skeletal muscle (muscle). The skeletal muscle (muscle) may be from an animal, but is preferably from a human.

[0088] Since training athletes and the like to maintain their health and build muscle strength has been attracting attention, the human subject is particularly preferably an athlete, a sportsperson, a manual laborer, etc. Also, since it is important to prevent muscle loss in elderly people, elderly people are also included as the human subject.

[0089] The present invention also relates to a muscle spray liquid; a cream, aqueous liquid, emulsion, or gel for muscle strengthening; a cream or gel for muscle massage; and a cream, aqueous liquid, emulsion, or gel for muscle strengthening, each of which contains the myokine production promoter. Such products (forms) are prepared in a conventional manner, if necessary, by blending, for example, the known blending ingredients described below.

[0090] The above-mentioned form is preferable as a form that can be suitably acted on skeletal muscles (muscles). The timing of application (administration) to skeletal muscles (muscles) may be before, during, or after exercise. It is preferable to apply (administer) a cream or gel for muscle massage to the skin above the muscles after exercise, which also serves as a massage.

[0091] <<Composition>> The myokine production promoter of the present invention, the agent having the above-mentioned functions, and the specific products (embodiments) described above can be appropriately supplemented with additives to adjust the form, physical properties, usability, etc. Such compounded products include water added externally other than the "main ingredient, cypress intracellular fluid"; compounding agents for creams and external preparations; fragrances; and the like. Specific examples include fragrances such as essential oils; aroma oils; cooling agents such as menthol, peppermint, and terpenes; stabilizers; humectants; emulsifiers; binders; isotonicity agents; thickening agents such as petrolatum; gelling agents; organic and inorganic fillers; preservatives; other medicinal ingredients; and the like.

[0092] The myokine production promoter of the present invention has a completely different smell from "so-called cypress essential oil for aromatherapy," even if the object 41 is the same cypress. Also, the strength of the smell is weaker than that of the essential oil. However, while an extremely small amount of vapor (gas) in the air is sufficient for aromatherapy (essential oils), the above-mentioned applications of the present invention use a large amount of the liquid 42 of the present invention, that is, the myokine production promoter. Therefore, the odor may become strong during use. Therefore, among the above-mentioned compounding ingredients, it is preferable to compound the fragrance or cooling agent as an "odor adjusting agent." EXAMPLES

[0093] The present invention will be described in more detail below with reference to examples and comparative examples. However, the present invention is not limited to these examples as long as it does not depart from the gist of the present invention. In the examples, the term "%" simply means "% by mass" unless otherwise specified.

[0094] Example 1 <Solid-liquid separation from cypress using low-temperature vacuum extraction method> As the target object (raw material) 41, “cypress”, 30 kg of branches and leaves from thinned, undried cypress trees felled in Oita Prefecture were used. The felled trees were cut into pieces so that the longest diameter of each piece was 3 cm to 30 cm, and the pieces were placed in a container 21 shown in FIGS.

[0095] 30 kg of cypress was subjected to solid-liquid separation using the apparatus shown in Figure 1 and Figure 2. The conditions for solid-liquid separation were as follows. (1) Temperature of object: 30~40℃ (2) Temperature setting inside the container: 35-40℃ (3) Pressure: 4~8kPa (4) Rotation speed of the stirring blade: 4 rpm (revolutions per minute)

[0096] Most of the liquid 42 accumulated in the recovery tank 34 was a homogeneous aqueous phase (aqueous layer). The entire liquid (total recovered liquid) was used as the target aqueous liquid 42. Even if an oily component was present, it was dissolved or finely dispersed in the aqueous phase (aqueous layer). The yield of the obtained aqueous liquid 42 was 20 kg. Finally, the solid residue 44 was collected from the discharge outlet 26. The amount of the solid residue 44 was 10 kg. The solid residue 44 was also effectively utilized.

[0097] The resulting aqueous liquid 42 is hereinafter abbreviated as "Cypress CE."

[0098] Example 2 <Solid-liquid separation from cedar using low-temperature vacuum extraction method> Object 41, "cedar," was made from 30 kg of branches and leaves from thinned cypress trees felled in Oita Prefecture. The felled trees were cut into pieces so that the longest diameter in each piece was 3 cm to 30 cm, and the pieces were placed in a container 21 shown in FIGS.

[0099] 30 kg of cedar was subjected to solid-liquid separation using the apparatus shown in Figures 1 and 2. The conditions for solid-liquid separation were the same as those in Example 1 (in the case of cypress) above.

[0100] Most of the liquid collected in the recovery tank 34 was a homogeneous aqueous phase (aqueous layer). The entire liquid (total recovered liquid) was used as the target aqueous liquid. Even if an oily component was present, it was dissolved or finely dispersed in the aqueous phase (aqueous layer). The yield of the obtained aqueous liquid was 20 kg. Finally, the solid residue 44 was recovered from the discharge outlet 26. The amount of the solid residue 44 was 10 kg. The solid residue 44 was also effectively utilized.

[0101] The resulting aqueous liquid is hereinafter abbreviated as "Sugi CE."

[0102] Comparative Example 1 <Extracted from Hinoki cypress using steam distillation method> The raw materials used were the same as those in Preparation Example 1. Using a conventional steam distillation apparatus, extraction was carried out using steam at 100° C. in a conventional manner, and then the water used as the extraction medium was separated. The product obtained is what is known as "essential oil," and the intracellular fluid of the cypress tree was never obtained in a pure form by itself.

[0103] Comparative Example 2 <Extracted from Hinoki using solvent extraction method> The raw materials used were the same as those in Preparation Example 1. The raw material was extracted in the usual manner using a water / ethanol mixed solvent at 40°C in an amount three times the amount of the raw material as the extraction medium.

[0104] From the obtained extract, the extraction medium was distilled off under reduced pressure at 40°C or lower to obtain an extract. During the vacuum distillation, the intracellular fluid of cypress dissipated (evaporated). The obtained extract did not contain the active ingredient in the present invention and contained extraction solvents such as water and ethanol added from the outside.

[0105] Evaluation Example 1 <Evaluation on Promotion of Myokine Production> After adding cypress CE or sugi CE to C2C12 cells at varying concentrations, electric pulse stimulation (hereinafter abbreviated as "EPS") was applied under the following conditions. Hereinafter, the treatment by electric pulse stimulation is abbreviated as "EPS treatment". "C2C12 cells" are mouse skeletal muscle cells that can create a state of in vitro exercise by applying EPS. For example, they can be prepared, adjusted, and used with reference to the descriptions in Hatakeyama H, Kanzaki M. Mol Biol Cell, 24(6), 809-817, (2013), etc.

[0106] <<C2C12 Cells>> C2C12 cells were seeded in a 4-well plate multi-dish (Nunclon Delta treated, 4-well square / flattened bottom) at 1.25×10 5 cells / well, and Dulbecco's modified Eagle's medium (DMEM, 4.5% / D-glucose) supplemented with 10% fetal bovine serum (FBS) and 30 μg / mL penicillin-streptomycin was used to grow the cells at 37°C and 5% CO 2 conditions.

[0107] Three days later, when the cells reached 80-90% confluence, Dulbecco's modified Eagle's medium (DMEM, 1.0% / D-glucose) supplemented with 2.0% horse serum (HS) and 30 μg / mL penicillin-streptomycin was used at 37°C and 5% CO 2Under the conditions, differentiation was carried out for 7 - 9 days.

[0108] <<Electrical Pulse Stimulation (EPS) Treatment>> First, after the differentiation of C2C12 cells, the medium was removed, and 5 mL of serum - free Eagle's Minimum Essential Medium (MEM) at 37 °C was added. After one and a half hours, the medium was removed, and again 5 mL of fresh serum - free Eagle's Minimum Essential Medium (MEM) at 37 °C was added.

[0109] Next, with sterile water, "Chamaecyparis obtusa CE", which is the liquid 42 obtained by solid - liquid separation of Chamaecyparis obtusa in Preparation Example 1 and adjusted to 500 mM, and "Cryptomeria japonica CE", which is the liquid obtained by solid - liquid separation of Cryptomeria japonica in Example 2, were added to the above - mentioned medium to a concentration of 1 mM and 2 mM. As a control, PBS was added, and it was left standing in an incubator.

[0110] Next, at 37 °C, 5% CO 2 Under the conditions, after 30 minutes, the C - Pace EM (IonOptix Corporation) C - Dish carbon electrode was placed in a 4 - well plate and left standing in an incubator (37 °C, 5% CO 2 ). EPS treatment was carried out for 120 minutes under the conditions of an applied voltage of 40 V, an applied frequency of 1 Hz, and a single - application time of 2 ms.

[0111] <<RT - PCR Analysis>> After EPS treatment, immediately the medium was removed, and the wells were washed twice with PBS at 37 °C. Total RNA was extracted using the RNeasy Micro Kit (Qiagen). Using the extracted RNA as a template, cDNA was synthesized using the Affinity Script QPCR cDNA synthesis kit (Agilent).

[0112] Next, using the synthesized cDNA as a template, PCR was carried out using Emerald Amp PCR Master (TAKARA). All PCR reactions consisted of initial denaturation at 95°C for 5 minutes, followed by 43-45 cycles of thermal denaturation at 95°C for 45 seconds, annealing at 50-55°C for 45 seconds, and extension reaction at 72°C for 1 minute, with the extension reaction being completed at 72°C for 10 minutes.

[0113] For PCR, specific gene primers (64 genes) were designed and used in RT-PCR reactions. The details of the specific primer designs used in this study are shown in Tables 1 and 2.

[0114] [Table 1]

[0115] [Table 2]

[0116] Evaluation example 2 <Confirmation of myokine production from solid-liquid separated liquids obtained in Examples 1 and 2> <<Identification of myokine secretion promoting genes by combined use of Hinoki CE or Cedar CE and exercise>> Cypress CE, cedar CE, and PBS (control) were added to C2C12 cells, which are motile cells that secrete myokines, and gene expression of various myokines was examined using the RT-PCR method described above. In addition, after EPS treatment for 0, 60, and 120 minutes, RNA was extracted and subjected to RT-PCR analysis.

[0117] In this study, the samples were added at concentrations of 1 mM and 2 mM. For many cytokines, increased gene expression was observed at a concentration of 2 mM compared to 1 mM, so data at a concentration of 2 mM is presented in this study. The results are shown in Figures 4 to 8. In Figures 4 to 8, significant differences are marked with "*" or "**" and are shown by encircling the bars in the bar graphs with ellipses.

[0118] <<Interleukin-6 (IL-6)>> When Hinoki CE or Cedar CE was added to C2C12 cells, an increase in IL-6 gene expression was confirmed compared to PBS, as shown in Figure 4. Immediately after the addition of the liquid (0 minutes), high IL-6 gene expression was confirmed with 10% Cedar CE, and significant gene expression was confirmed with 10% Hinoki CE (Figure 4). Additionally, 60 minutes after addition of either liquid, no effect was observed on the level of IL-6 gene expression, but 120 minutes after addition, a significant (5%) increase in the level of IL-6 gene expression was observed with 5% and 10% Hinoki CE (Figure 4).

[0119] IL-6 is known as a cytokine with various functions, but since IL-6 levels increase in the blood after exercise, it was previously thought that "intense exercise causes inflammation of skeletal muscles, which is secreted by immune cells, resulting in an increase in IL-6 levels in the blood."

[0120] However, recent studies have shown that exercise does not increase IL-6 mRNA expression, suggesting that the increase in blood IL-6 levels is due to secretion from skeletal muscle cells (Ullum H, Haahr PM, Diamant M, Palmo J, Halkjaer-Kristensen J, Pedersen BK. Bicycle exercise enhances plasma IL-6 but does not change IL-1 alpha, IL-1 beta, IL-6, or TNF-alpha pre-mRNA in BMNC. J Appl Physiol, 77(1):93-7,1985.).

[0121] Furthermore, recent advances in culture techniques have revealed that IL-6 is a myokine secreted from skeletal muscle cells in response to contraction (Nedachi T, Fujita H, Kanzaki M. Contractile C2C12 myotube model for studying exercise-inducible responses in skeletal muscle. Am J Physiol Endocrinol Metab,5(5):E1191-204, 2008.).

[0122] The physiological effects of IL-6 include the promotion of glucose uptake in skeletal muscles, insulin secretion promotion, whole-body glucose metabolism, promotion of muscle formation, and anti-inflammatory effects (Munoz-Canoves P, Scheele C, Pedersen BK, Serrano AL. Interleukin-6 myokinesignaling in skeletal muscle: a double-edged sword FEBS J, 280(17):4131-48,2013.), maintenance of the function of muscle satellite cells, and even the breakdown of adipose tissue.

[0123] The above results (FIG. 4) suggest that cypress CE and cedar CE promote the secretion of IL-6, and therefore have all of the physiological actions of IL-6. In other words, because Hinoki CE promotes the secretion of IL-6, it was suggested that it is (has) a function as an anti-inflammatory agent for skeletal muscle, an agent that promotes skeletal muscle formation, and an agent that repairs skeletal muscle damage.

[0124] <<Recombinant Murine IP-2(CXCL2)> > CXCL2 is a proinflammatory cytokine and a chemotactic cytokine that attracts leukocytes to the site of injury. It is also a potent neutrophil chemoattractant and is known to be involved in many immune responses, such as wound healing, cancer metastasis, and angiogenesis (Al-Alwan LA, Chang Y, Mogas A, Halayko AJ, Baglole CJ, Martin JG, Rousseau S, Eidelman DH, Hamid Q. Differential roles of CXCL2 and CXCL3 and their receptors in regulating normal and asthmatic airway smooth muscle cell migration. J Immunol, 191(5):2731-2741, 2013.).

[0125] As shown in FIG. 5, the addition of Hinoki CE to C2C12 cells resulted in a significant increase in CXCL2 after 120 minutes, as compared to PBS.

[0126] On the other hand, although an increasing tendency was observed for cedar CE at 60 and 120 minutes, the increase was not concentration-dependent and there was variation in the standard deviation. Considering the above results, it is certain that the addition of Hinoki CE stimulates CXCL2, suggesting that using Hinoki CE after exercise may have some positive effect on the immune response.

[0127] Since Hinoki CE promotes the secretion of CXCL2, it was suggested that it has all of the physiological actions mentioned above that CXCL2 has. In other words, it was suggested that Hinoki CE promotes the secretion of CXCL2 and thus functions as an inhibitor of post-exercise immune decline.

[0128] It is known that moderate exercise improves immune function, while intense exercise lowers immunity. This suggests that treatments using massage creams containing Hinoki cypress CE (ingredients) can help suppress and prevent the decline in immunity after exercise, leading to improved immunity.

[0129] <<Recombinant Murine IP-10(CXCL10)> > Like CXCL2, CXCL10 is a cytokine that exhibits immune, inflammatory, hematopoietic, and angiogenic activities, as well as anticancer activities (Sgadari, C., AL Angiolillo, BW Cherney, SE Pike, JM Farber, LG Koniaris, P. Vanguri, PR Burd, N. Sheikh, G. Gupta, J. Teruya-Feldstein, and G. Tosato. Interferon-inducible protein-10 identified as a mediator of tumor necrosis in vivo. Proc Natl Acad Sci USA 93: 13791-13796, 1996.).

[0130] It has also been suggested that CXCL10 is a myokine that controls exercise-dependent immune function. Recent research has shown that while myokines secreted from normal skeletal muscles promote secretion in response to muscle contraction during exercise, CXCL10 is said to be a myokine that inhibits secretion, and it has been reported that it exhibits an effect similar to that of myostatin, the expression of which is reduced by exercise.

[0131] By binding to its receptor CXCR3, CXCL10 increases the proliferation and differentiation of satellite cells, skeletal muscle stem cells that play a central role in muscle regeneration. Furthermore, it has been reported that expression of CXCL10 in damaged muscles is involved in muscle regeneration, such as by increasing satellite cell proliferation and increasing muscle growth (Tidball JG, Flores I, Welc SS, Wehling-Henricks M, Ochi. Aging of the immune system and impaired muscle regeneration: A failure of immunomodulation of adult myogenesis. Exp Gerontol, 145:111200, 2021.).

[0132] As can be seen from Figure 6, the increase in CXCL10 expression by the addition of Hinoki CE was an experiment at the cellular level, but when applied to humans, it is suggested that Hinoki CE and Cedar CE not only improve immunity, but also repair the fine structure of muscles damaged by exercise.

[0133] Hinoki CE promotes the secretion of CXCL10, suggesting that it has all of the physiological actions described above that CXCL10 has. In other words, because Hinoki CE promotes the secretion of CXCL10, it was suggested that it is (has) a function as a repair agent for damaged skeletal muscle and an inhibitor of post-exercise immune depression.

[0134] <<Rps6kb1(RPS6KB1)> > There are several pathways mediated by RPS6KB1 in the PI3K-Akt signaling pathway, an intracellular signaling pathway involved in biological processes, and it has been suggested that RPS6KB1 is involved in cell proliferation, angiogenesis, glucose metabolism, etc. (Pende M, Kozma SC, Jaquet M, Oorschot V, Burcelin R, Le Marchand-Brustel Y, Klumperman J, Thorens B, Thomas G. Hypoinsulinaemia, glucose intolerance and diminished beta-cell size in S6K1-deficient mice. Nature, 408(6815):994-997, 2000.).

[0135] In addition, while RPS6KB1 has been reported to be involved in muscle hypertrophy, there are also reports that it is involved in increasing muscle strength rather than muscle size (Marabita M, Baraldo M, Solagna F, Ceelen JJM, Sartori R, Nolte H, Nemazanyy I, Pyronnet S, Kruger M, Pende M, Blaauw B. S6K1 Is Required for Increasing Skeletal Muscle Force during Hypertrophy. Cell Rep. 4;17(2):501-513. 2016).

[0136] Based on these reports, considering the effects of exercise and Hinoki CE and Cedar CE, it can be seen that efficient training and the use of Hinoki CE and Cedar CE can provide particularly efficient training effects for muscle hypertrophy.

[0137] As can be seen from FIG. 7, Hinoki CE promotes the secretion of RPS6KB1, suggesting that it has all of the above-mentioned physiological actions of RPS6KB1. In other words, it was suggested that Hinoki CE promotes the secretion of RPS6KB1 and thus functions as a promoter of skeletal muscle formation and hypertrophy of skeletal muscle.

[0138] <<Musclin(OSTN)> > OSTN is expressed in skeletal muscle and its physiological role has been reported to be the regulation of glucose metabolism (Nishizawa H, Matsuda M, Yamada Y, Kawai K, Suzuki E, Makishima M, Kitamura T, Shimomura I. Musclin, a novel skeletal muscle-derived secretory factor. J Biol Chem. 7;279 (19):19391-19395. 2004.), suggesting its possible role in insulin resistance. OSTN is a relatively new myokine that has come to be known as a secretory factor derived from skeletal muscle.

[0139] In recent years, it has also been reported that Musclin (OSTN) promotes mitochondrial biogenesis and contributes to improved endurance (ibid.). As can be seen from FIG. 8, Hinoki CE promotes the secretion of Musclin (OSTN), suggesting that it has all of the physiological actions described above and below that Musclin (OSTN) has. In other words, it was suggested that Hinoki CE promotes the secretion of Musclin (OSTN), and thus is (has) a function as a promoter of skeletal muscle formation.

[0140] In particular, the physiological effect of Hinoki CE reaches its maximum value 60 minutes after administration (Figure 8), so if Hinoki CE is administered approximately one hour after exercise, it can improve insulin resistance, and is therefore thought to be effective even in people with lifestyle-related diseases.

[0141] Evaluation example 3 <Evaluation of the liquids obtained by extraction in Comparative Example 1 and Comparative Example 2> The essential oil from Hinoki obtained in Comparative Example 1 is an essential oil, and is neither an intracellular liquid nor an aqueous liquid. In addition, no promotion of myokine production was observed in C2C12 cells.

[0142] The liquid from cypress obtained in Comparative Example 2 was not an intracellular liquid of cypress, nor was it an aqueous liquid obtained by solid-liquid separation. In addition, no promotion of myokine production was observed in C2C12 cells.

[0143] Evaluation example 4 <Summary of Hinoki CE and Sugi CE evaluated in Evaluation Example 1 and Evaluation Example 2> Addition of Hinoki CE to C2C12 cells resulted in increased gene expression of Interleukin-6 (IL-6), Recombinant Murine IP-2 (CXCL2), Recombinant Murine IP-10 (CXCL10), Rps6kb1 (RPS6KB1), and / or Musclin (OSTN) (Figures 4-8).

[0144] Since it has been made clear from papers and other sources that each of the above-mentioned myokines has the above-mentioned effects, Hinoki CE produces all of the above-mentioned myokines in C2C12 cells, and it is clear that the function of all of these myokines makes Hinoki CE (function as) an anti-inflammatory agent, a formation promoter, a damage repair agent, a hypertrophy agent, and an agent to suppress post-exercise immune decline in skeletal muscle.

[0145] In addition, since cedar CE also promotes the production of some of the myokines mentioned above (Figures 4-8), it was found that cedar CE also has some of the functions mentioned above. Therefore, it was inferred that the inclusion of at least cypress CE, and the use of cedar CE in combination, would result in an even more excellent myokine production promoter.

[0146] These results show that applying creams, oils, aqueous liquids, emulsions, gels, etc. containing Hinoki CE during exercise, and especially after exercise, can reduce skeletal muscle inflammation, promote skeletal muscle formation, repair damaged skeletal muscle, enlarge skeletal muscle, and suppress post-exercise immune decline. It was also predicted that creams or gels for muscle massage may have some other health benefits, such as promoting blood flow, in addition to the above-mentioned effects. [Industrial Applicability]

[0147] The cypress solid-liquid separated liquid contained in the myokine production promoter of the present invention contains the intracellular fluid of cypress in its original state, and has a different component composition from conventional cypress extracts. In addition, the solid-liquid separation is performed at low temperature under reduced pressure, and since no external extraction medium is mixed in, there is no thermal decomposition (components), and all of the components are derived from cypress (natural products), making it extremely safe.

[0148] Therefore, the myokine production promoter of the present invention is useful for both healthy individuals and those with diseases, and is particularly effective for athletes, and will be widely used in fields such as physical education, training (exercise), nursing care, medicine, chiropractic and massage. It is also widely used in the manufacturing and use of medical products, quasi-drugs, massage products, cosmetics, bath additives, toiletries, etc. It is also widely used in the forestry industry, where cypress and cedar trees are cut down, in the waste treatment industry, and in the effective use of waste. [Explanation of symbols]

[0149] 21 Container 22 Cooler 23 Mixing blade 24 Heating device 25 Exhaust port 26 Outlet 27 Piping 32 Vacuum gauge 33 Thermometer 34 Collection tank 35 Pressure Reducing Device 41 Object (Raw Material) 42 (aqueous) liquid 44 (solid) residue

Claims

1. A method for producing a myokine production promoter, comprising the steps of: maintaining a temperature of a solid-liquid separation object placed in a container at 45°C or lower; reducing the pressure inside the container to separate the object into solid and liquid without using an extraction liquid or extraction steam as an extraction medium; and recovering the liquid contained in the object as an active ingredient, A method for producing a myokine production promoter, characterized in that the object is a cypress product that is not completely dried and is still undried.

2. The method for producing a myokine production promoter according to claim 1, wherein the myokine is Interleukin 6 (IL6), Recombinant Murine IP-2 (CXCL2), Recombinant Murine IP-10 (CXCL10), Rps6kb1 (RPS6KB1), or Musclin (OSTN).

3. 2. A method for producing a myokine production promoter according to claim 1, wherein the target object, the cypress, is cypress thinning material, cypress branches and leaves, cypress driftwood, or cypress bark.

4. 2. The method for producing a myokine production promoter according to claim 1, wherein the cypress is cut and then placed in the container for solid-liquid separation.

5. 2. The method for producing a myokine production promoter according to claim 1, wherein the pressure inside the vessel is maintained at 20 kPa or less at least during main solid-liquid separation.

6. 2. A method for producing a myokine production promoter as described in claim 1, wherein the target object further comprises undried cedar that is not completely dried.

7. A myokine production promoter, which is produced by using the method for producing a myokine production promoter according to any one of claims 1 to 6.

8. An anti-inflammatory agent for skeletal muscle, comprising the myokine production promoter according to claim 7.

9. A skeletal muscle formation promoter comprising the myokine production promoter according to claim 7.

10. A skeletal muscle damage repair agent comprising the myokine production promoter according to claim 7.

11. A skeletal muscle hypertrophy agent comprising the myokine production promoter according to claim 7.

12. A post-exercise immune-depression suppressant comprising the myokine production promoter according to claim 7.

13. A spray liquid for muscle application, comprising the myokine production promoter according to claim 7.

14. A muscle-building cream, aqueous liquid, emulsion, or gel, comprising the myokine production promoter according to claim 7.

15. A cream or gel for muscle massage, comprising the myokine production promoter according to claim 7.

16. A muscle-building cream, aqueous liquid, emulsion, or gel, comprising the skeletal muscle formation promoter according to claim 9.

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