Myokine receptor expression promoter and cell growth promoter

Abstract

Provided is a new myokine receptor expression promoter that promotes the expression of a myokine receptor.　This myokine receptor expression promoter contains, as an active ingredient, at least one selected from the group consisting of Panax ginseng extract, Angelica root extract, and royal jelly extract.

Description

Myokine receptor expression promoter and cell proliferation promoter

[0001] The present invention relates to a myokine receptor expression promoter and a cell proliferation promoter.

[0002] Myokines are a general term for physiologically active substances secreted from skeletal muscles, and research into myokines has been active in recent years.

[0003] For example, Patent Document 1 reports a method for assessing skin conditions using blood myokine concentrations as indicators.

[0004] Furthermore, Patent Document 2 discloses a pharmaceutical composition containing growth differentiation factor 11 (hereinafter abbreviated as "GDF11"), a type of myokine. Patent Document 2 discloses that GDF11 can promote the proliferation of fibroblasts and is effective in improving skin wrinkles, regenerating skin, and treating wounds. Furthermore, GDF11 is a member of the TGF-β superfamily, and is known to be expressed in many tissues, including the skin, heart, skeletal muscle, and the developing nervous system. On the other hand, it is also known that the expression level of GDF11 decreases with age.

[0005] Furthermore, Non-Patent Document 1 reports a study on the anti-aging effect of GDF11 on the skin.

[0006] JP 2020-046200 A U.S. Patent Application Publication No. 2019-0111108

[0007] Luc Rochette et al., "Anti-Aging Effects of GDF11 on Skin", Int. J. Mol. Sci. ,2020,21,2598

[0008] Although many studies have been conducted to date that have focused on myokines or on GDF11, a type of myokine, no studies have focused on myokine receptors, or more specifically, on the GDF11 receptor, for example.

[0009] The present invention is intended to improve the above situation, and its object is to provide a novel myokine receptor expression promoter that promotes the expression of myokine receptors.

[0010] Another object of the present invention is to provide a cell proliferation promoter containing a novel myokine receptor expression promoter.

[0011] The present invention that achieves the above object is as follows.

[0012] Aspect 1: A myokine receptor expression promoter comprising as an active ingredient at least one selected from the group consisting of Panax ginseng extract, Angelica acutiloba extract, and royal jelly extract. Aspect 2: The myokine receptor expression promoter of Aspect 1, wherein the myokine is GDF11. Aspect 3: The myokine receptor expression promoter of Aspect 1 or 2, wherein the myokine receptor comprises at least one of ALK5 and ACVR2B. Aspect 4: The myokine receptor expression promoter of any one of Aspects 1 to 3, wherein the myokine receptor is in fibroblasts or keratinocytes. Aspect 5: The myokine receptor expression promoter of any one of Aspects 1 to 4, which is capable of promoting cell proliferation via expression of the myokine receptor. Aspect 6: A cell proliferation promoter comprising the myokine receptor expression promoter of any one of Aspects 1 to 5 and a myokine.

[0013] According to the present invention, a novel myokine receptor expression promoter that promotes the expression of a myokine receptor can be provided.

[0014] Furthermore, the present invention can provide a cell proliferation promoter containing a novel myokine receptor expression promoter.

[0015] Figure 1 is a schematic diagram illustrating the effect of the myokine receptor expression promoter of the present invention. Figure 2 is a diagram showing the myokine receptor expression promoting effect of Example 1. Figure 3 is a diagram showing the myokine receptor expression promoting effect of Example 2. Figure 4 is a diagram showing the myokine receptor expression promoting effect of Example 3. Figure 5 is a diagram showing the cell proliferation promoting effect of Example 4.

[0016] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described in detail below with reference to the preferred embodiments. However, the present invention is not limited to the following embodiments, and various modifications can be made within the scope of the present invention.

[0017] <<Myokine Receptor Expression Promoter>> The myokine receptor expression promoter of the present invention contains at least one selected from the group consisting of Panax ginseng extract, Angelica acutiloba extract, and royal jelly extract as an active ingredient.

[0018] The present inventors, unlike conventional approaches, have focused on myokine receptors and succeeded in developing a myokine receptor expression promoter capable of promoting myokine receptor expression. Specifically, through intensive research, the present inventors have discovered that the myokine receptor expression promoter of the present invention can promote myokine receptor expression by containing as an active ingredient at least one selected from the group consisting of Panax ginseng extract, Angelica acutiloba extract, and royal jelly extract. It has also been found that the myokine receptor expression promoter of the present invention can promote cell proliferation when used in combination with a myokine. This cell proliferation is believed to be mediated by myokine receptor expression. Thus, the myokine receptor expression promoter of the present invention can promote myokine receptor expression and further promote cell proliferation, thereby maximizing the effects of myokines on cells.

[0019] "Enhanced myokine receptor expression" refers to, for example, a statistically significant increase (e.g., Student's t-test) in the expression level of a myokine receptor gene when administered with a myokine receptor expression promoter, compared to a control when no myokine receptor expression promoter is administered. Alternatively, "enhanced myokine receptor expression" refers to an increase in the expression level of a myokine receptor gene by, for example, 5% or more, 10% or more, 15% or more, 18% or more, 20% or more, 22% or more, or 24% or more when administered with a myokine receptor expression promoter, compared to a control when no myokine receptor expression promoter is administered. The expression level of a myokine receptor gene can be determined by any method known in the art.

[0020] More specifically, for example, the myokine receptor promoter of the present invention promotes the expression of the receptor for GDF11, a type of myokine, and also promotes the proliferation of skin cells, etc., and as a result, it is thought that the effects of GDF11 on the skin, such as cell proliferation and wrinkle improvement, can be maximized.

[0021] The effects of the myokine receptor expression promoter of the present invention will be described below by way of example with reference to Figure 1. However, the present invention is not limited to Figure 1.

[0022] Figure 1 is a schematic diagram illustrating the effect of the myokine receptor expression promoter of the present invention. As shown in Figures 1(a) and 1(b), administration of myokine receptor expression promoter 100 of the present invention promotes myokine receptor expression in cell 2, transforming the initial myokine receptor 10r into myokine receptors 10r and 11r, allowing it to receive more myokine 1. Furthermore, as shown in Figure 1(c), through the expression of myokine receptors, cell 2 proliferates to become cells 2 and 3, allowing it to receive even more myokine 1. Although not shown, the myokines may include those produced by muscle stimulation or those administered externally.

[0023] <Panax ginseng extract> Panax ginseng extract, also known as ginseng extract, is commercially available. In the myokine receptor expression promoter of the present invention, the Panax ginseng extract may be extracted with an extraction solvent such as water, alcohol, or a mixture thereof. The alcohol used as the extraction solvent is not particularly limited, and may be, for example, a lower alcohol such as ethanol, or a polyhydric alcohol such as 1,3-butylene glycol.

[0024] The myokine receptor expression promoter of the present invention may contain ginseng extract at, for example, 0.001% (v / v) or more, 0.005% (v / v) or more, 0.01% (v / v) or more, 0.05% (v / v) or more, 0.1% (v / v) or more, 0.2% (v / v) or more, 0.3% (v / v) or more, 0.4% (v / v) or more, or 0.5% (v / v) or more. , and may contain 1.0% (v / v) or less, 0.9% (v / v) or less, 0.8% (v / v) or less, 0.7% (v / v) or less, 0.6% (v / v) or less, 0.5% (v / v) or less, 0.4% (v / v) or less, 0.3% (v / v) or less, 0.2% (v / v) or less, 0.1% (v / v) or less, 0.05% (v / v) or less, or 0.01% (v / v) or less.

[0025] By including ginseng extract in the myokine receptor expression promoter of the present invention, myokine receptor expression can be promoted, and the expression-promoting effect is particularly pronounced for the GDF11 receptor, and more specifically, the expression-promoting effect can be even more pronounced for the GDF11 receptor ALK5.

[0026] <Angelica Root Extract> Angelica Root extract, also known as an extract of Angelica Root (or "Toki"), is commercially available. In the myokine receptor expression promoter of the present invention, the Angelica Root extract may be extracted with an extraction solvent such as water, alcohol, or a mixture thereof. The alcohol used as the extraction solvent is not particularly limited, and may be, for example, a lower alcohol such as ethanol, or a polyhydric alcohol such as 1,3-butylene glycol.

[0027] The myokine receptor expression promoter of the present invention may contain Angelica acutiloba extract at a concentration of, for example, 0.001% (v / v) or more, 0.005% (v / v) or more, 0.01% (v / v) or more, 0.05% (v / v) or more, 0.1% (v / v) or more, 0.2% (v / v) or more, 0.3% (v / v) or more, 0.4% (v / v) or more, or 0.5% (v / v) or more. Alternatively, it may contain 1.0% (v / v) or less, 0.9% (v / v) or less, 0.8% (v / v) or less, 0.7% (v / v) or less, 0.6% (v / v) or less, 0.5% (v / v) or less, 0.4% (v / v) or less, 0.3% (v / v) or less, 0.2% (v / v) or less, 0.1% (v / v) or less, 0.05% (v / v) or less, or 0.01% (v / v) or less.

[0028] By including Angelica acutiloba extract in the myokine receptor expression promoter of the present invention, myokine receptor expression can be promoted, and the expression-promoting effect on the GDF11 receptor is particularly pronounced, and more specifically, the expression-promoting effect on the GDF11 receptor ACVR2B can be even more pronounced.

[0029] <Royal Jelly Extract> Royal jelly extract, also known as royal jelly extract, is commercially available. In the myokine receptor expression promoter of the present invention, the royal jelly extract may be extracted with an extraction solvent such as water, alcohol, or a mixture thereof. The alcohol used as the extraction solvent is not particularly limited and may be, for example, a lower alcohol such as ethanol, or a polyhydric alcohol such as 1,3-butylene glycol.

[0030] The myokine receptor expression promoter of the present invention may contain royal jelly extract at, for example, 0.001% (v / v) or more, 0.005% (v / v) or more, 0.01% (v / v) or more, 0.05% (v / v) or more, 0.1% (v / v) or more, 0.2% (v / v) or more, 0.3% (v / v) or more, 0.4% (v / v) or more, or 0.5% (v / v) or more. , and may contain 1.0% (v / v) or less, 0.9% (v / v) or less, 0.8% (v / v) or less, 0.7% (v / v) or less, 0.6% (v / v) or less, 0.5% (v / v) or less, 0.4% (v / v) or less, 0.3% (v / v) or less, 0.2% (v / v) or less, 0.1% (v / v) or less, 0.05% (v / v) or less, or 0.01% (v / v) or less.

[0031] By including royal jelly extract in the myokine receptor expression promoter of the present invention, myokine receptor expression can be promoted, and the expression-promoting effect is particularly pronounced for the GDF11 receptor, and more specifically, the expression-promoting effect can be even more pronounced for the GDF11 receptor, ALK5.

[0032] In the present invention, the myokine is not particularly limited and may be, for example, GDF11. When the myokine is GDF11, the myokine receptor expression promoter of the present invention promotes the expression of the GDF11 receptor, thereby having effects such as promoting cell proliferation. As a result, the promoter may also have effects such as improved skin firmness, transparency, and moisture, as well as anti-aging effects.

[0033] In the present invention, the myokine receptor is not particularly limited and may be appropriately selected depending on the type of myokine of interest. For example, when the myokine is GDF11, the myokine receptor may include at least one of ALK5 and ACVR2B. ALK5 is a TGF-β type I receptor and may be present in, for example, fibroblasts or keratinocytes. ACVR2B is an activin A type IIB receptor and may be present in, for example, fibroblasts.

[0034] In the present invention, the myokine receptor may be, for example, in a fibroblast or a keratinocyte.

[0035] <Other Components> In addition to the active ingredients described above, the myokine receptor expression promoter of the present invention may further contain other components as long as the effects of the present invention are not impaired.

[0036] Other ingredients that may be used include, but are not limited to, water, oil, moisturizers, surfactants, other plant extracts, antioxidants, preservatives, disinfectants, powder ingredients, thickeners, pigments, pH adjusters, etc.

[0037] <Cell proliferation promoter> The myokine receptor expression promoter of the present invention can promote cell proliferation through the expression of myokine receptors. Thus, the present invention can provide a cell proliferation promoter.

[0038] The cell proliferation promoter of the present invention comprises the myokine receptor expression promoter of the present invention described above and a myokine.

[0039] The myokine receptor expression promoter in the cell proliferation promoter of the present invention may contain at least one active ingredient selected from the group consisting of the above-mentioned Panax ginseng extract, Angelica acutiloba extract, and royal jelly extract.

[0040] Furthermore, the concentration of myokine in the cell proliferation promoter of the present invention is not particularly limited and may be, for example, 1.0 ng / ml or more, 5.0 ng / ml or more, 10 ng / ml or more, 15 ng / ml or more, 20 ng / ml or more, or 25 ng / ml or more, and may be 1000 ng / ml or less, 500 ng / ml or less, 300 ng / ml or less, 100 ng / ml or less, 50 ng / ml or less, 30 ng / ml or less, or 25 ng / ml or less.

[0041] The cell proliferation promoter of the present invention may further contain other ingredients in addition to the myokine receptor expression promoter of the present invention and myokine described above, as long as the effects of the present invention are not impaired.

[0042] Other ingredients that may be used include, but are not limited to, water, oil, moisturizers, surfactants, other plant extracts, antioxidants, preservatives, disinfectants, powder ingredients, thickeners, pigments, pH adjusters, etc.

[0043] In the present invention, cell proliferation can be evaluated by, for example, alamarBlue assay.

[0044] <<Uses>> The myokine receptor expression promoter and cell proliferation promoter of the present invention can be used independently as an external skin preparation, a pharmaceutical product, or a food product.

[0045] Examples of topical skin preparations that can be used include, but are not limited to, emulsions, creams, lotions, ointments, etc. Furthermore, examples of the dosage form of topical preparations include, but are not limited to, aqueous systems, solubilized systems, emulsions, powder systems, oil systems, gel systems, ointments, aerosol systems, two-layer systems such as water-oil systems, and three-layer systems such as water-oil-powder systems.

[0046] The pharmaceutical may be one that can be administered in the form of a conventional pharmaceutical preparation by mixing with a solid or liquid non-toxic pharmaceutical carrier suitable for administration methods such as oral administration, parenteral ingestion, rectal administration, injection, etc. In addition, examples of pharmaceutical dosage forms include, but are not limited to, solid preparations such as powders, dustings, granules, tablets, and capsules; liquid preparations such as solutions, suspensions, and emulsions; freeze-dried preparations; and topical preparations.

[0047] The food may be, for example, a beauty food, a nutritional supplement, a functional food, a health food, a food for specified health uses, etc. The form of the food may be, for example, solid, semi-solid, jelly-like, or liquid, but is not limited to these.

[0048] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0049] <Common Items> Unless otherwise specified, the examples were carried out based on the following common items: <Reagents> Commercially available royal jelly extract (Royal Jelly Extract B: Koei Kogyo Co., Ltd.), Panax Ginseng extract (Falcorex Ninjin B: Ichimaru Pharcos Co., Ltd.), and Angelica Root extract (Falcorex Angelica Root B: Ichimaru Pharcos Co., Ltd.) were used.

[0050] <Culture Medium> A serum-free medium was prepared by adding 5 ml of GlutaMAX (GlutaMAX (100X): Thermo Fisher Scientific), 10 ml of HEPES (HEPES (1M): Thermo Fisher Scientific), and 5 ml of an antibacterial agent (Antibiotic-Antimycotic (100X): Thermo Fisher Scientific) to a commercially available cell culture medium (Dulbecco's Modified Eagle's Medium (1.0 g / L glucose) (containing pyruvic acid, not containing L-glutamine or phenol red) (liquid): Nakarai).

[0051] FBS (charcoal treated FBS: Biowest) was inactivated by heating at 56°C for 30 minutes, and then mixed with the above serum-free medium to make FBS concentrations of 0.5% and 10%, respectively, to prepare 0.5% FBS-DMEM and 10% FBS-DMEM.

[0052] <Cell Culture (Examples 1 and 2)> Commercially available normal adult skin-derived fibroblasts (Human Dermal Fibroblasts: LIFELINE) were seeded on a 100 mm collagen-coated dish and cultured in 10% FBS-DMEM at 37°C and 5% CO. ２ The cells were cultured in PBS until they reached subconfluence. The cells were harvested by trypsinization and plated in a 24-well plate at 5 × 10 ４ The cells were seeded at 1000 cells / well and incubated at 37°C, 5% CO ２ The cells were cultured in PBS until they reached confluence.

[0053] Example 1 In Example 1, the effect of a myokine receptor expression promoter containing ginseng extract as an active ingredient on myokine receptors in fibroblasts was examined.

[0054] More specifically, after confirming that the cells had reached confluence, the medium was changed to 0.5% FBS-DMEM and cultured overnight. Subsequently, ginseng extract was added to the medium to a concentration of 0.1% (v / v), and the culture was continued. A control group was cultured in 0.5% FBS-DMEM alone without the extract.

[0055] 24 hours after the addition of the ginseng extract, the medium was removed, and the cells were lysed and RNA was extracted using a commercially available RNA extraction device (QIAGEN EZ2 connect: Qiagen Inc.) and its reagents (EZ2 RNA / miRNA Tissue / Cells Kit: Qiagen Inc.).

[0056] Evaluation of myokine receptor expression levels by quantitative PCR: cDNA was synthesized using the extracted RNA as a template according to a commercially available kit and protocol (PrimeScript RT reagent Kit with gDNA Eraser: Takara Bio), and quantitative PCR was performed using the following reagents and equipment.

[0057] Reagent: TB Green Premix Ex Taq II (Tli RNase H Plus, manufactured by Takara Bio Inc.) Quantitative PCR device: Mx-3005P, manufactured by Agilent

[0058] At this time, the gene expression level of ALK5 was measured as a myokine receptor. The expression level of the RPLP0 gene (SEQ ID NOs: 1 and 2), which has a nearly constant expression level, was also measured simultaneously as an internal standard. A commercially available gene-specific PCR primer (HA346508, manufactured by Takara Bio Inc.) was used as the PCR primer. The results are shown in Figure 2. RPLP0 forward: 5'-TTCGACAATGGCAGCAGCATCTACAA-3' (SEQ ID NO: 1) RPLP0 reverse: 5'-CTGCAGACAGACACTGGCAACA-3' (SEQ ID NO: 2)

[0059] As shown in Figure 2, compared to culture in medium alone (control), the myokine receptor expression promoter of Example 1 containing ginseng extract as an active ingredient was confirmed to have a significant effect of promoting ALK5 expression. More specifically, the myokine receptor expression promoter of Example 1 was found to enhance the expression level of ALK5 by 15% compared to the control.

[0060] Example 2 In Example 2, the effect of a myokine receptor expression promoter containing Angelica acutiloba extract as an active ingredient on myokine receptors in fibroblasts was investigated.

[0061] The same experiment as in Example 1 was carried out, except that the Angelica acutiloba extract was added to the medium at a concentration of 0.1% (v / v) and the gene expression level of the myokine receptor ACVR2B was measured. A commercially available gene-specific PCR primer (HA345172, manufactured by Takara Bio Inc.) was used as the PCR primer. The results are shown in Figure 3.

[0062] As shown in Figure 3, the myokine receptor expression promoter of Example 2 containing Angelica acutiloba extract as an active ingredient was confirmed to have a significant ACVR2B expression promoting effect compared to culture in medium alone (control). More specifically, the myokine receptor expression promoter of Example 2 was found to enhance ACVR2B expression by 24% compared to the control.

[0063] Example 3 In Example 3, the effect of a myokine receptor expression promoter containing royal jelly extract as an active ingredient on myokine receptors in keratinocytes was investigated.

[0064] A commercially available royal jelly extract (Royal Jelly Extract B: Koei Kogyo Co., Ltd.) was dissolved in 1×PBS(−) and used.

[0065] Epidermal cell culture: Commercially available normal adult skin-derived keratinocytes (Kurabo) were used at 5 x 10 ５ The cells were seeded into a T75 flask at 100 cells / flask and incubated at 37°C in 5% CO in a keratinocyte medium (Humedia-KG2, Kurabo).２ The keratinocytes were cultured under atmospheric conditions until they reached semi-confluence and used as seed cells. The keratinocytes were harvested by trypsinization and placed in a 24-well plate at 5 × 10 ４ The cells were seeded at 100 cells / well and incubated at 37°C in 5% CO in a keratinocyte medium (Humedia-KG2, Kurabo). ２ The cells were cultured in PBS until they reached confluence.

[0066] Royal jelly extract was added to the medium to a concentration of 0.1% (v / v) to epidermal cells cultured until confluent, and the culture was continued. As a control, a group of cells cultured in PBS (as above) alone was prepared.

[0067] 24 hours after the addition of the royal jelly extract, the medium was removed, and cells were lysed and RNA was extracted using a commercially available RNA extraction reagent (RNeasy Mini Kit, Qiagen).

[0068] <Evaluation> As in the above-mentioned examples, the expression level of myokine receptor was evaluated by quantitative PCR. The results are shown in Figure 4.

[0069] As shown in Figure 4, the myokine receptor expression promoter of Example 3 containing royal jelly extract as an active ingredient was confirmed to have an ALK5 expression promoting effect compared to culture in medium alone (control). More specifically, the myokine receptor expression promoter of Example 3 was found to enhance ALK5 expression by 19% compared to the control.

[0070] Example 4 In Example 4, the effects on fibroblast proliferation of a myokine receptor expression promoter containing ginseng extract as an active ingredient and a cell proliferation promoter containing myokine were investigated.

[0071] GDF11 was used as the myokine. Commercially available GDF11 (Human / Mouse / Rat GDF11 Recombinant Protein #86907: Cell Signaling Technology) was dissolved and diluted with purified water to a concentration of 1 μg / ml. The medium was prepared in the same manner as in Example 1.

[0072] Cell culture: Commercially available normal adult skin-derived fibroblasts were seeded on a 100 mm collagen-coated dish and incubated in 10% FBS-DMEM at 37°C and 5% CO ２ The cells were cultured in PBS until they reached subconfluence. The cells were harvested by trypsinization and plated in a 24-well plate at 4 × 10 ３ The cells were seeded at 100 cells / well.

[0073] The day after seeding the cells, the medium was changed to 0.5% FBS-DMEM, and GDF11 and ginseng extract were added at the following concentrations, followed by continued culturing: (Concentrations) FBS-DMEM as control 1: 0.5% (v / v) Ginseng extract as control 2: 0.2% (v / v) GDF11 as control 3: 25 ng / ml In Example 4, GDF11: 25 ng / ml, ginseng extract: 0.2% (v / v)

[0074] Evaluation by AlamarBlue Assay Three days after adding the reagent, AlamarBlue™ Cell Viability Reagent (Life Technologies (Invitrogen)) was added in an amount of 1 / 10 of the medium volume and incubated for 2 hours. Thereafter, fluorescence at 590 nm was measured using a plate reader (SYNERGY H1: BioTek) with an excitation wavelength of 560 nm. The respective results are shown in FIG. 5.

[0075] As shown in FIG. 5, it was confirmed that the cell proliferation promoting agent of Example 4, which contains both GDF11 and ginseng extract, significantly promoted cell proliferation.

[0076] 1 Myokine 2, 3 Cell 10r, 11r, 12r, 13r Myokine receptor 100 Myokine receptor expression promoter

Claims

1. A myokine receptor expression promoter comprising as an active ingredient at least one selected from the group consisting of ginseng extract, angelica extract, and royal jelly extract.

2. The myokine receptor expression promoter according to claim 1, wherein the myokine is GDF11.

3. The myokine receptor expression promoter according to claim 1, wherein the myokine receptor includes at least one of ALK5 and ACVR2B.

4. The myokine receptor expression promoter according to claim 1, wherein the myokine receptor is in fibroblasts or keratinocytes.

5. A myokine receptor expression promoter as described in claim 1, which can promote cell proliferation through the expression of the myokine receptor.

6. A cell proliferation promoter comprising the myokine receptor expression promoter according to any one of claims 1 to 5 and a myokine.

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