Composition comprising a high-pressure treatment of a mixture containing sturgeon eggs

Abstract

The anti-inflammatory composition promotes proliferation of macrophage and suppresses inflammation caused by inflammatory cytokine and / or NO synthase, and promotes proliferation of human fibroblast or human epidermal keratinocyte and promotes synthesis of hyaluronic acid in epidermis and dermis. To provide a skin-beautifying and promoting composition which keeps the tension of the skin, strengthens the bonding force of the skin, improves the barrier function of the skin, enhances the strength and water-retaining function of the skin surface, or promotes the anti-aging of the skin, to provide a hair-growing composition which promotes the proliferation of human epidermal keratinocyte cells, improves the strength of the skin surface, and promotes hair growth, and to provide cosmetic compositions thereof.SOLUTION: The composition comprises a high-pressure treated product of a mixture of sturgeon eggs and 1, 3-butylene glycol.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present invention relates to a composition comprising a high-pressure treated mixture containing sturgeon roe, and more particularly to a composition comprising a high-pressure treated mixture of sturgeon roe and 1,3-butylene glycol, the composition being one or more compositions selected from the group consisting of anti-inflammatory compositions, skin-beautifying compositions, and hair-growth compositions. [Background technology]

[0002] Sturgeon is the common name for many species of the sturgeon family (Acipenseridae), including the genera Acipenser, Huso, Scaphirhynchus, and Pseudoscaphirhynchus. Caviar, a prized luxury food, generally refers to the salted unfertilized eggs or roe from the ovaries of female sturgeons belonging to the genera Acipenser or Huso.

[0003] Cosmetics made from fish roe have recently been used. Many of these are hydrolysates or extracts using organic solvents. Sturgeon roe, a luxury food ingredient, is also used as such fish roe. For example, there is a sturgeon roe extract extracted using a mixed solvent of 1,3-butylene glycol and water, and a sturgeon roe extract extracted using 1,3-butylene glycol after removing phospholipids using an organic solvent that is not miscible in water at any ratio at 30°C (Patent Documents 1 and 2). Furthermore, there is a placenta extract obtained by high-pressure treatment of placenta, although it is not a cosmetic made from fish roe (Patent Document 3). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 5210513 [Patent Document 2] Patent No. 5561912 [Patent Document 3] Patent No. 6117456 Summary of the Invention [Problem to be solved by the invention]

[0005] However, Patent Documents 1 and 2 do not disclose or suggest, even as comparative examples, a sturgeon roe extract or sturgeon roe extract liquid extracted using only 1,3-butylene glycol as a solvent. Therefore, even if Patent Document 1 or 2 is combined with Patent Document 3, it is not possible to arrive at a composition consisting of a high-pressure treated mixture of sturgeon roe and 1,3-butylene glycol.

[0006] The present invention aims to provide a composition comprising a high-pressure treated mixture of sturgeon eggs and 1,3-butylene glycol, which is one or more compositions selected from the group consisting of a composition for promoting macrophage proliferation, an anti-inflammatory composition, a composition for promoting human fibroblast proliferation, a composition for promoting human epidermal keratinocyte proliferation, a composition for promoting skin beauty, and a hair growth composition, as well as a cosmetic composition. In particular, the present invention provides a composition comprising a high-pressure treated mixture of sturgeon eggs and 1,3-butylene glycol, which promotes macrophage proliferation and inhibits inflammatory cytokines. The present invention aims to provide an anti-inflammatory composition that suppresses inflammation caused by acetylcholine and / or nitric oxide synthase; a skin-beautifying composition that promotes the proliferation of human fibroblasts or human epidermal keratinocytes and stimulates the synthesis of hyaluronic acid in the epidermis and dermis, thereby maintaining skin firmness, strengthening the skin's cohesiveness, improving the skin's barrier function, increasing the strength and moisture-retaining function of the skin surface, or promoting anti-aging of the skin; a hair-growth composition that promotes the proliferation of human epidermal keratinocytes, improves the strength of the skin surface, and promotes hair growth; and cosmetic compositions containing these. [Means for solving the problem]

[0007] As a result of extensive research, the present inventors have found that a composition comprising a high-pressure treated mixture of sturgeon eggs and 1,3-butylene glycol has macrophage proliferation promoting effects, anti-inflammatory effects, human fibroblast proliferation promoting effects, human epidermal keratinocyte proliferation promoting effects, skin beautifying effects, and hair growth effects, and have completed the following inventions.

[0008] (1) A composition comprising a high-pressure treated mixture of sturgeon roe and 1,3-butylene glycol, the composition being one or more compositions selected from the group consisting of (a), (b), and (c) below; (a) an anti-inflammatory composition; (b) Beautiful skin promoting composition; (c) a hair growth composition.

[0009] (2) A composition comprising a high-pressure treated mixture of sturgeon roe and 1,3-butylene glycol, the composition being one or more compositions selected from the group consisting of (d), (e), and (f) below; (d) a macrophage proliferation-promoting composition; (e) a human fibroblast proliferation-promoting composition; (f) A composition for promoting the proliferation of human epidermal keratinocytes.

[0010] (3) The composition according to (1) or (2), which is a cosmetic composition. [Effects of the Invention]

[0011] The compositions and cosmetic compositions comprising the high-pressure treated products of the present invention can promote the proliferation of macrophages, human fibroblasts, or human epidermal keratinocytes, and can suppress inflammation caused by inflammatory cytokines and / or NO synthase, promote the synthesis of hyaluronic acid in the epidermis and dermis, maintain skin firmness, strengthen the skin's cohesive strength, improve the skin's barrier function, increase the strength and moisture retention of the skin surface, promote anti-aging of the skin, or improve the strength of the skin surface and promote hair growth. [Brief explanation of the drawings]

[0012] [Figure 1A] This is a bar graph showing the proliferation rate (%) of macrophages (RAW264.7 cells) for the control and high-pressure treated samples (1) Sample 1, (2) Sample 2, (3) Sample 3, and (4) Sample 4. [Figure 1B] FIG. 1 is a bar graph (%) showing the proliferation rate of macrophages (RAW264.7 cells) for the control and the high-pressure-treated samples (1) Sample 5 and (2) Sample 6. [Figure 2] This is a bar graph showing the proliferation rate of macrophages (RAW264.7 cells) for the control and non-high-pressure treated samples (1) Sample 7, (2) Sample 8, and (3) Sample 9. [Figure 3] FIG. 1 is a line graph showing the relationship between the concentration (μg / mL) and the macrophage proliferation rate (%) for the control, sample 2, sample 3, and sample 5. [Figure 4] FIG. 1 is a bar graph showing (1) the expression levels of TNF-α mRNA, (2) the expression levels of iNOS mRNA, and (3) the expression levels of IL-6 mRNA in macrophages (RAW264.7 cells) for the control, LPS (lipopolysaccharide; lipopolysaccharide derived from Escherichia coli), sample 2, sample 3, sample 5, LPS+sample 2, LPS+sample 3, and LPS+sample 5. [Figure 5] FIG. 1 is a line graph showing the relationship between the concentration (μg / mL) of a control, sample 2, sample 3, and sample 5 and the proliferation rate (%) of human fibroblasts. [Figure 6] FIG. 1 is a bar graph showing (1) the expression level of HAS1 mRNA and (2) the expression level of HAS3 mRNA in human fibroblasts for the control, sample 2, sample 3, and sample 5. [Figure 7] FIG. 1 is a bar graph showing (1) the expression level of COL1A1 mRNA and (2) the expression level of COL7A1 mRNA in human fibroblasts for the control, sample 2, sample 3, and sample 5. [Figure 8]FIG. 1 is a line graph showing the relationship between the concentration (μg / mL) of a control, sample 2, sample 3, and sample 5 and the proliferation rate (%) of human epidermal keratinocytes. [Figure 9] FIG. 1 is a bar graph showing (1) the expression level of OCLN mRNA and (2) the expression level of FLG mRNA in human epidermal keratinocytes for the control, sample 2, sample 3, and sample 5. [Figure 10] FIG. 1 is a bar graph showing (1) the expression level of AQP3 mRNA and (2) the expression level of TGM1 mRNA in human epidermal keratinocytes for the control, sample 2, sample 3, and sample 5. [Figure 11] FIG. 1 is a bar graph showing the expression levels of SIRT1 mRNA in human epidermal keratinocytes for the control, sample 2, sample 3, and sample 5. [Figure 12] FIG. 1 is a bar graph showing the expression levels of COL17A1 mRNA in human epidermal keratinocytes for the control, sample 2, sample 3, and sample 5. DETAILED DESCRIPTION OF THE INVENTION

[0013] The agent according to the present invention will be described in detail below. The composition according to the present invention is a composition comprising a high-pressure treated mixture of sturgeon eggs and 1,3-butylene glycol, and is one or more compositions selected from the group consisting of a macrophage proliferation-promoting composition, an anti-inflammatory composition, a human fibroblast proliferation-promoting composition, a human epidermal keratinocyte proliferation-promoting composition, a skin-beautifying composition, and a hair-growth composition.

[0014] The sturgeon eggs used in the present invention may be eggs of any fish classified in the Acipenseridae family of the Acipenseriformes order, including, for example, eggs of fish belonging to the genera Acipenser, Huso, Scaphirhynchus, and Pseudoscaphirhynchus. These eggs may be either wild-caught or farmed, although farmed eggs are preferred in light of compliance with the Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES). Furthermore, the method by which the eggs are extracted from the sturgeon is not important, and eggs extracted by a method selectable by those skilled in the art, such as eggs extracted by dissecting the sturgeon or eggs extracted from the sturgeon, may be used. Furthermore, the composition of the present invention may be prepared using eggs extracted from the sturgeon as is, or eggs washed with physiological saline or processed to a degree that does not impair the characteristics and effects of the present invention. Alternatively, the eggs may be frozen and stored, and then thawed for use.

[0015] The 1,3-butylene glycol used in the present invention is also known as 1,3-butanediol or 1,3-dihydroxybutane, and is a dihydric alcohol sometimes abbreviated as "BG." Its molecular formula is CH 10 O2 and can be synthesized from acetaldehyde. 1,3-Butylene glycol is used in cosmetics and pharmaceuticals (topical preparations). In cosmetics, it is used for moisturizing by increasing the moisture content of the stratum corneum, as an antiseptic agent, and as a solvent. In pharmaceuticals (topical preparations), it is used as a base, antioxidant, moisturizing, viscous, solvent, and dissolution aid.

[0016] In the present invention, the "mixture of sturgeon roe and 1,3-butylene glycol at a concentration of 100% (v / v)" preferably includes a mixture of sturgeon roe and 1,3-butylene glycol at a concentration of 100% (v / v) (a mixture of sturgeon roe and 1,3-butylene glycol at a concentration of 100% (v / v)), as well as a mixture of sturgeon roe, 1,3-butylene glycol at a concentration of 100% (v / v), and a substance that can be added to the extent that the features and effects of the present invention are not impaired. In such cases, the substance that can be added to the extent that the features and effects of the present invention are not impaired is not particularly limited, and examples thereof include monohydric alcohols such as ethanol, butanol, and propanol; dihydric alcohols such as propylene glycol, 1,2-hexanediol, and 1,4-pentanediol; polyhydric alcohols such as glycerin; and water.

[0017] In the present invention, the term "high-pressure treated product" refers to a substance obtained by high-pressure treating the above-mentioned specified mixture. In the present invention, "high-pressure treatment" refers to a treatment in which high pressure is applied. The "high pressure" in the present invention is not particularly limited as long as it is a pressure of 50 MPa or higher, but is preferably 50 MPa to 400 MPa, more preferably 60 MPa to 300 MPa, even more preferably 70 MPa to 250 MPa, even more preferably 80 MPa to 200 MPa, and most preferably 90 MPa to 150 MPa. In the examples of the present specification, a pressure of 100 MPa was selected as the preferred pressure.

[0018] The temperature conditions for "high-pressure treatment" are not particularly limited as long as they are above room temperature and do not cause irreversible protein aggregation, but are preferably 30°C or higher and lower than 80°C, more preferably 35°C or higher and 75°C or lower, even more preferably 40°C or higher and 70°C or lower, still more preferably 45°C or higher and 65°C or lower, and most preferably 50°C or higher and 60°C or lower. In the examples of the present specification, a temperature of 55°C was selected as the preferred temperature condition.

[0019] Furthermore, the time required for the "high pressure treatment" can be appropriately selected depending on the amount of the mixture to be treated and the amount of the treated product to be obtained.

[0020] The term "anti-inflammatory composition" refers to a composition that suppresses inflammation, and in the present invention, particularly refers to a composition that suppresses inflammation caused by inflammatory cytokines and / or NO synthase. Examples of such anti-inflammatory compositions include compositions that promote the proliferation of macrophages and suppress the expression in macrophages of one or more mRNAs selected from the group consisting of TNF-α (Tumor Necrosis Factor-α) mRNA, iNOS (inducible Nitric Oxide Synthase; NOS2) mRNA, and IL-6 (Interleukin-6) mRNA, thereby suppressing inflammation caused by one or more cytokines selected from the group consisting of TNF-α, iNOS (NOS2), and IL-6 and / or NO synthase.

[0021] The term "skin-beautifying composition" refers to a composition that promotes the maintenance or formation of beautiful skin, and in the present invention particularly refers to one or more compositions selected from the group consisting of compositions that promote the synthesis of hyaluronic acid in the epidermis or dermis, compositions that maintain skin firmness, compositions that strengthen the skin's cohesive strength, compositions that improve the skin's barrier function, compositions that increase the strength of the skin surface, compositions that enhance the skin's moisture-retaining function, and compositions that promote anti-aging of the skin. Such compositions may also be compositions that have the effect of reducing or preventing facial blemishes, freckles, dark circles, and dullness, for example. Examples of such skin-beautifying compositions include compositions that promote the proliferation of human fibroblasts, promote the expression of one or more mRNAs selected from the group consisting of HAS1 (Hyaluronan Synthase I) mRNA, HAS3 (Hyaluronan Synthase III) mRNA, COL1A1 (Collagen Type I Alpha 1) mRNA, and COL7A1 (Collagen Type VII Alpha 1) mRNA in human fibroblasts, and produce one or more hyaluronic acid synthases and / or collagens selected from the group consisting of HAS1, HAS3, COL1A1, and COL7A1 in the skin, as well as compositions that promote the proliferation of human epidermal keratinocytes, and promote the expression of OCLN (Occludin) mRNA, FLG (Filaggrin) mRNA, AQP3 (Aquaporin III) mRNA, TGM1 (Transglutaminase I) mRNA, and SIRT1 (Silent Information Regulatory I) A composition that can promote the expression of one or more mRNAs selected from the group consisting of mRNAs, and produce in the skin one or more proteins and / or enzymes selected from the group consisting of OCLN, FLG, AQP3, TGM1, and SIRT1.

[0022] The term "hair growth composition" refers to a composition that promotes the growth of existing hair, including vellus hair, or a composition that maintains existing hair, as well as a composition that promotes the production of new hair or a composition that prevents existing hair from falling out. In the present invention, the term particularly refers to a composition that improves the strength of the skin surface and promotes hair growth. Examples of such hair growth compositions include a composition that promotes the proliferation of human epidermal keratinocytes and promotes the expression of COL17A1 (Collagen Type XVII Alpha 1) mRNA in human hair follicle keratinocytes, thereby producing COL17A1 in the skin.

[0023] The terms "macrophage proliferation-promoting composition," "human fibroblast proliferation-promoting composition," and "human epidermal keratinocyte proliferation-promoting composition" refer to a composition that promotes the proliferation of macrophages, a composition that promotes the proliferation of human fibroblasts, and a composition that promotes the proliferation of human epidermal keratinocytes, respectively.

[0024] The cosmetic composition of the present invention may be appropriately blended with additives commonly used in the field of cosmetics, provided that the characteristics of the present invention are not impaired. Examples of such additives include one or more additives selected from the group consisting of moisturizers, preservatives, pearlescent materials, antioxidants, pigments, thickeners, and pH adjusters. Furthermore, fragrances, antibacterial agents, various oily components, ultraviolet absorbers, active oxygen scavengers, antioxidants, antimicrobial agents, minerals, amino acids, and the like may also be added. Furthermore, known whitening ingredients may also be added.

[0025] The method for producing the composition of the present invention comprises the following steps (i) to (iii): (i) a step of mixing sturgeon eggs with 1,3-butylene glycol to prepare a mixture (mixture preparation step), (ii) a step of high-pressure treating the mixture prepared in the mixture preparation step (i) to prepare a high-pressure treated product (high-pressure treated product preparation step), and (iii) a step of filtering the high-pressure treated product prepared in the high-pressure treated product preparation step (ii) to remove residue and obtain a filtrate (filtration step).

[0026] The mixing of sturgeon eggs and 1,3-butylene glycol in the mixture preparation step (i) can be carried out by any suitable method known to those skilled in the art, and, if desired, any substances that can be added can be added and mixed within a range that does not impair the characteristics and effects of the present invention.

[0027] The high-pressure treatment in the high-pressure-treated product preparation step (ii) can be carried out on the mixture prepared in the mixture preparation step (i) under the pressure and temperature conditions described above by a method known to those skilled in the art. Such high-pressure treatment is generally carried out using a high-pressure treatment device, and examples of such high-pressure treatment devices include various whole extractors (Toyo Koatsu Co., Ltd.).

[0028] The filtration in the filtration step (iii) can be carried out on the high-pressure-treated product prepared in the high-pressure-treated product preparation step (ii) by a suitable method known to those skilled in the art. Such filtration is generally carried out using a filtering device, such as a stocking-type draining bag or various meshes.

[0029] At the time of filing, in order to directly identify the composition of the present invention by its structure or properties, a high-pressure-treated mixture of sturgeon eggs and 1,3-butylene glycol must first be fractionated. For example, fractionation by chromatography requires significant time and expense, requiring the selection of appropriate separation resins and developing solvents from a vast array of options. Then, from the numerous fractions, fractions containing active ingredients must be identified using indicators such as anti-inflammatory activity, skin-beautifying activity, hair growth activity, macrophage proliferation promotion activity, human fibroblast proliferation promotion activity, and / or human epidermal keratinocyte proliferation promotion activity. Furthermore, experiments to obtain target fractions using these activities must be repeated until the target active ingredient is isolated as a pure product. However, since the active ingredient may not be a single ingredient but may be multiple ingredients, these experiments require significant effort.

[0030] Even if the active ingredient is successfully isolated, its chemical structure must be determined, at least roughly, using NMR, IR, Mass, chemical decomposition reactions, etc., and then, rather than selecting the determined chemical structure from among those claimed in the present invention, the assumed compound must be chemically synthesized by a completely different means and the pure product must be isolated.

[0031] Furthermore, the chemical structure of the desired high-pressure treated product can be determined only when it is determined that the isolated pure product obtained from the high-pressure treated product and the chemically synthesized pure product having the same chemical structure as the isolated pure product obtained from the high-pressure treated product are completely identical in terms of the degree of anti-inflammatory effect, skin beauty promoting effect, hair growth promoting effect, macrophage proliferation promoting effect, human fibroblast proliferation promoting effect, and / or human epidermal keratinocyte proliferation promoting effect.

[0032] The above-mentioned experiments would normally require several years for even a competent person skilled in the art, which would require an unrealistic number of experiments and involve significantly excessive economic expenditure (see Patent and Utility Model Examination Handbook, Part II, Chapter 2, Requirements for the Description of the Claims, "2205 Determination of 'Impossible or Impractical Circumstances' in Examination When a Claim for an Invention of a Product Recites a Manufacturing Process for That Product," p. 16, bottom line to p. 17, lines 1-4). In other words, directly identifying the high-pressure-treated product by its structure or properties would require a person skilled in the art at the time of filing an application with an uneconomical amount of time and expense. Requiring such identification work would be prohibitive for applicants seeking the earliest possible filing date under the first-to-file principle, given the rapid advances in technology and fierce international competition for patents (Supreme Court Case 2012 (Judgment) 1204, p. 10, or Supreme Court Case 2012 (Judgment) 2658, p. 10-11, etc.). [Example]

[0033] The composition comprising the high-pressure treated product according to the present invention will be described below based on examples. However, the technical scope of the present invention is not limited to the embodiments shown by these examples.

[0034] 1. Preparation of Specimens and Test Samples [1-1] Mixtures of high-pressure-treated sturgeon eggs and various solvents Sturgeon eggs (frozen, from China) and the solvents 70% (v / v, hereafter the same) ethanol, 100% 1,3-butylene glycol (hereafter referred to as "100% BG"), 50% 1,3-butylene glycol (hereafter referred to as "50% BG"), pure water (ion-exchanged water), or enzyme water were each placed in a retort pouch, sealed, and degassed to obtain a mixture, which was then high-pressure treated at 55°C and 100 MPa for 24 hours. Two different mixtures of sturgeon eggs and enzyme water were prepared, and after high-pressure treatment, the enzymes were inactivated in both cases by boiling in water at 85-90°C for 10 minutes. The enzyme used in these enzyme waters was a proteolytic complex enzyme (Marugoto Enzyme PrE; Supercritical Technology Research Institute, Inc.), the water used in these enzyme waters was pure water (ion-exchanged water), and the high-pressure treatment for each was performed using the Marugoto Extraction Device TFS-100 (Toyo Koatsu Co., Ltd.).

[0035] After high-pressure treatment of the six mixtures, each mixture was filtered using a stocking-type draining bag (DCM Co., Ltd.) to remove most of the residue. The mixture was then further filtered using a 200 μm mesh to remove the remaining residue, yielding each filtrate. The resulting filtrate was divided into 1.5 mL tubes and stored at -20°C.

[0036] To test the six stored filtrates, 300 μL of each thawed filtrate was added to two 5 mL Petri dishes (60 × 15 mm) each, for a total of 12 dishes. These dishes were then placed in a dryer and dried at 72 °C for 48 hours. The solids content (%) was calculated from the weights of the dishes before, after, and after the filtrate was added. Based on the calculated solids content (%), each filtrate was diluted with autoclave water (hereinafter referred to as "DDW") to a concentration of 11,000 μg / mL. The six filtrates were then sterilized by filtration using a 0.45 μm filter to prepare samples 1 to 6. Each filtrate was then diluted with DDW to 10 μg / mL, 100 μg / mL, and 300 μg / mL to prepare the test samples. The composition of Samples 1 to 6 is shown in Table 1 below.

[0037] [Table 1]

[0038] [1-2] Mixtures of non-high pressure treated sturgeon eggs and various solvents Sturgeon eggs (frozen, from China), pure water (ion-exchanged water), 100% BG, or enzyme water were each placed in a retort pouch, sealed, degassed, and left to stand for 24 hours without high-pressure treatment. The mixture of sturgeon eggs and enzyme water was then inactivated by boiling in hot water at 85-90°C for 10 minutes, just like the high-pressure-treated product. Each mixture was filtered using a stocking-type draining bag (DCM Co., Ltd.) to remove most of the residue, and then further filtered using a 200 μm mesh to remove the remaining residue, yielding each filtrate. Six 5 mL Petri dishes (60 x 15 mm) were prepared by adding 300 μL of each filtrate to two of each dish. These dishes were then dried at 72°C for 48 hours. The solids content (%) was calculated from the weights of the dishes before, after, and after drying. Based on the calculated solids content (%), each filtrate was diluted with DDW to 11,000 μg / mL. These three filtrates were sterilized by filtration using a 0.45 μm filter to prepare Samples 7–9. For cell experiments, the final concentrations in the medium were adjusted appropriately to 10 μg / mL, 100 μg / mL, and 300 μg / mL, respectively, to prepare the test samples. The composition of Samples 7–9 is shown in Table 2.

[0039] [Table 2]

[0040] 2. Exam [2-1] Macrophage proliferation activity test A total of 27 test samples obtained from samples 1 to 9 prepared in [1-1] and [1-2] above were co-cultured with macrophages (RAW264.7 cells; hereinafter referred to as "RAW cells") for 72 hours, and then the cell viability (%) was determined by the MTT method.

[0041] Specifically, RAW cells were seeded in a 96-well plate at 100 μL / well (n=3) and cultured in a CO2 incubator (37°C, 5% CO2) for 24 hours. Then, 10 μL / well of each of the 27 test samples obtained from Samples 1 to 9 was added, and the cells were cultured for an additional 72 hours in a CO2 incubator. The culture medium used was D-MEM (high glucose) medium containing 2% Fetal Bovine Serum (hereinafter referred to as "FBS") and 1% penicillin. The culture supernatants were removed, and 50 μL / well of the cell culture reagent PBS(-) (Fujifilm Wako Pure Chemical Industries, Ltd.) and 10 μL / well of CellTiter reagent (CellTiter 96® AQueous One Solution Cell Proliferation Assay; Promega Corporation) were added. After 3 hours of incubation in a CO2 incubator, the absorbance at 490 nm was measured using a microplate reader. Cell proliferation rates were calculated using the absorbance at 490 nm of cells without test sample as the control. Statistical analysis was performed using the Dunnett test. *P<0.05, **P<0.01 were considered significant compared to the control. The results are shown in Figures 1A, 1B, and 2.

[0042] 1A and 1B show the results for test samples 1 to 6, and FIG. 2 shows the results for test samples 7 to 9. As shown in FIGS. 1A, 1B, and 2, significant macrophage proliferation was observed compared to the control in the test samples of Sample 2 diluted at 10 μg / mL, 100 μg / mL, and 300 μg / mL, the test sample of Sample 3 diluted at 300 μg / mL, and the test samples of Sample 5 diluted at 100 μg / mL and 300 μg / mL. These results demonstrate that Sample 2 (i.e., a composition consisting of a high-pressure-treated mixture of sturgeon eggs and 100% BG), Sample 3 (i.e., a composition consisting of a high-pressure-treated mixture of sturgeon eggs and 50% BG), and Sample 5 (i.e., a composition consisting of a high-pressure-treated mixture of sturgeon eggs and an aqueous solution of a complex protease) have macrophage proliferation activity. Therefore, subsequent proliferation activity tests and gene expression tests in various cells were carried out using Samples 2, 3, and 5.

[0043] [2-2] Macrophage proliferation activity test and gene expression test (1) Proliferation activity test The results of the macrophage proliferation activity test were as described in [2-1] above. Sample 2, i.e., a composition consisting of a high-pressure-treated mixture of sturgeon roe and 100% BG; Sample 3, i.e., a composition consisting of a high-pressure-treated mixture of sturgeon roe and 50% BG; and Sample 5, i.e., a composition consisting of a high-pressure-treated mixture of sturgeon roe and an aqueous solution of proteolytic complex enzymes, all exhibited macrophage proliferation activity. These results are shown in Figure 3. Figure 3 is a line graph showing the macrophage proliferation rates (%) for Samples 2, 3, and 5. Statistical calculations were performed using the Dunnett test, with *P<0.05 and **P<0.01 indicating significant differences compared to the control.

[0044] (2) Gene expression test Macrophages, i.e., RAW cells, were seeded in a 6-well plate at 3 mL / well and cultured for 2 days. After that, 300 μL each of Sample 2, Sample 3, and Sample 5 at a final concentration of 100 μg / mL was added to each of 2 wells and cultured for 2 hours. After setting the final concentration of each sample to 100 μg / mL, in order to promote the production of inflammatory cytokines (TNF-α (Tumor Necrosis Factor-α), NO synthase iNOS (inducible Nitric Oxide Synthase; NOS2), inflammatory cytokine IL-6 (Interleukin-6)) in RAW cells, LPS (lipopolysaccharide; lipopolysaccharide derived from Escherichia coli, FUJIFILM Wako Pure Chemical Corporation) was added to a final concentration of 0.1 μg / mL and cultured for 4 hours. After culturing, RNA of the measured genes TNF-α, iNOS (NOS2), and IL-6 was extracted using an RNA extraction kit (RNeasy Mini Kit and QIAshredder (QIAGEN)). After preparing cDNA from the extracted RNA using a High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems), the expression level of mRNA was measured using 5 mL of TaqMan(R) Fast Advanced Master Mix (Applied Biosystems) and a Step One Real-time PCR System (Applied Biosystems), and relative values were calculated by the comparative quantification method (comparative Ct method / ΔΔCt method). GAPDH was used as the control (housekeeping gene). The results are shown in Fig. 4, and the primers used in Real-time PCR are shown below. In Fig. 4, statistical calculations were performed by Student T.test, and †P<0.5, *P<0.05, **P<0.01 were considered to have significant differences.

[0045] <Primers used in Real-time PCR> All of these are TaqMan® Gene Expression Assays (Thermo Fisher Scientific) and are identified by the following TaqMan® Gene Expression Assays IDs: TNF-α mRNA GAPDH:Mm99999915_g1 TNF-α:Mm00443258_m1 iNOS mRNA GAPDH:Mm99999915_g1 iNOS:Mm0044052_m1 IL-6 mRNA GAPDH:Mm99999915_g1 IL-6:Mm00446190_m1

[0046] When RAW cells are stimulated with LPS, they produce the inflammatory cytokine TNF-α, the NO synthase iNOS (NOS2), and the inflammatory cytokine IL-6. As shown in Figure 4, Samples 2, 3, and 5 were found to suppress the expression of TNF-α mRNA, iNOS (NOS2) mRNA, and IL-6 mRNA in RAW cells. In particular, Sample 2, a composition consisting of a high-pressure-treated mixture of sturgeon eggs and 100% BG, showed the highest activity in suppressing the expression of these mRNAs in RAW cells. The functions involved in the measured genes are shown in Table 3 below.

[0047] [Table 3]

[0048] (3) Conclusion Based on the results of the proliferation activity test and gene expression test in macrophages (1) and (2) above, it can be said that Sample 2, i.e., a composition consisting of a high-pressure-treated mixture of sturgeon eggs and 100% BG, has macrophage proliferation activity and clearly suppresses the expression of mRNA for the inflammatory cytokine TNF-α, the NO synthase iNOS (NOS2), and the inflammatory cytokine IL-6 in macrophages. Therefore, it can be said that the composition consisting of a high-pressure-treated mixture of sturgeon eggs and BG is an anti-inflammatory composition.

[0049] [2-3] Proliferation activity test and gene expression test in human fibroblasts (1) Proliferation activity test For samples 2, 3, and 5 prepared in [1-1] above, a proliferation activity test was performed on human fibroblasts using the same method as in [2-1] above, except that fibroblast medium (PromoCell) containing 2% FBS and 1% penicillin was used instead of D-MEM (high glucose) medium containing 2% FBS and 1% penicillin, and trypsin-EDTA (Fujifilm Wako Pure Chemical Industries, Ltd.) was used to detach and recover the cultured cells. The results are shown in Figure 5. Statistical calculations were performed using the Dunnett test, and *P<0.05 and **P<0.01 were considered significant compared to the control.

[0050] As shown in Figure 5, significant human fibroblast proliferation was observed compared to the control in the sample in which Sample 2 was added to the medium at a final concentration of 10 μg / mL and the sample in which Sample 3 was added to the medium at a final concentration of 10 μg / mL. These results demonstrate that Sample 2, i.e., a composition consisting of a high-pressure-treated mixture of sturgeon eggs and 100% BG, and Sample 3, i.e., a composition consisting of a high-pressure-treated mixture of sturgeon eggs and 50% BG, have the activity of proliferating human fibroblasts.

[0051] (2) Gene expression test For Samples 2, Sample 3, and Sample 5 prepared in [1-1] above, except that they were cultured without adding LPS, RNA of the measured genes HAS1 (Hyaluronan Synthase I), HAS3 (Hyaluronan Synthase III), COL1A1 (Collagen Type I Alpha 1), and COL7A1 (Collagen Type VII Alpha 1) was extracted, and the primers used in Real-time PCR were changed according to the measured genes, a gene expression test in human fibroblasts was conducted by the same method as in [2-2](2) above. The results are shown in FIGS. 6 and 7, and the primers used in Real-time PCR are shown below. In FIGS. 6 and 7, statistical calculations were performed by the Dunnett test, and *P<0.05 and **P<0.01 were considered to have significant differences compared to the control.

[0052] <Primers used in Real-time PCR> All are those of TaqMan(R) Gene Expression Assays (Thermo Fisher Scientific) and are identified by the following IDs of TaqMan(R) Gene Expression Assays. HAS1mRNA GAPDH:Hs02786624_g1 HAS1:Hs00758053_m1 HAS3mRNA GAPDH:Hs02786624_g1 HAS3:Hs00193436_m1 COL1A1mRNA GAPDH:Hs02786624_g1 COL1A1:Hs00164004_m1 COL7A1mRNA GAPDH:Hs02786624_g1 COL7A1:Hs00164310_m1

[0053] As shown in Figures 6 and 7, when Sample 2 was added to the medium at a final concentration of 100 μg / mL, it was revealed that the sample induced significantly higher expression of HAS1 mRNA, HAS3 mRNA, COL1A1 mRNA, and COL7A1 mRNA in human fibroblasts compared to the control. The functions, etc., associated with the measured genes are shown in Table 4 below.

[0054] [Table 4]

[0055] (3) Conclusion Based on the results of the proliferation activity test and gene expression test in human fibroblasts in (1) and (2) above, it can be said that sample 2, i.e., a composition consisting of a high-pressure treated mixture of sturgeon eggs and 100% BG, has proliferation activity in human fibroblasts and also promotes the expression in human fibroblasts of the mRNA for HAS1, a hyaluronic acid synthase abundant in the epidermis, the mRNA for HAS3, a hyaluronic acid synthase abundant in the dermis, the mRNA for COL1A1, a type I collagen that maintains skin firmness, and the mRNA for COL7A1, a type VII collagen that strengthens the skin's binding strength. Therefore, it can be said that the composition consisting of a high-pressure treated mixture of sturgeon eggs and BG is a skin-beautifying composition.

[0056] [2-4] Proliferation activity test in human epidermal keratinocytes, and gene expression test in human epidermal keratinocytes and human hair follicle keratinocytes (1) Proliferation activity test Samples 2, 3, and 5 prepared in [1-1] above were subjected to a proliferation activity test in human epidermal keratinocytes using the same method as in [2-1] above, except that trypsin-EDTA (Fujifilm Wako Pure Chemical Industries, Ltd.) was used to detach and recover the cultured cells. The results are shown in Figure 8. In Figure 8, statistical calculations were performed using the Dunnett test, with *P<0.05 and **P<0.01 indicating a significant difference compared to the control.

[0057] As shown in Fig. 8, significant growth of human epidermal keratinocytes compared to the control was observed in the samples with final concentrations of 10 μg / mL, 100 μg / mL, and 300 μg / mL when Sample 2 was added to the medium, and in the sample with a final concentration of 100 μg / mL when Sample 3 was added to the medium. From these results, it became clear that the composition consisting of the high-pressure-treated product of Sample 2, which is a mixture of shark egg and 100% BG, and the composition consisting of the high-pressure-treated product of Sample 3, which is a mixture of shark egg and 50% BG, have the growth activity of human epidermal keratinocytes.

[0058] (2) Gene expression test Regarding Sample 2, Sample 3, and Sample 5 prepared in [1-1] described above, except that they were cultured without adding LPS, RNA of the measured genes OCLN (Occludin), FLG (Filaggrin), AQP3 (Aquaporin III), TGM1 (Transglutaminase I), SIRT1 (Silent Information Regulator I), and COL17A1 (Collagen Type XVII Alpha 1) was extracted, and the primers used in Real-time PCR were changed according to the measured genes. The gene expression test in human epidermal keratinocytes and human hair follicle keratinocytes was performed by the same method as [2-2](2) described above. The results are shown in Figs. 9 to 12, and the primers used in Real-time PCR are shown below. In Figs. 9 to 12, statistical calculations were performed by the Dunnett test, and *P < 0.05 and **P < 0.01 were considered to have a significant difference compared to the control.

[0059] <Primers used in Real-time PCR> All of them are from TaqMan(R) Gene Expression Assays (Thermo Fisher Scientific) and are identified by the following IDs of TaqMan(R) Gene Expression Assays. OCLN mRNA GAPDH:Hs02786624_g1 OCLN:Hs05465837_g1 FLG mRNA GAPDH:Hs02786624_g1 FLG:Hs00856927_g1 AQP3 mRNA GAPDH:Hs02786624_g1 AQP3:Hs00185020_m1 TGM1 mRNA GAPDH:Hs02786624_g1 TGM1:Hs00165929_m1 SIRT1 mRNA GAPDH:Hs02786624_g1 SIRT1:Hs01009006_m1 COL17A1 mRNA GAPDH:Hs02786624_g1 COL17A1:Hs00990036_m1

[0060] As shown in Figures 9 to 11, when sample 2 was added to the medium at a final concentration of 100 μg / mL, it significantly increased the expression of OCLN mRNA, FLG mRNA, AQP3 mRNA, TGM1 mRNA, and SIRT1 mRNA in human epidermal keratinocytes compared to the control. Furthermore, when sample 3 was added to the medium at a final concentration of 100 μg / mL and when sample 5 was added to the medium at a final concentration of 100 μg / mL, it significantly increased the expression of SIRT1 mRNA in human epidermal keratinocytes compared to the control. Furthermore, as shown in Figure 12, when sample 2 was added to the medium at a final concentration of 100 μg / mL, it significantly increased the expression of COL17A1 mRNA in human hair follicle keratinocytes compared to the control. The functions involved in the measured genes are shown in Tables 5 and 6 below.

[0061] [Table 5]

[0062] [Table 6]

[0063] (3) Conclusion Based on the results of the proliferation activity tests in human epidermal keratinocytes (1) and (2) above, as well as the gene expression tests in human epidermal keratinocytes and human hair follicle keratinocytes, it was determined that Sample 2, i.e., a composition consisting of a high-pressure-treated mixture of sturgeon eggs and 100% BG, has proliferation activity in human epidermal keratinocytes and promotes the expression in human epidermal keratinocytes of the mRNA for OCLN, a tight junction component protein with barrier function; FLG, a stratum corneum component protein with moisturizing function; AQP3, a water channel protein involved in maintaining skin moisture retention and elasticity and improving barrier function; TGM1, an epidermal enzyme that acts on protein cross-linking and increases the strength and moisture retention function of the skin surface; and SIRT1, an NAD-dependent deacetylase with anti-aging properties. Therefore, it can be said that the composition consisting of a high-pressure-treated mixture of sturgeon eggs and BG is a skin-beautifying composition.

[0064] Furthermore, Sample 2, i.e., a composition consisting of a high-pressure-treated mixture of sturgeon eggs and 100% BG, and Sample 3, i.e., a composition consisting of a high-pressure-treated mixture of sturgeon eggs and 50% BG, have the proliferation activity of human epidermal keratinocytes and promote the expression of mRNA for COL17A1, a type XVII collagen that improves the strength of the skin surface and has hair growth functions, in human hair follicle keratinocytes.Therefore, the composition consisting of a high-pressure-treated mixture of sturgeon eggs and BG can be said to be a hair growth composition.

Claims

1. A composition comprising a high-pressure treated mixture of sturgeon roe and 1,3-butylene glycol, the composition comprising one or more compositions selected from the group consisting of the following (a), (b) and (c); (a) an anti-inflammatory composition; (b) Beautiful skin promoting composition, (c) a hair growth composition.

2. The composition according to claim 1 , which is a cosmetic composition.

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