WO2022050318A1 - Use of placenta extract - Google Patents
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- WO2022050318A1 WO2022050318A1 PCT/JP2021/032175 JP2021032175W WO2022050318A1 WO 2022050318 A1 WO2022050318 A1 WO 2022050318A1 JP 2021032175 W JP2021032175 W JP 2021032175W WO 2022050318 A1 WO2022050318 A1 WO 2022050318A1
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- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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Definitions
- the present invention relates to the use of placenta extract.
- placenta mammalian placenta (placenta) and its extracts are attracting attention from a wide range of age groups, regardless of gender, as new materials that promote both health and beauty (see, for example, Patent Document 1).
- placenta is used mainly in the form of extracts in the field of health foods for the purpose of supporting health and in the field of cosmetics for the purpose of supporting beauty, and daily health and skin condition. It has been well received by users in terms of.
- the present invention has been made in view of such circumstances, and provides a new method of using the placenta extract for a clearer purpose.
- a component that suppresses or promotes melanin production in cosmetics or functional foods a hyaluronic acid production promoting component, a collagen production promoting component, an elastase activity inhibiting component, and an antiallergic substance. It was decided to use placenta extract as an additive material for any of the components selected from the activity promoting component and the tyrosinase activity inhibiting component.
- the present invention also has the following features.
- the placenta extract comprises a group of components exhibiting solubility in a non-aqueous specific solvent consisting of a mixture of any one or two or more selected from dimethyl sulfoxide, ethanol, methanol and hexane. matter.
- the placenta extract is composed of a group of components exhibiting solubility in an aqueous solvent containing water as a main component.
- the present invention is selected from a component that suppresses or promotes melanin production in cosmetics or functional foods, a hyaluronic acid production promoting component, a collagen production promoting component, an elastase activity inhibiting component, an antiallergic activity promoting component, and a tyrosinase activity inhibiting component. Since the placenta extract is used as an additive material for any of the above components, it is possible to provide a new method of using the placenta extract for a clearer purpose.
- the placenta extract comprises a group of components exhibiting solubility in a non-aqueous specific solvent consisting of a mixture of any one or two or more selected from dimethyl sulfoxide, ethanol, methanol and hexane. If so, it can exhibit an action of promoting melanin production, and also exhibits a more remarkable effect of inhibiting elastase activity than the elastase activity-inhibiting component contained in the component group exhibiting solubility in an aqueous solvent. Can be done.
- the placenta extract is composed of a group of components exhibiting solubility in an aqueous solvent containing water as a main component, it has an action of suppressing melanin production and a solubility in a non-aqueous specific solvent. It is possible to exhibit more remarkable actions as compared with the hyaluronic acid production promoting component and the collagen production promoting component contained in the indicated component group.
- the present invention relates to a method of using the placenta extract, and more specifically, in health foods and cosmetics which have been used in some cases without necessarily clarifying the significance of the addition of the placenta extract. It provides a new usage of placenta extract for various purposes.
- a component that suppresses or promotes melanin production in cosmetics or functional foods a hyaluronic acid production promoting component, a collagen production promoting component, an elastase activity inhibiting component, an antiallergic activity promoting component, and a tyrosinase activity. It is characteristic in that placenta extract is used as an additive material for any of the components selected from the inhibitory components.
- the placenta extract is an extract of the placenta (placenta) of mammals.
- placenta placenta
- the placenta extract contains the extract or the dried product of the extract.
- placenta from which the placenta extract is extracted is not particularly limited, and placenta of various mammals including pigs, horses, and humans can be adopted.
- cosmetics include basic cosmetics, hair tonics, perfumes, toothpastes, shampoos, conditioners, soaps and cleaning agents used for body cleaning, and toiletry products such as bath salts. It also includes medicated cosmetics that claim a preventive effect.
- makeup cosmetics include foundations, eyebrows, mascara, eye shadows, eyelines, lipsticks, glosses, blushers, white powders, and manicures.
- Basic cosmetics include, for example, lotions and milky lotions. Examples include facial cleansers, cleansers, serums and creams.
- functional food is a concept that includes all foods that do not contain pharmaceutical ingredients and are thought to contribute to the maintenance and promotion of health. It also includes foods, foods for specified health use, foods with nutritional function, and foods with health claims such as foods with functional claims.
- the placenta extract is selected from a component that suppresses or promotes melanin production, a hyaluronic acid production promoting component, a collagen production promoting component, an elastase activity inhibiting component, an antiallergic activity promoting component, and a tyrosinase activity inhibiting component. It is used as an additive material for the ingredients.
- the use as an additive material means that the placenta extract is added to cosmetics and functional foods so as to contain each of the above components, and the placenta extract is used as an extraction material for each of the above components. That is also included. In the latter case, each extracted component will be added to cosmetics and functional foods.
- placenta extract is made from any of the components selected from a component that suppresses or promotes melanin production, a hyaluronic acid production promoting component, a collagen production promoting component, an elastase activity inhibiting component, an antiallergic activity promoting component, and a tyrosinase activity inhibiting component.
- the purpose is to be an additive material.
- placenta extract itself exerts its function as these components.
- the action may be substantially exerted by a single substance contained in the placenta extract.
- the action may be exerted by a plurality of substances.
- ingredients that suppress or promote melanin production are used externally in the form of cosmetics and / or orally in the form of functional foods (hereinafter referred to as external / oral use modes) in the skin. It suffices if the production of melanin can be suppressed or promoted. Such components can be confirmed by a melanin production test described later.
- the hyaluronic acid production promoting component and the collagen production promoting component need only be able to promote the production of hyaluronic acid and collagen when used in an external or oral use mode, and can be confirmed by, for example, a collagen or hyaluronic acid production test described later.
- the elastase activity-inhibiting component only needs to be able to inhibit the activity of elastase and decompose elastin when used in an external or oral use mode, and can be confirmed by, for example, an elastase activity-inhibiting test described later.
- the anti-allergic activity promoting component may be used as long as it can suppress allergic symptoms when used externally or orally. For example, it is confirmed by measuring ⁇ -hexosaminidase activity as in the anti-allergic activity test described later. be able to.
- the tyrosinase activity-inhibiting component may inhibit the activity of tyrosinase and prevent the colonization of melanin when used in an external or oral use mode, and can be confirmed by, for example, a tyrosinase inhibitory activity test described later.
- the placenta extract may also consist of a group of components exhibiting solubility in a non-aqueous specific solvent consisting of a mixture of any one or two or more selected from dimethyl sulfoxide, ethanol, methanol and hexane. good.
- the placenta extract can be an extract extracted by a non-aqueous specific solvent.
- the placenta extract consisting of a group of components exhibiting solubility in a non-aqueous specific solvent as each of the above-mentioned components, it is possible to exhibit an action of promoting melanin production, and also in an aqueous solvent. It is possible to exhibit a more remarkable elastase activity inhibitory action as compared with the elastase activity inhibitory component contained in the component group showing solubility.
- the placenta extract may consist of a group of components exhibiting solubility in an aqueous solvent containing water as a main component.
- the placenta extract can be an extract extracted with an aqueous solvent.
- the aqueous solvent is a solvent containing water as a main component, and in addition to water itself, for example, a solvent containing water at a ratio of 50 w / w% or more is included.
- the components other than water constituting the aqueous solvent may be, for example, a solid component (powder or the like) that is soluble in water, or a liquid that is miscible with water such as alcohol.
- the placenta extract consisting of the component group exhibiting solubility in an aqueous solvent as each of the above-mentioned components, the action of suppressing melanin production and the solubility in a non-aqueous specific solvent are exhibited. It can exhibit more remarkable actions as compared with the hyaluronic acid production promoting component and the collagen production promoting component contained in the component group.
- the solvent may be a mixed solvent of an aqueous solvent and a non-aqueous specific solvent. That is, the above-mentioned placenta extraction is performed by using a mixed solvent containing water at a ratio of less than 50 w / w% and using a non-aqueous specific solvent or a solid (powder, etc.) that is soluble in the mixed solvent to be prepared. It is possible to prepare a placenta extract having a composition ratio different from that of the product.
- the present application also provides a method for using the component found from the placenta extract by the present invention as an active ingredient in cosmetics or a health functional ingredient in functional foods for the purpose of promoting the production of melanin. be.
- melanin can be obtained from the above-mentioned components. It is possible to provide new cosmetics and functional foods that promote or suppress the production of melanin.
- placenta extract a placenta extract extracted from horse-derived placenta (placenta)
- placenta horse-derived placenta
- placenta extract In recent years, various natural ingredients have been used as functional materials to be blended in cosmetics and functional foods. Among them, placenta, which is the placenta of mammals, is attracting attention as a material that contributes to beauty and health maintenance.
- placenta extract was first prepared in order to confirm the effectiveness by conducting several functional evaluation tests with horse placenta as a representative example.
- Horse placenta powder was used as an extraction raw material for horse placenta extract.
- Horse placenta powder is a lyophilized powder of horse placenta.
- DMSO dimethyl sulfoxide
- the tube was allowed to stand at room temperature, and the obtained supernatant was used as a horse placenta extract.
- This horse placenta extract was prepared by adding 1 ml of an extraction solvent to 100 mg of placenta powder, and was referred to as a 100 mg / ml sample.
- the diluted sample prepared by appropriately diluting the 100 mg / ml sample is, for example, 1 mg / ml sample for a 100-fold diluted sample and 10 ⁇ g / ml for a 10000-fold diluted sample. Express like a sample.
- Melanin production / suppression ability confirmation test (1st test) Melanin is a general term for substances in which phenolic substances are polymerized into pigments. Melanin present in human skin is in the form of polymers formed by various indole compounds synthesized from tyrosine.
- melanin The most important role of melanin is UV protection, which suppresses sunlight damage and the development of malignant tumors caused by UV rays.
- Other known functions of melanin include absorption of reactive oxygen species that are liable to the living body and uptake of metals and drugs.
- Cells containing melanin gradually dissociate into the upper stratum corneum, and the adhesion between cells is decomposed by the action of lipid-metabolizing enzymes (lipase, etc.) and proteolytic enzymes (elastase, etc.), and finally the stratum corneum is exfoliated and shed.
- lipid-metabolizing enzymes lipase, etc.
- proteolytic enzymes elastase, etc.
- human melanoma cells (G361 cell line) were used as model cells, and cells and samples were co-cultured, and melanin produced in the culture medium and cell viability were measured.
- human melanoma cells (G361 cell line) were precultured using McCoy's 5A medium (including 1% penicillin-streptomycin and 10% fetal bovine serum (FBS)).
- McCoy's 5A medium including 1% penicillin-streptomycin and 10% fetal bovine serum (FBS)
- 0.5 ml was seeded on a 24-well plate at a concentration of 1.0 ⁇ 10 5 cells / ml and cultured overnight in a CO 2 incubator (37 ° C, 5% CO 2 ).
- each sample of 2.5 ⁇ l of placenta extract diluted to a predetermined concentration (hereinafter also referred to as each diluted sample) are added, and the mixture is returned to the CO 2 incubator.
- the cells were cultured for 72 hours.
- the ability to produce and suppress melanin was confirmed by confirming the amount of melanin produced.
- the medium was removed from the plate that had been cultured for 72 hours, each well was washed with PBS (Phosphate Buffered Saline; phosphate buffered saline), and then 1 ml of 1 M NaOH solution was added to each well.
- the reaction was carried out at room temperature for 4 hours, and the absorbance at 405 nm was measured with a plate reader to determine the amount of melanin produced.
- the cell viability was measured by the MTT method. Remove medium from plates that have been cultured for 72 hours and add 1 ml of fresh medium to each well. Then, 200 ⁇ l of MTT stain (5 mg / ml in PBS) was added to each well, and the cells were statically cultured in a CO 2 incubator for 4 hours.
- FIG. 1 is a graph showing the melanin production ratio with respect to the control
- FIG. 1 (a) shows the melanin production ratio by the placenta extract (hereinafter, also referred to as placenta water-based extract) extracted with an aqueous solvent
- FIG. (B) shows the melanin production ratio by the placenta extract (hereinafter referred to as placenta non-aqueous extract) extracted with a non-aqueous specific solvent.
- the placenta aqueous extract contains a component that suppresses melanin production, in other words, a component that contributes to skin whitening.
- the placenta non-aqueous extract contains a component that promotes melanin production and has an effect of maintaining the black color of hair.
- the placenta water-based extract and the placenta non-water-based extract have the opposite properties of "suppression” and “promotion” in the production of melanin, respectively. It can be said that it is a really interesting point that became clear.
- Collagen is a structural protein that forms connective tissues such as animal skin. Collagen contributes to the maintenance of the structure of the skin and is important for keeping the skin youthful and preventing wrinkles.
- Collagen is produced by fibroblasts present in the dermis of the skin, and if the fibroblasts can promote collagen production, youthful skin can be maintained. Therefore, the collagen production promoting component of fibroblasts is considered to be useful as an anti-aging cosmetic material.
- Hyaluronic acid is mainly produced by fibroblasts, which are the main cells of the dermis of the skin, and cells of other connective tissues. It is the main component of the extracellular matrix and is involved in various cell-cell interactions. In addition, it is present in synovial fluid at a high concentration and is responsible for normal water retention and lubrication of joints.
- FIG. 2 is a graph showing the hyaluronic acid production ratio with respect to the control
- FIG. 2 (a) shows the hyaluronic acid production ratio by the placenta aqueous extract
- FIG. 2 (b) shows the hyaluronic acid production ratio by the placenta non-aqueous extract. Is shown.
- FIG. 3 is a graph showing the collagen production ratio to the control
- FIG. 3 (a) shows the collagen production ratio by the placenta aqueous extract
- FIG. 3 (b) shows the collagen production ratio by the placenta non-aqueous extract.
- both the aqueous and non-aqueous placenta extracts contained a component that promotes collagen production.
- the placenta aqueous extract contains a component that promotes hyaluronic acid and collagen production
- the placenta non-aqueous extract contains a component that promotes collagen production. rice field.
- Elastase activity inhibition test The cross-linked structure of collagen and elastin present in the dermis plays an important role in maintaining the elasticity of the skin. Elastase, which is an elastin-degrading enzyme, is induced by stimulation with ultraviolet rays or the like, and when elastin is decomposed, the crosslinked structure becomes brittle and the elasticity of the skin is lost. In this test, the skin elasticity retention function of placenta extract is evaluated by measuring the elastase inhibitory activity.
- cytolytic solution was used in the experiment.
- human skin fibroblasts (NHDF-Ad) precultured until they became confluent were washed with PBS and resuspended in 1.0 ⁇ 10 6 cells / ml with the cytolytic solution. Then, sonication was performed, and the mixture was centrifuged at 4 ° C. and 13,000 rpm for 15 minutes, and the supernatant was collected as a cytolytic solution.
- the absorbance at 405 nm was measured with a microplate reader, and the elastase activity was calculated.
- phosphoramidone final concentration 0.04 mM
- elastase inhibitor was used as a positive control.
- FIG. 4 is a graph showing the elastase activity ratio with respect to the control
- FIG. 4 (a) shows the elastase activity ratio with the placenta aqueous extract
- FIG. 4 (b) shows the elastase activity ratio with the placenta non-aqueous extract.
- aqueous and non-aqueous placenta extracts contained a component that inhibits elastase activity.
- the placenta aqueous extract is probably used at a concentration of 100 ⁇ g / ml or more, and based on the result shown in FIG. 4 (a), it is used at a concentration of 400 ⁇ g / ml or more, and the placenta non-aqueous extract is used. Therefore, it is expected that the use of the placenta extract at a concentration of 40 ⁇ g / ml or more, more effectively at a concentration of 400 ⁇ g / ml or more, can contribute to the maintenance of skin elasticity using the placenta extract as an active ingredient for inhibiting elastase activity. ..
- rat basophil leukemia cells (RBL-2H3) were used as basophil model cells, and the granule release inhibitory activity (anti-allergic activity) by the placenta extract was evaluated.
- the granules contain ⁇ -hexosaminidase, and the amount of enzyme released from the granules is measured by measuring the amount of enzyme in the cell supernatant, and the amount of enzyme is the enzyme in the cell supernatant. It was measured by the intensity of activity.
- rat basophil leukemia cells (RBL-2H3) were prepared at a concentration of 5 ⁇ 10 5 cells / ml, and 100 ⁇ l was seeded on a 96-well plate. Two such plates, one for measuring ⁇ -hexosaminidase activity and one for MTT test, were prepared and precultured in a CO 2 incubator (37 ° C, 5% CO 2 ) for 40 hours.
- p-Nitrophenyl-N-acetyl- ⁇ -glucosaminide p-Nitrophenyl-N-acetyl-beta-
- 50 ⁇ l of the supernatant was separated.
- 50 ⁇ l of a glucosamine) solution was added as a substrate, and the mixture was reacted at room temperature for 1 to 3 hours after being shielded from light with an aluminum foil.
- 100 ⁇ l of the reaction terminator was added to all the wells to stop the enzyme reaction, and the absorbance at 405 nm was measured with a plate reader to measure the ⁇ -hexosaminidase activity.
- MTT stain 5 mg / ml in medium
- 100 ⁇ l of MTT stain 5 mg / ml in medium
- the cells were statically cultured at 37 ° C. for 4 hours.
- the medium was removed, 1 ml of hydrochloric acid-isopropanol solution was added to each well, the light was shielded, and the mixture was left at room temperature for 4 hours.
- the absorbance at 570 nm was measured with a microplate reader and used as an index of cell viability.
- FIG. 5 is a graph showing the ⁇ -hexosaminidase activity ratio to the control. As can be seen from FIG. 5, the ⁇ -hexosaminidase activity decreased in both the aqueous and non-aqueous placenta extracts when the 400 ⁇ g / ml sample, which was in the high concentration range, was added.
- the placenta extract according to this embodiment contains a component showing antiallergic activity.
- Tyrosinase inhibitory activity test The melanin pigment that causes age spots and freckles on the skin is produced by the action of an enzyme called tyrosinase in melanocytes. Tyrosinase is activated by ultraviolet rays to produce melanin, and it is known that inhibition of the action of this enzyme prevents the fixation of melanin and obtains a whitening effect.
- the test method includes 33.3 ⁇ l of each diluted sample, 333 ⁇ l of 2.5 mM substrate (L-tyrosine or L-DOPA), 600 ⁇ l of 0.1 M phosphate buffer (pH 6.8), and 33 ⁇ l of tyrosinase (1380 unit / ml). ) was mixed, and immediately after that, the change over time in the absorbance at 475 nm was recorded by a spectrophotometer. The absorbance at 475 nm was attributed to the formation of dopachrome after the enzyme-substrate reaction, and the increase in absorbance per hour was calculated to determine the enzyme activity value (L-tyrosine: 10 minutes measurement, L-DOPA: 3 minutes). measurement).
- the relative tyrosinase activity at the time of adding each diluted sample was calculated with the tyrosinase activity value at the time of adding the control (solvent) as 100%.
- Kojic acid final concentration: 17 ⁇ g / ml was used as a positive control.
- FIG. 6A is a graph showing the effect of the aqueous or non-aqueous placenta extract of the tyrosinase activity rate using L-DOPA as a substrate
- FIG. 6B is a graph showing the effect of the tyrosinase activity rate using L-tyrosine as a substrate. It is a graph which showed the influence of the water-based or non-water-based placenta extract of.
- the aqueous or non-aqueous placenta extract according to the present embodiment contains a component showing tyrosinase inhibitory activity, and this effect tends to be more remarkable in the placenta non-aqueous placenta extract. It was suggested that it was in.
- the components isolated by the present inventors from the placenta non-aqueous extract are as follows.
- HS-6 Represented by 3 ⁇ , 6 ⁇ -dihydroxycholest-5-ene (3 ⁇ , 6 ⁇ -dihydroxycholest-5-ene).
- test method is the same as in the first test described above, but the sample to be added is not a placenta extract, but is added to the culture solution after dissolving in a dimethyl sulfoxide solution so that each of these components has a final concentration of 100 ⁇ M. did.
- FIG. 7 (a) is a graph showing the cell viability in each sample
- FIG. 7 (b) is the extracellular melanin production ratio per cell to the control
- FIG. 7 (c) is the intracellular melanin production per cell to the control. It is a graph which shows the ratio.
- Table 1 shows a table of extracellular / intracellular melanin production ratios per cell, including cell viability. In the "type” shown in Table 1, "A + " indicates that the amount of melanin produced increases at a rate of more than 20% with respect to the control, and "A-" indicates that the amount of melanin produced increases by 20% or less with respect to the control.
- HS-3 (3 ⁇ -hydroxycholesta-4-ene-6-on), HS-7 (cholesta-3,5-diene-7-on), HS-10 (palmitic acid) and HSE-2. It was confirmed that (thymine) significantly increased cell viability compared to control (DMSO) (HS-3: 109.4%, HS-7: 113.3%, HS-10: 110.3%, HSE- 2: 114.3%). Based on these results, it can be confirmed that HS-3, HS-6, HS-7, HS-8, HS-10, HSE-2 and HAS-1 do not show cytotoxicity to B16 melanoma cells. rice field.
- the intracellular melanin production ratio per cell is controlled by the addition of HS-3, HS-4, HS-8, HS-10, HSE-2 and HAS-1 (DMSO).
- HS-3, HS-4, HS-8, HS-10, HSE-2 and HAS-1 DMSO
- HSE-2 HSE-2
- HAS-1 HAS-1
- the addition of HS-1, HS-2, HS-6, HS-7 and HAS-2 increased the extracellular melanin production ratio per cell as compared with control (DMSO).
- HS-1 cholesterol
- HS-2 cholesterol-7-on
- HS-3 (3 ⁇ -hydroxycholesta-4-en-6-one) showed a decrease in melanin production at a rate of more than 20% relative to the control both intracellularly and extracellularly. In particular, extracellularly, a decrease in melanin production equivalent to that of arbutin (100 ⁇ M) was observed.
- HS-4 (5 ⁇ , 6 ⁇ -epoxy-reductase-3 ⁇ -ol) showed a decrease in melanin production both intracellularly and extracellularly, albeit at a rate of 20% or less with respect to the control.
- HS-6 (3 ⁇ , 6 ⁇ -dihydroxycholesta-5-ene) showed a decrease in melanin production at a rate of more than 20% relative to the control extracellularly, especially to the same extent as arbutin (100 ⁇ M). On the contrary, in the cells, an increase in melanin production was observed, albeit at a rate of 20% or less with respect to the control.
- HS-7 (Cholesta-3,5-diene-7-on) showed an increase in melanin production of more than 20% relative to the control both intracellularly and extracellularly.
- HS-8 cholesterol esterate
- HS-10 palmitic acid showed a decrease in melanin production at a rate of more than 20% relative to the control, both intracellularly and extracellularly.
- HSE-2 thymine
- HAS-1 uridine showed a decrease in melanin production both intracellularly and extracellularly, both intracellularly and extracellularly, at a rate of 20% or less with respect to the control.
- HAS-2 uridine monophosphate
- cholesterol (HS-1), cholesterol-7-one (HS-2), 3 ⁇ , 6 ⁇ -dihydroxycholesta-5-ene (HS-6), cholesterol-3 , 5-Diene-7-one (HS-7), uridine monophosphate (HAS-2) components have been shown to promote melanin production compared to control (DMSO). More specifically, the components of cholesterol (HS-1), cholesterol-7-on (HS-2), and cholesterol-3,5-diene-7-on (HS-7) are both intracellular and extracellular. In addition, it was shown that each component of 3 ⁇ , 6 ⁇ -dihydroxycholester-5-ene (HS-6) and uridine monophosphate (HAS-2) promotes the production of melanin in the cell. This can be said to indicate that these ingredients are useful as active ingredients and health functional ingredients for the purpose of promoting the production of melanin in cosmetics and functional foods.
- 3 ⁇ -hydroxycholesta-4-ene-6-one (HS-3), 5 ⁇ , 6 ⁇ -epoxycholestane-3 ⁇ -ol (HS-4), 3 ⁇ , 6 ⁇ -dihydroxycholesta-5- En (HS-6), cholesteryl oleate (HS-8), palmitic acid (HS-10), thymine (HSE-2), uridine (HAS-1), uridine monophosphate (HAS-2) are melanin It was shown to suppress the production of.
- 3 ⁇ -hydroxycholesta-4-en-6-one (HS-3), 5 ⁇ , 6 ⁇ -epoxycholestane-3 ⁇ -ol (HS-4), cholesteryl oleart (HS-8),
- the components of palmitic acid (HS-10), timine (HSE-2), and uridine (HAS-1) can suppress the production of melanin both intracellularly and extracellularly, especially HS-3, HS-8, and HS.
- HSE-2, HAS-1 significantly suppress the production of extracellular melanin
- 3 ⁇ , 6 ⁇ -dihydroxycholesta-5-ene (HS-6), uridine monophosphate (HAS-2).
- Each component of is shown to suppress the production of melanin extracellularly. This can be said to indicate that these ingredients are useful as active ingredients and health functional ingredients for the purpose of suppressing the production of melanin and, by extension, whitening in cosmetics and functional foods.
- HS-6 6 ⁇ -dihydroxycholesta-5-ene
- HAS-2 uridine monophosphate
- a component that suppresses or promotes melanin production in cosmetics or functional foods a hyaluronic acid production promoting component, a collagen production promoting component, an elastase activity inhibiting component, and an antiallergic substance. Since the placenta extract is used as an additive material for any of the components selected from the activity promoting component and the tyrosinase activity inhibiting component, it is necessary to provide a new method of using the placenta extract for a clearer purpose. Can be done.
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Abstract
To provide a new method for using placenta extract for a clearer purpose than in the past. The use of placenta extract as an additive material for any component selected from components that suppress or promote melanin production, components that promote hyaluronic acid production, components that promote collagen production, components that inhibit elastase activity, components that promote anti-allergy activity, and components that inhibit tyrosinase activity in a cosmetic or a functional food. The placenta extract also has the characteristic of being composed of a component group that exhibits solubility in a nonaqueous specific solvent or being composed of a component group that exhibits solubility in an aqueous solvent having water as the main component.
Description
本発明は、プラセンタ抽出物の使用に関する。
The present invention relates to the use of placenta extract.
健康や美容は、時代にかかわらず多くの人々の関心事である。それゆえ、健康に良いものや美容に良いものとして、これまでに様々な素材が提案されている。
Health and beauty are of concern to many people regardless of age. Therefore, various materials have been proposed so far as good for health and good for beauty.
中でも、哺乳類の胎盤(プラセンタ)やその抽出物は、健康と美容の両側面において増進をもたらす新たな素材として、男女問わず幅広い年代層から注目されている(例えば、特許文献1参照。)。
Among them, mammalian placenta (placenta) and its extracts are attracting attention from a wide range of age groups, regardless of gender, as new materials that promote both health and beauty (see, for example, Patent Document 1).
上述のようにプラセンタは、健康のサポートを目的として健康食品に、また、美容のサポートを図るべく化粧料分野において、主に抽出物の状態でそれぞれ利用されており、日々の健康や肌の調子の面において使用者に好評を得ている。
As mentioned above, placenta is used mainly in the form of extracts in the field of health foods for the purpose of supporting health and in the field of cosmetics for the purpose of supporting beauty, and daily health and skin condition. It has been well received by users in terms of.
しかしながら、これまで提案されていた健康食品や化粧料は、プラセンタの配合の意義が必ずしも明確にされることなく使用される場合があった。
However, the health foods and cosmetics that have been proposed so far may be used without necessarily clarifying the significance of the placenta formulation.
本発明は、斯かる事情に鑑みてなされたものであって、より明確な目的の下でのプラセンタ抽出物の新たな使用方法を提供する。
The present invention has been made in view of such circumstances, and provides a new method of using the placenta extract for a clearer purpose.
上記従来の課題を解決するために本発明では、(1)化粧料又は機能性食品におけるメラニン生成を抑制又は促進する成分、ヒアルロン酸産生促進成分、コラーゲン産生促進成分、エラスターゼ活性阻害成分、抗アレルギー活性促進成分、チロシナーゼ活性阻害成分から選ばれるいずれかの成分の添加材料として、プラセンタ抽出物を使用することとした。
In order to solve the above-mentioned conventional problems, in the present invention, (1) a component that suppresses or promotes melanin production in cosmetics or functional foods, a hyaluronic acid production promoting component, a collagen production promoting component, an elastase activity inhibiting component, and an antiallergic substance. It was decided to use placenta extract as an additive material for any of the components selected from the activity promoting component and the tyrosinase activity inhibiting component.
また本発明では、以下の点にも特徴を有する。
(2)前記プラセンタ抽出物は、ジメチルスルホキシド、エタノール、メタノール、ヘキサンから選ばれるいずれか1つ若しくはいずれか2つ以上の混合液からなる非水系特定溶媒に対して溶解性を示す成分群よりなること。
(3)前記プラセンタ抽出物は、水を主成分とする水系溶媒に対して溶解性を示す成分群よりなること。 The present invention also has the following features.
(2) The placenta extract comprises a group of components exhibiting solubility in a non-aqueous specific solvent consisting of a mixture of any one or two or more selected from dimethyl sulfoxide, ethanol, methanol and hexane. matter.
(3) The placenta extract is composed of a group of components exhibiting solubility in an aqueous solvent containing water as a main component.
(2)前記プラセンタ抽出物は、ジメチルスルホキシド、エタノール、メタノール、ヘキサンから選ばれるいずれか1つ若しくはいずれか2つ以上の混合液からなる非水系特定溶媒に対して溶解性を示す成分群よりなること。
(3)前記プラセンタ抽出物は、水を主成分とする水系溶媒に対して溶解性を示す成分群よりなること。 The present invention also has the following features.
(2) The placenta extract comprises a group of components exhibiting solubility in a non-aqueous specific solvent consisting of a mixture of any one or two or more selected from dimethyl sulfoxide, ethanol, methanol and hexane. matter.
(3) The placenta extract is composed of a group of components exhibiting solubility in an aqueous solvent containing water as a main component.
また本発明では、(4)下記一般式(i):
で表されるコレステロール(Cholesterol)、又は下記一般式(ii):
で表されるコレステロール-7-オン(Cholesterol-7-one)、又は下記一般式(iii):
で表されるコレスタ-3,5-ジエン-7-オン(Cholesta-3,5-diene-7-one)、又は下記一般式(iv):
で表されるウリジン一リン酸(Uridine Monophosphate)を、化粧料中又は機能性食品中におけるメラニンの生成促進を目的とした有効成分又は保健機能成分として使用することとした。
Further, in the present invention, (4) the following general formula (i):
Cholesterol represented by, or the following general formula (ii):
Cholesterol-7-one represented by, or the following general formula (iii):
Cholesta-3,5-diene-7-one represented by, or the following general formula (iv):
It was decided to use Uridine Monophosphate represented by (1) as an active ingredient or a health functional ingredient for the purpose of promoting the production of melanin in cosmetics or functional foods.
また本発明では、(5)下記一般式(iv):
で表されるウリジン一リン酸(Uridine Monophosphate)、又は下記一般式(v):
で表される3β-ヒドロキシコレスタ-4-エン-6-オン(3β-hydroxycholest-4-ene-6-one)、又は下記一般式(vi):
で表される3β,6β-ジヒドロキシコレスタ-5-エン(3β,6β-dihydroxycholest-5-ene)、又は下記一般式(vii):
で表されるコレステリルオレアート(Cholestereyl oleate)、又は下記一般式(viii):
で表されるパルミチン酸(Palmitic acid)、又は下記一般式(ix):
で表されるチミン(Thymine)を、化粧料中又は機能性食品中におけるメラニンの生成抑制を目的とした有効成分又は保健機能成分として使用することとした。
Further, in the present invention, (5) the following general formula (iv):
Uridine monophosphate represented by, or the following general formula (v):
3β-hydroxycholest-4-ene-6-one represented by 3β-hydroxycholest-4-ene-6-one, or the following general formula (vi):
3β, 6β-dihydroxycholest-5-ene (3β, 6β-dihydroxycholest-5-ene) represented by, or the following general formula (vii):
Cholestereyl oleate represented by, or the following general formula (viii):
Palmitic acid represented by, or the following general formula (ix):
It was decided to use thymine represented by (3) as an active ingredient or a health functional ingredient for the purpose of suppressing the production of melanin in cosmetics or functional foods.
本発明によれば、化粧料又は機能性食品におけるメラニン生成を抑制又は促進する成分、ヒアルロン酸産生促進成分、コラーゲン産生促進成分、エラスターゼ活性阻害成分、抗アレルギー活性促進成分、チロシナーゼ活性阻害成分から選ばれるいずれかの成分の添加材料として、プラセンタ抽出物を使用することとしたため、より明確な目的の下でのプラセンタ抽出物の新たな使用方法を提供することができる。
According to the present invention, it is selected from a component that suppresses or promotes melanin production in cosmetics or functional foods, a hyaluronic acid production promoting component, a collagen production promoting component, an elastase activity inhibiting component, an antiallergic activity promoting component, and a tyrosinase activity inhibiting component. Since the placenta extract is used as an additive material for any of the above components, it is possible to provide a new method of using the placenta extract for a clearer purpose.
また、前記プラセンタ抽出物は、ジメチルスルホキシド、エタノール、メタノール、ヘキサンから選ばれるいずれか1つ若しくはいずれか2つ以上の混合液からなる非水系特定溶媒に対して溶解性を示す成分群よりなることとすれば、メラニン生成を促進する作用を呈することができ、また、水性溶媒に対して溶解性を示す成分群に含まれるエラスターゼ活性阻害成分に比してより顕著なエラスターゼ活性阻害作用を呈することができる。
Further, the placenta extract comprises a group of components exhibiting solubility in a non-aqueous specific solvent consisting of a mixture of any one or two or more selected from dimethyl sulfoxide, ethanol, methanol and hexane. If so, it can exhibit an action of promoting melanin production, and also exhibits a more remarkable effect of inhibiting elastase activity than the elastase activity-inhibiting component contained in the component group exhibiting solubility in an aqueous solvent. Can be done.
また、前記プラセンタ抽出物は、水を主成分とする水系溶媒に対して溶解性を示す成分群よりなることとすれば、メラニン生成を抑制する作用や、非水系特定溶媒に対して溶解性を示す成分群に含まれるヒアルロン酸産生促進成分やコラーゲン産生促進成分に比してより顕著な各作用を呈することができる。
Further, if the placenta extract is composed of a group of components exhibiting solubility in an aqueous solvent containing water as a main component, it has an action of suppressing melanin production and a solubility in a non-aqueous specific solvent. It is possible to exhibit more remarkable actions as compared with the hyaluronic acid production promoting component and the collagen production promoting component contained in the indicated component group.
また、下記一般式(i):
で表されるコレステロール(Cholesterol)、又は下記一般式(ii):
で表されるコレステロール-7-オン(Cholesterol-7-one)、又は下記一般式(iii):
で表されるコレスタ-3,5-ジエン-7-オン(Cholesta-3,5-diene-7-one)、又は下記一般式(iv):
で表されるウリジン一リン酸(Uridine Monophosphate)を、化粧料中又は機能性食品中におけるメラニンの生成促進を目的とした有効成分又は保健機能成分として使用すれば、上述の成分によりメラニンの生成を促進する新たな化粧料や機能性食品を提供することができる。
In addition, the following general formula (i):
Cholesterol represented by, or the following general formula (ii):
Cholesterol-7-one represented by, or the following general formula (iii):
Cholesta-3,5-diene-7-one represented by, or the following general formula (iv):
When uridine monophosphate represented by (1) is used as an active ingredient or a health functional ingredient for the purpose of promoting the production of melanin in cosmetics or functional foods, the above-mentioned ingredients produce melanin. It is possible to provide new cosmetics and functional foods that promote.
また、下記一般式(iv):
で表されるウリジン一リン酸(Uridine Monophosphate)、又は下記一般式(v):
で表される3β-ヒドロキシコレスタ-4-エン-6-オン(3β-hydroxycholest-4-ene-6-one)、又は下記一般式(vi):
で表される3β,6β-ジヒドロキシコレスタ-5-エン(3β,6β-dihydroxycholest-5-ene)、又は下記一般式(vii):
で表されるコレステリルオレアート(Cholestereyl oleate)、又は下記一般式(viii):
で表されるパルミチン酸(Palmitic acid)、又は下記一般式(ix):
で表されるチミン(Thymine)を、化粧料中又は機能性食品中におけるメラニンの生成抑制を目的とした有効成分又は保健機能成分として使用すれば、上述の成分によりメラニンの生成を抑制する新たな化粧料や機能性食品を提供することができる。
In addition, the following general formula (iv):
Uridine monophosphate represented by, or the following general formula (v):
3β-hydroxycholest-4-ene-6-one represented by 3β-hydroxycholest-4-ene-6-one, or the following general formula (vi):
3β, 6β-dihydroxycholest-5-ene (3β, 6β-dihydroxycholest-5-ene) represented by, or the following general formula (vii):
Cholestereyl oleate represented by, or the following general formula (viii):
Palmitic acid represented by, or the following general formula (ix):
When thymine represented by (3) is used as an active ingredient or a health functional ingredient for the purpose of suppressing the production of melanin in cosmetics or functional foods, a new ingredient that suppresses the production of melanin by the above-mentioned ingredients is used. It is possible to provide cosmetics and functional foods.
本発明は、プラセンタ抽出物の使用方法に関し、より詳細には、これまでプラセンタ抽出物の添加の意義が必ずしも明確にされることなく使用されることもあった健康食品や化粧料において、より明確な目的の下でのプラセンタ抽出物の新たな使用方法を提供するものである。
The present invention relates to a method of using the placenta extract, and more specifically, in health foods and cosmetics which have been used in some cases without necessarily clarifying the significance of the addition of the placenta extract. It provides a new usage of placenta extract for various purposes.
特に、本実施形態に係る使用では、化粧料又は機能性食品におけるメラニン生成を抑制又は促進する成分、ヒアルロン酸産生促進成分、コラーゲン産生促進成分、エラスターゼ活性阻害成分、抗アレルギー活性促進成分、チロシナーゼ活性阻害成分から選ばれるいずれかの成分の添加材料として、プラセンタ抽出物を使用する点において特徴的である。
In particular, in the use according to the present embodiment, a component that suppresses or promotes melanin production in cosmetics or functional foods, a hyaluronic acid production promoting component, a collagen production promoting component, an elastase activity inhibiting component, an antiallergic activity promoting component, and a tyrosinase activity. It is characteristic in that placenta extract is used as an additive material for any of the components selected from the inhibitory components.
プラセンタ抽出物は、哺乳類の胎盤(プラセンタ)の抽出物であり、例えば溶媒を用いて抽出した場合は、その抽出液であったり、抽出液の乾燥物がプラセンタ抽出物に含まれる。
The placenta extract is an extract of the placenta (placenta) of mammals. For example, when it is extracted using a solvent, the placenta extract contains the extract or the dried product of the extract.
プラセンタ抽出物の抽出元であるプラセンタは、特に限定されるものではなく、ブタ、ウマ、ヒトをはじめ種々の哺乳類のプラセンタを採用することができる。
The placenta from which the placenta extract is extracted is not particularly limited, and placenta of various mammals including pigs, horses, and humans can be adopted.
化粧料は、いわゆるメーキャップ化粧品の他、基礎化粧品やヘアトニック、香水、歯磨き、シャンプー、リンス、身体の洗浄等に用いられる石鹸や洗浄料、入浴剤などのトイレタリー製品も含むものであり、また、予防効果等を謳う、薬用化粧品も含まれる。
In addition to so-called makeup cosmetics, cosmetics include basic cosmetics, hair tonics, perfumes, toothpastes, shampoos, conditioners, soaps and cleaning agents used for body cleaning, and toiletry products such as bath salts. It also includes medicated cosmetics that claim a preventive effect.
また、メーキャップ化粧品としては、ファンデーションや眉墨(アイブロー)、マスカラ、アイシャドー、アイライン、口紅、グロス、頬紅(チーク)、白粉、マニキュアなどが挙げられ、基礎化粧品としては、例えば化粧水や乳液、洗顔料、クレンジング、美容液、クリームなどが挙げられる。
Examples of makeup cosmetics include foundations, eyebrows, mascara, eye shadows, eyelines, lipsticks, glosses, blushers, white powders, and manicures. Basic cosmetics include, for example, lotions and milky lotions. Examples include facial cleansers, cleansers, serums and creams.
また機能性食品は、医薬品成分を含まない健康の保持増進に寄与するとされる食品全般を包含する概念であり、例えば、栄養補助食品や健康補助食品、栄養調整食品のほか、所謂サプリメントなどの一般食品であったり、特定保健用食品や栄養機能食品、機能性表示食品の如き保健機能食品も含まれる。
In addition, functional food is a concept that includes all foods that do not contain pharmaceutical ingredients and are thought to contribute to the maintenance and promotion of health. It also includes foods, foods for specified health use, foods with nutritional function, and foods with health claims such as foods with functional claims.
なお、上述した化粧料や機能性食品についての説明は、本発明の理解に供すべくこれらに相当する物品等の一例を列挙したものであり、各語句の解釈はこれら列挙された物品等に限定されるものではない。ただし、本出願人が本願を権利化するにあたり、本発明をこれら物品等に限定することを妨げない。
In addition, the above-mentioned explanation about cosmetics and functional foods is a list of examples of articles and the like corresponding thereto for the understanding of the present invention, and the interpretation of each term is limited to these listed articles and the like. It is not something that will be done. However, this does not prevent the applicant from limiting the present invention to these articles, etc. in order to acquire the rights of the present application.
また本実施形態では、プラセンタ抽出物を、メラニン生成を抑制又は促進する成分、ヒアルロン酸産生促進成分、コラーゲン産生促進成分、エラスターゼ活性阻害成分、抗アレルギー活性促進成分、チロシナーゼ活性阻害成分から選ばれるいずれかの成分の添加材料として使用する。
Further, in the present embodiment, the placenta extract is selected from a component that suppresses or promotes melanin production, a hyaluronic acid production promoting component, a collagen production promoting component, an elastase activity inhibiting component, an antiallergic activity promoting component, and a tyrosinase activity inhibiting component. It is used as an additive material for the ingredients.
ここで、添加材料としての使用とは、上記各成分を含有させるべくプラセンタ抽出物を化粧料や機能性食品に添加することを意味するほか、プラセンタ抽出物を上記各成分の抽出材料として使用することも含まれる。後者の場合、抽出された各成分が化粧料や機能性食品に添加されることとなる。
Here, the use as an additive material means that the placenta extract is added to cosmetics and functional foods so as to contain each of the above components, and the placenta extract is used as an extraction material for each of the above components. That is also included. In the latter case, each extracted component will be added to cosmetics and functional foods.
プラセンタ抽出物の使用は、メラニン生成を抑制又は促進する成分、ヒアルロン酸産生促進成分、コラーゲン産生促進成分、エラスターゼ活性阻害成分、抗アレルギー活性促進成分、チロシナーゼ活性阻害成分から選ばれるいずれかの成分の添加材料であることを目的とする。
The use of placenta extract is made from any of the components selected from a component that suppresses or promotes melanin production, a hyaluronic acid production promoting component, a collagen production promoting component, an elastase activity inhibiting component, an antiallergic activity promoting component, and a tyrosinase activity inhibiting component. The purpose is to be an additive material.
これら各成分は追って詳述するが、本発明者らの鋭意研究により明らかとなったものであり、プラセンタ抽出物自体がこれらの成分としての機能を発揮する。ただし、プラセンタ抽出物を構成する全ての成分が同機能を発揮する必要はなく、実質的には、プラセンタ抽出物に含まれる一部の単一の物質により作用が発揮されても良いし、また、複数の物質によって作用が発揮されても良い。
Each of these components will be described in detail later, but it has been clarified by the diligent research of the present inventors, and the placenta extract itself exerts its function as these components. However, not all the components constituting the placenta extract need to exert the same function, and the action may be substantially exerted by a single substance contained in the placenta extract. , The action may be exerted by a plurality of substances.
メラニン生成を抑制又は促進する成分は、化粧料の態様にて外用的に、及び/または機能性食品の態様にて経口的に(以下、外用・経口使用態様と称する。)使用して皮膚におけるメラニンの生成を抑制又は促進できれば良い。このような成分は、後述するメラニン生成試験にて確認を行うことができる。
Ingredients that suppress or promote melanin production are used externally in the form of cosmetics and / or orally in the form of functional foods (hereinafter referred to as external / oral use modes) in the skin. It suffices if the production of melanin can be suppressed or promoted. Such components can be confirmed by a melanin production test described later.
ヒアルロン酸産生促進成分やコラーゲン産生促進成分は、外用・経口使用態様での使用でヒアルロン酸やコラーゲンの産生を促進できれば良く、例えば後述するコラーゲン、ヒアルロン酸産生試験にて確認を行うことができる。
The hyaluronic acid production promoting component and the collagen production promoting component need only be able to promote the production of hyaluronic acid and collagen when used in an external or oral use mode, and can be confirmed by, for example, a collagen or hyaluronic acid production test described later.
エラスターゼ活性阻害成分は、外用・経口使用態様での使用でエラスターゼの活性を阻害してエラスチンの分解をすることができれば良く、例えば後述するエラスターゼ活性阻害試験にて確認を行うことができる。
The elastase activity-inhibiting component only needs to be able to inhibit the activity of elastase and decompose elastin when used in an external or oral use mode, and can be confirmed by, for example, an elastase activity-inhibiting test described later.
抗アレルギー活性促進成分は、外用・経口使用態様での使用でアレルギー症状を抑制することができれば良く、例えば後述する抗アレルギー活性試験の如くβ-ヘキソサミニダーゼ活性を測定することで確認を行うことができる。
The anti-allergic activity promoting component may be used as long as it can suppress allergic symptoms when used externally or orally. For example, it is confirmed by measuring β-hexosaminidase activity as in the anti-allergic activity test described later. be able to.
チロシナーゼ活性阻害成分は、外用・経口使用態様での使用でチロシナーゼの活性を阻害し、メラニンの定着を防止できれば良く、例えば後述するチロシナーゼ阻害活性試験にて確認を行うことができる。
The tyrosinase activity-inhibiting component may inhibit the activity of tyrosinase and prevent the colonization of melanin when used in an external or oral use mode, and can be confirmed by, for example, a tyrosinase inhibitory activity test described later.
またプラセンタ抽出物は、ジメチルスルホキシド、エタノール、メタノール、ヘキサンから選ばれるいずれか1つ若しくはいずれか2つ以上の混合液からなる非水系特定溶媒に対して溶解性を示す成分群よりなることとしても良い。
The placenta extract may also consist of a group of components exhibiting solubility in a non-aqueous specific solvent consisting of a mixture of any one or two or more selected from dimethyl sulfoxide, ethanol, methanol and hexane. good.
すなわち、プラセンタ抽出物は、非水系特定溶媒によって抽出された抽出物とすることができる。
That is, the placenta extract can be an extract extracted by a non-aqueous specific solvent.
そして、非水系特定溶媒に対して溶解性を示す成分群よりなるプラセンタ抽出物を前述の各成分として使用することにより、メラニン生成を促進する作用を呈することができ、また、水性溶媒に対して溶解性を示す成分群に含まれるエラスターゼ活性阻害成分に比してより顕著なエラスターゼ活性阻害作用を呈することができる。
Then, by using the placenta extract consisting of a group of components exhibiting solubility in a non-aqueous specific solvent as each of the above-mentioned components, it is possible to exhibit an action of promoting melanin production, and also in an aqueous solvent. It is possible to exhibit a more remarkable elastase activity inhibitory action as compared with the elastase activity inhibitory component contained in the component group showing solubility.
またプラセンタ抽出物は、水を主成分とする水系溶媒に対して溶解性を示す成分群よりなることとしても良い。換言すれば、プラセンタ抽出物は水系溶媒によって抽出された抽出物とすることができる。
Further, the placenta extract may consist of a group of components exhibiting solubility in an aqueous solvent containing water as a main component. In other words, the placenta extract can be an extract extracted with an aqueous solvent.
ここで水系溶媒は水を主成分とした溶媒であり、水そのものの他に、例えば50w/w%以上の割合で水を含有する溶媒が含まれる。水系溶媒を構成する水以外の成分としては、例えば、水に対して溶解性を示す固体(粉末等)の成分であったり、アルコールの如き水と混和性を示す液体とすることができる。
Here, the aqueous solvent is a solvent containing water as a main component, and in addition to water itself, for example, a solvent containing water at a ratio of 50 w / w% or more is included. The components other than water constituting the aqueous solvent may be, for example, a solid component (powder or the like) that is soluble in water, or a liquid that is miscible with water such as alcohol.
このように、水系溶媒に対して溶解性を示す成分群よりなるプラセンタ抽出物を前述の各成分として使用することにより、メラニン生成を抑制する作用や、非水系特定溶媒に対して溶解性を示す成分群に含まれるヒアルロン酸産生促進成分やコラーゲン産生促進成分に比してより顕著な各作用を呈することができる。
As described above, by using the placenta extract consisting of the component group exhibiting solubility in an aqueous solvent as each of the above-mentioned components, the action of suppressing melanin production and the solubility in a non-aqueous specific solvent are exhibited. It can exhibit more remarkable actions as compared with the hyaluronic acid production promoting component and the collagen production promoting component contained in the component group.
また溶媒は、水系溶媒と非水系特定溶媒との混合溶媒としても良い。すなわち、50w/w%未満の割合で水を含み、残余を非水系特定溶媒や、調製する混合溶媒に対して溶解性を示す固体(粉末等)とした混合溶媒することで、上述のプラセンタ抽出物とは異なる組成割合を備えたプラセンタ抽出物を調製することができる。
Further, the solvent may be a mixed solvent of an aqueous solvent and a non-aqueous specific solvent. That is, the above-mentioned placenta extraction is performed by using a mixed solvent containing water at a ratio of less than 50 w / w% and using a non-aqueous specific solvent or a solid (powder, etc.) that is soluble in the mixed solvent to be prepared. It is possible to prepare a placenta extract having a composition ratio different from that of the product.
また本願は、本発明者がプラセンタ抽出物より見出した成分の、メラニンの生成促進を目的とした化粧料中の有効成分又は機能性食品中の保健機能成分としての使用方法についても提供するものである。
The present application also provides a method for using the component found from the placenta extract by the present invention as an active ingredient in cosmetics or a health functional ingredient in functional foods for the purpose of promoting the production of melanin. be.
具体的には、下記一般式(i):
で表されるコレステロール(Cholesterol)、又は下記一般式(ii):
で表されるコレステロール-7-オン(Cholesterol-7-one)、又は下記一般式(iii):
で表されるコレスタ-3,5-ジエン-7-オン(Cholesta-3,5-diene-7-one)、又は下記一般式(iv):
で表されるウリジン一リン酸(Uridine Monophosphate)の、メラニンの生成促進を目的とした使用であり、これら(i)~(iv)に示す物質のメラニンの生成促進能については、追ってデータを示して説明する。
Specifically, the following general formula (i):
Cholesterol represented by, or the following general formula (ii):
Cholesterol-7-one represented by, or the following general formula (iii):
Cholesta-3,5-diene-7-one represented by, or the following general formula (iv):
Uridine Monophosphate, which is represented by, is used for the purpose of promoting the production of melanin, and the data on the ability of the substances shown in (i) to (iv) to promote the production of melanin will be shown later. I will explain.
また本願は、下記一般式(iv):
で表されるウリジン一リン酸(Uridine Monophosphate)、又は下記一般式(v):
で表される3β-ヒドロキシコレスタ-4-エン-6-オン(3β-hydroxycholest-4-ene-6-one)、又は下記一般式(vi):
で表される3β,6β-ジヒドロキシコレスタ-5-エン(3β,6β-dihydroxycholest-5-ene)、又は下記一般式(vii):
で表されるコレステリルオレアート(Cholestereyl oleate)、又は下記一般式(viii):
で表されるパルミチン酸(Palmitic acid)、又は下記一般式(ix):
で表されるチミン(Thymine)の、メラニンの生成抑制を目的とした使用であり、これら(iv)~(ix)に示す物質のメラニンの生成抑制能については、追ってデータを示して説明する。
Further, in the present application, the following general formula (iv):
Uridine monophosphate represented by, or the following general formula (v):
3β-hydroxycholest-4-ene-6-one represented by 3β-hydroxycholest-4-ene-6-one, or the following general formula (vi):
3β, 6β-dihydroxycholest-5-ene (3β, 6β-dihydroxycholest-5-ene) represented by, or the following general formula (vii):
Cholestereyl oleate represented by, or the following general formula (viii):
Palmitic acid represented by, or the following general formula (ix):
The use of thymine represented by (3) for the purpose of suppressing the production of melanin, and the ability of the substances shown in (iv) to (ix) to suppress the production of melanin will be described later by showing data.
そして、これら(i)~(ix)の成分を、化粧料中又は機能性食品中におけるメラニンの生成促進や生成抑制を目的とした有効成分又は保健機能成分として使用すれば、上述の成分によりメラニンの生成を促進又は抑制する新たな化粧料や機能性食品を提供することができる。
Then, if these components (i) to (ix) are used as an active ingredient or a health functional ingredient for the purpose of promoting or suppressing the production of melanin in cosmetics or functional foods, melanin can be obtained from the above-mentioned components. It is possible to provide new cosmetics and functional foods that promote or suppress the production of melanin.
以下、本実施形態に係る各使用方法について、実験結果を参照しながら更に説明する。なお、以下ではウマ由来のプラセンタ(胎盤)より抽出したプラセンタ抽出物(以下、ウマプラセンタ抽出物という。)を用いた例について説明するが、プラセンタの由来はウマに限定されるものではない。
Hereinafter, each usage method according to this embodiment will be further described with reference to the experimental results. In the following, an example using a placenta extract (hereinafter referred to as horse placenta extract) extracted from horse-derived placenta (placenta) will be described, but the origin of placenta is not limited to horses.
〔1.プラセンタ抽出物の調製〕
近年、化粧料や機能性食品に配合する機能性素材として、様々な天然成分が使用されている。中でも、哺乳類の胎盤であるプラセンタは、美容や健康維持に寄与する素材として注目されている。 [1. Preparation of placenta extract]
In recent years, various natural ingredients have been used as functional materials to be blended in cosmetics and functional foods. Among them, placenta, which is the placenta of mammals, is attracting attention as a material that contributes to beauty and health maintenance.
近年、化粧料や機能性食品に配合する機能性素材として、様々な天然成分が使用されている。中でも、哺乳類の胎盤であるプラセンタは、美容や健康維持に寄与する素材として注目されている。 [1. Preparation of placenta extract]
In recent years, various natural ingredients have been used as functional materials to be blended in cosmetics and functional foods. Among them, placenta, which is the placenta of mammals, is attracting attention as a material that contributes to beauty and health maintenance.
しかし、先に述べたように、プラセンタやその抽出物が持つ機能性について科学的に検討された例は未だ少ないのが現状である。そこでここでは、ウマプラセンタを代表例とし、幾つかの機能性評価試験を実施して有効性を確認すべく、まずはプラセンタ抽出物の調製を行った。
However, as mentioned earlier, there are still few examples of scientific studies on the functionality of placenta and its extracts. Therefore, here, placenta extract was first prepared in order to confirm the effectiveness by conducting several functional evaluation tests with horse placenta as a representative example.
ウマプラセンタ抽出物の抽出原料として、ウマプラセンタ粉末を使用した。ウマプラセンタ粉末は、ウマ胎盤の凍結乾燥粉末である。
Horse placenta powder was used as an extraction raw material for horse placenta extract. Horse placenta powder is a lyophilized powder of horse placenta.
まず、100mgのウマプラセンタ粉末を1.5mlチューブに量り取り、1mlの抽出溶媒を添加して、室温にて30分間撹拌を行った。抽出溶媒は、水系溶媒として滅菌超純水、非水系特定溶媒としてジメチルスルホキシド(Dimethyl sulfoxide : 以下、DMSOともいう。)をそれぞれ使用した。
First, 100 mg of horse placenta powder was weighed in a 1.5 ml tube, 1 ml of an extraction solvent was added, and the mixture was stirred at room temperature for 30 minutes. As the extraction solvent, sterilized ultrapure water was used as the aqueous solvent, and dimethyl sulfoxide (hereinafter, also referred to as DMSO) was used as the non-aqueous specific solvent.
撹拌後、チューブを室温に静置し、得られた上清をウマプラセンタ抽出物とした。なお、このウマプラセンタ抽出物は、100mgのプラセンタ粉末に1mlの抽出溶媒を添加して調製したことに由来し、100mg/mlサンプルと称することとした。また、以下の各試験において、100mg/mlサンプルを適宜希釈して調製した希釈サンプルに関し、例えば100倍希釈されたサンプルであれば1mg/mlサンプル、10000倍希釈されたサンプルであれば10μg/mlサンプルのように表現する。
After stirring, the tube was allowed to stand at room temperature, and the obtained supernatant was used as a horse placenta extract. This horse placenta extract was prepared by adding 1 ml of an extraction solvent to 100 mg of placenta powder, and was referred to as a 100 mg / ml sample. In each of the following tests, the diluted sample prepared by appropriately diluting the 100 mg / ml sample is, for example, 1 mg / ml sample for a 100-fold diluted sample and 10 μg / ml for a 10000-fold diluted sample. Express like a sample.
〔2.メラニン生成・抑制能確認試験(第1試験)〕
メラニンはフェノール類物質が高分子化して色素になった物質の総称である。ヒトの皮膚に存在するメラニンは、チロシンから合成された様々なインドール化合物がポリマーを形成した形態をとっている。 [2. Melanin production / suppression ability confirmation test (1st test)]
Melanin is a general term for substances in which phenolic substances are polymerized into pigments. Melanin present in human skin is in the form of polymers formed by various indole compounds synthesized from tyrosine.
メラニンはフェノール類物質が高分子化して色素になった物質の総称である。ヒトの皮膚に存在するメラニンは、チロシンから合成された様々なインドール化合物がポリマーを形成した形態をとっている。 [2. Melanin production / suppression ability confirmation test (1st test)]
Melanin is a general term for substances in which phenolic substances are polymerized into pigments. Melanin present in human skin is in the form of polymers formed by various indole compounds synthesized from tyrosine.
メラニンの最も重要な役割は、紫外線防御であり、紫外線による日光障害や悪性腫瘍の発症を抑制する。メラニンのそのほかの機能としては、生体に有蓋な活性酸素種の吸収、金属や薬剤の取り込みなどが知られている。
The most important role of melanin is UV protection, which suppresses sunlight damage and the development of malignant tumors caused by UV rays. Other known functions of melanin include absorption of reactive oxygen species that are liable to the living body and uptake of metals and drugs.
メラニンを含む細胞は徐々に角質上層に異同し、脂質代謝酵素(リパーゼなど)やタンパク質分解酵素(エラスターゼなど)の作用によって細胞間の接着が分解され、最終的に角質は剥離、脱落する。
Cells containing melanin gradually dissociate into the upper stratum corneum, and the adhesion between cells is decomposed by the action of lipid-metabolizing enzymes (lipase, etc.) and proteolytic enzymes (elastase, etc.), and finally the stratum corneum is exfoliated and shed.
本試験では、ヒトメラノーマ細胞(G361細胞株)をモデル細胞として、細胞とサンプルとを共培養し、培養液中に生成されたメラニンと細胞生存率を測定した。
In this test, human melanoma cells (G361 cell line) were used as model cells, and cells and samples were co-cultured, and melanin produced in the culture medium and cell viability were measured.
細胞培養の方法としては、ヒトメラノーマ細胞(G361細胞株)はMcCoy's 5A medium(含1%ペニシリン-ストレプトマイシン及び10%ウシ胎児血清(FBS))を用いて前培養した。
As a cell culture method, human melanoma cells (G361 cell line) were precultured using McCoy's 5A medium (including 1% penicillin-streptomycin and 10% fetal bovine serum (FBS)).
その後、24穴プレートに1.0×105cells/mlの濃度で0.5mlを播種し、CO2インキュベータ(37℃、5% CO2)にてオーバーナイト培養した。
Then, 0.5 ml was seeded on a 24-well plate at a concentration of 1.0 × 10 5 cells / ml and cultured overnight in a CO 2 incubator (37 ° C, 5% CO 2 ).
オーバーナイト後、培地を除去し、0.5mlの新しい培地と所定の濃度に希釈された2.5μlのプラセンタ抽出物の各サンプル(以下、各希釈サンプルともいう。)を添加し、再びCO2インキュベータにて72時間培養を行った。
After overnight, the medium is removed, 0.5 ml of fresh medium and each sample of 2.5 μl of placenta extract diluted to a predetermined concentration (hereinafter also referred to as each diluted sample) are added, and the mixture is returned to the CO 2 incubator. The cells were cultured for 72 hours.
また、メラニン生成・抑制能の確認は、産生されたメラニン量を確認することで行った。具体的には、72時間の培養が行われたプレートから培地を除去し、各ウェルをPBS(Phosphate Buffered Saline; リン酸緩衝食塩水)にて洗浄した後に各ウェルに1mlの1M NaOH溶液を加え、室温で4時間反応させ、プレートリーダにて405nmにおける吸光度を測定することでメラニン産生量とした。
In addition, the ability to produce and suppress melanin was confirmed by confirming the amount of melanin produced. Specifically, the medium was removed from the plate that had been cultured for 72 hours, each well was washed with PBS (Phosphate Buffered Saline; phosphate buffered saline), and then 1 ml of 1 M NaOH solution was added to each well. The reaction was carried out at room temperature for 4 hours, and the absorbance at 405 nm was measured with a plate reader to determine the amount of melanin produced.
また、細胞生存率の測定は、MTT法により行った。72時間の培養が行われたプレートから培地を除去し、各ウェルに1mlの新しい培地を添加する。次いで、各ウェルにMTT染色液(5mg/ml in PBS)を200μl添加し、CO2インキュベータにて4時間に亘り静置培養した。
The cell viability was measured by the MTT method. Remove medium from plates that have been cultured for 72 hours and add 1 ml of fresh medium to each well. Then, 200 μl of MTT stain (5 mg / ml in PBS) was added to each well, and the cells were statically cultured in a CO 2 incubator for 4 hours.
そして、培地を除去し、各ウェルに塩酸-イソプロパノール溶液を1mlずつ加えて、ピペッティングにてホルマザンを完全に溶解させ、マイクロプレートリーダで570nmにおける吸光度を測定することで、細胞生存率の算出を行った。
Then, the medium was removed, 1 ml of hydrochloric acid-isopropanol solution was added to each well, formazan was completely dissolved by pipetting, and the absorbance at 570 nm was measured with a microplate reader to calculate the cell viability. went.
図1はコントロールに対するメラニン生産比を示したグラフであり、図1(a)は水系溶媒にて抽出したプラセンタ抽出物(以下、プラセンタ水系抽出物ともいう。)によるメラニン生産比を示し、図1(b)は非水系特定溶媒にて抽出したプラセンタ抽出物(以下、プラセンタ非水系抽出物という。)によるメラニン生産比を示している。
FIG. 1 is a graph showing the melanin production ratio with respect to the control, and FIG. 1 (a) shows the melanin production ratio by the placenta extract (hereinafter, also referred to as placenta water-based extract) extracted with an aqueous solvent, and FIG. (B) shows the melanin production ratio by the placenta extract (hereinafter referred to as placenta non-aqueous extract) extracted with a non-aqueous specific solvent.
図1(a)に示すように、プラセンタ水系抽出物に関し、400μg/mlサンプル、100μg/mlサンプル、10μg/mlサンプルについて確認を行ったところ、検討された全ての濃度において約20%程度のメラニン生成抑制効果が認められた。
As shown in FIG. 1 (a), when the placenta aqueous extract was confirmed with respect to 400 μg / ml sample, 100 μg / ml sample, and 10 μg / ml sample, about 20% of melanin was examined at all the examined concentrations. A production inhibitory effect was observed.
このことより、プラセンタ水系抽出物には、メラニン生成を抑制する成分、換言すれば肌の美白に寄与する成分が存在するものと考えられた。
From this, it was considered that the placenta aqueous extract contains a component that suppresses melanin production, in other words, a component that contributes to skin whitening.
またこれに対し、プラセンタ非水系抽出物に関し各希釈サンプルについて確認を行ったところ、図1(b)に示すように、検討された全ての濃度において約20~40%程度のメラニン生成促進効果が認められた。
On the other hand, when the placenta non-aqueous extract was confirmed for each diluted sample, as shown in FIG. 1 (b), the melanin production promoting effect of about 20 to 40% was obtained at all the examined concentrations. Admitted.
このことより、プラセンタ非水系抽出物には、メラニン生成を促進する成分が含まれており、毛髪の黒色を維持するなどの効果があるものと考えられた。
From this, it was considered that the placenta non-aqueous extract contains a component that promotes melanin production and has an effect of maintaining the black color of hair.
また、これら水系、非水系のプラセンタ抽出物についての結果を踏まえると、プラセンタ水系抽出物とプラセンタ非水系抽出物とでは、メラニンの産生においてそれぞれ「抑制」と「促進」という正反対の性質を持つ点が明らかとなったのは、実に興味深い点であると言える。
In addition, based on the results of these water-based and non-water-based placenta extracts, the placenta water-based extract and the placenta non-water-based extract have the opposite properties of "suppression" and "promotion" in the production of melanin, respectively. It can be said that it is a really interesting point that became clear.
〔3.コラーゲン・ヒアルロン酸産生試験〕
コラーゲンは、動物の皮膚などの結合組織を形成する構造タンパク質である。コラーゲンは皮膚の構造維持に貢献しており、皮膚を若々しく保ち、シワを作らないために重要である。 [3. Collagen / hyaluronic acid production test]
Collagen is a structural protein that forms connective tissues such as animal skin. Collagen contributes to the maintenance of the structure of the skin and is important for keeping the skin youthful and preventing wrinkles.
コラーゲンは、動物の皮膚などの結合組織を形成する構造タンパク質である。コラーゲンは皮膚の構造維持に貢献しており、皮膚を若々しく保ち、シワを作らないために重要である。 [3. Collagen / hyaluronic acid production test]
Collagen is a structural protein that forms connective tissues such as animal skin. Collagen contributes to the maintenance of the structure of the skin and is important for keeping the skin youthful and preventing wrinkles.
コラーゲンは、皮膚の真皮に存在する繊維芽細胞により産生され、繊維芽細胞のコラーゲン産生を促進することができれば、若々しい皮膚を維持することができる。それゆえ、繊維芽細胞のコラーゲン産生促進成分は、抗老化の化粧品素材として有用であると考えられる。
Collagen is produced by fibroblasts present in the dermis of the skin, and if the fibroblasts can promote collagen production, youthful skin can be maintained. Therefore, the collagen production promoting component of fibroblasts is considered to be useful as an anti-aging cosmetic material.
またヒアルロン酸は、主に皮膚真皮の主要な細胞である繊維芽細胞やその他結合組織の細胞によって産生される。細胞外マトリックスの主成分となっており、様々な細胞間相互作用に関与している。またその他、高濃度で滑液中に存在し、関節の正常な水分保持と潤滑を担っている。
Hyaluronic acid is mainly produced by fibroblasts, which are the main cells of the dermis of the skin, and cells of other connective tissues. It is the main component of the extracellular matrix and is involved in various cell-cell interactions. In addition, it is present in synovial fluid at a high concentration and is responsible for normal water retention and lubrication of joints.
本試験では、繊維芽細胞培養系への本実施形態に係るプラセンタ抽出物の添加によるコラーゲンやヒアルロン酸の産生能を測定し、プラセンタ抽出物における肌の水分保持効果に関する評価を行う。
本試験方法としては、モデル細胞として成人皮膚繊維芽細胞(NHDF-Ad)を用いた。
マイクロウェルプレートに1.0×104cells/wellずつ播種し、CO2インキュベータ(37℃、5% CO2)にて24時間に亘り前培養した。 In this test, the ability to produce collagen and hyaluronic acid by adding the placenta extract according to the present embodiment to the fibroblast culture system is measured, and the skin water retention effect of the placenta extract is evaluated.
As a test method, adult skin fibroblasts (NHDF-Ad) were used as model cells.
1.0 × 10 4 cells / well were seeded on microwell plates and precultured in a CO 2 incubator (37 ° C, 5% CO 2 ) for 24 hours.
本試験方法としては、モデル細胞として成人皮膚繊維芽細胞(NHDF-Ad)を用いた。
マイクロウェルプレートに1.0×104cells/wellずつ播種し、CO2インキュベータ(37℃、5% CO2)にて24時間に亘り前培養した。 In this test, the ability to produce collagen and hyaluronic acid by adding the placenta extract according to the present embodiment to the fibroblast culture system is measured, and the skin water retention effect of the placenta extract is evaluated.
As a test method, adult skin fibroblasts (NHDF-Ad) were used as model cells.
1.0 × 10 4 cells / well were seeded on microwell plates and precultured in a CO 2 incubator (37 ° C, 5% CO 2 ) for 24 hours.
前培養の後、培地を除去し、新しい培地と所定の濃度に希釈された各希釈サンプルを添加し、再びCO2インキュベータにて72時間培養を行い、培養液中に産生されたコラーゲン量とヒアルロン酸量を、市販のELISAキットを用いて測定した。同様に、同じ細胞を用いてMTT試験を行った。
After pre-culture, remove the medium, add new medium and each diluted sample diluted to the specified concentration, and incubate again in the CO 2 incubator for 72 hours to produce the amount of collagen and hyaluron in the culture medium. The amount of acid was measured using a commercially available ELISA kit. Similarly, the MTT test was performed using the same cells.
図2はコントロールに対するヒアルロン酸産生比を示したグラフであり、図2(a)はプラセンタ水系抽出物によるヒアルロン酸産生比を示し、図2(b)はプラセンタ非水系抽出物によるヒアルロン酸産生比を示している。
FIG. 2 is a graph showing the hyaluronic acid production ratio with respect to the control, FIG. 2 (a) shows the hyaluronic acid production ratio by the placenta aqueous extract, and FIG. 2 (b) shows the hyaluronic acid production ratio by the placenta non-aqueous extract. Is shown.
図2(a)に示すように、プラセンタ水系抽出物の添加により、ヒアルロン酸産生の上昇が認められた。このことから、プラセンタ水系抽出物には、ヒアルロン酸の産生を促進する成分が含まれていることが示唆された。
As shown in FIG. 2 (a), an increase in hyaluronic acid production was observed by the addition of the placenta aqueous extract. This suggests that the placenta aqueous extract contains a component that promotes the production of hyaluronic acid.
これに対し、プラセンタ非水系抽出物に関し各希釈サンプルについて確認を行ったところ、図2(b)に示すように、検討されたいずれの濃度においても、ヒアルロン酸産生の上昇は認められなかった。すなわち、プラセンタ非水系抽出物には、ヒアルロン酸の産生の促進効果は有しないものと考えられた。
On the other hand, when the placenta non-aqueous extract was confirmed for each diluted sample, as shown in FIG. 2 (b), no increase in hyaluronic acid production was observed at any of the examined concentrations. That is, it was considered that the placenta non-aqueous extract had no effect of promoting the production of hyaluronic acid.
図3はコントロールに対するコラーゲン産生比を示したグラフであり、図3(a)はプラセンタ水系抽出物によるコラーゲン産生比を示し、図3(b)はプラセンタ非水系抽出物によるコラーゲン産生比を示している。
FIG. 3 is a graph showing the collagen production ratio to the control, FIG. 3 (a) shows the collagen production ratio by the placenta aqueous extract, and FIG. 3 (b) shows the collagen production ratio by the placenta non-aqueous extract. There is.
図3(a)に示すように、プラセンタ水系抽出物において、顕著なコラーゲン産生の上昇が認められた。また、プラセンタ非水系抽出物においても、100μg/mlサンプルの添加によるコラーゲン産生の上昇が認められた。
As shown in FIG. 3 (a), a remarkable increase in collagen production was observed in the placenta aqueous extract. In addition, in the placenta non-aqueous extract, an increase in collagen production was observed by adding a 100 μg / ml sample.
これらのことから、水系、非水系のプラセンタ抽出物のいずれにおいても、コラーゲンの産生を促進する成分が含まれていることが示唆された。
From these facts, it was suggested that both the aqueous and non-aqueous placenta extracts contained a component that promotes collagen production.
また本試験結果を踏まえると、プラセンタ水系抽出物には、ヒアルロン酸及びコラーゲン産生を促進させる成分が、また、プラセンタ非水系抽出物にはコラーゲン産生を促進させる成分が含まれていることが示唆された。
Based on the results of this test, it is suggested that the placenta aqueous extract contains a component that promotes hyaluronic acid and collagen production, and the placenta non-aqueous extract contains a component that promotes collagen production. rice field.
〔4.エラスターゼ活性阻害試験〕
真皮に存在するコラーゲン・エラスチンの架橋構造は、肌の弾力を保持する重要な役割を果たしている。エラスチン分解酵素であるエラスターゼは、紫外線等による刺激によって誘導され、エラスチンが分解されると、架橋構造が脆くなり肌の弾力が失われる。本試験では、エラスターゼ阻害活性を測定することで、プラセンタ抽出物が有する肌弾力保持機能を評価する。 [4. Elastase activity inhibition test]
The cross-linked structure of collagen and elastin present in the dermis plays an important role in maintaining the elasticity of the skin. Elastase, which is an elastin-degrading enzyme, is induced by stimulation with ultraviolet rays or the like, and when elastin is decomposed, the crosslinked structure becomes brittle and the elasticity of the skin is lost. In this test, the skin elasticity retention function of placenta extract is evaluated by measuring the elastase inhibitory activity.
真皮に存在するコラーゲン・エラスチンの架橋構造は、肌の弾力を保持する重要な役割を果たしている。エラスチン分解酵素であるエラスターゼは、紫外線等による刺激によって誘導され、エラスチンが分解されると、架橋構造が脆くなり肌の弾力が失われる。本試験では、エラスターゼ阻害活性を測定することで、プラセンタ抽出物が有する肌弾力保持機能を評価する。 [4. Elastase activity inhibition test]
The cross-linked structure of collagen and elastin present in the dermis plays an important role in maintaining the elasticity of the skin. Elastase, which is an elastin-degrading enzyme, is induced by stimulation with ultraviolet rays or the like, and when elastin is decomposed, the crosslinked structure becomes brittle and the elasticity of the skin is lost. In this test, the skin elasticity retention function of placenta extract is evaluated by measuring the elastase inhibitory activity.
本試験では、細胞溶解液を実験に用いた。細胞溶解液を調製は、まず、コンフルエントになるまで前培養したヒト皮膚繊維芽細胞(NHDF-Ad)をPBSで洗浄し、細胞溶解液にて1.0×106cells/mlに再懸濁した。その後、超音波処理を行い、4℃、13,000rpmで15分間遠心し、上清を細胞溶解液として回収した。
In this test, cytolytic solution was used in the experiment. To prepare the cytolytic solution, first, human skin fibroblasts (NHDF-Ad) precultured until they became confluent were washed with PBS and resuspended in 1.0 × 10 6 cells / ml with the cytolytic solution. Then, sonication was performed, and the mixture was centrifuged at 4 ° C. and 13,000 rpm for 15 minutes, and the supernatant was collected as a cytolytic solution.
次に、このようにして調製した細胞溶解液12.5μlに対し、35.5μlの0.2Mトリス塩酸緩衝液(pH8.0)と、2μlの各希釈サンプルとを混合し、37℃にて15分間インキュベートした。その後、エラスターゼの基質として50μlの5mM N-スクシニル-Ala-Ala-Ala-p-ニトロアニリド(N-Succinyl-Ala-Ala-Ala-p-nitroanilide)を添加し、37℃にて3時間インキュベートした。そして、マイクロプレートリーダで405nmにおける吸光度を測定し、エラスターゼ活性を算出した。なお、ポジティブコントロールとして、エラスターゼ阻害剤であるホスホラミドン(終濃度0.04mM)を使用した。
Next, 35.5 μl of 0.2 M Tris-hydrochloric acid buffer (pH 8.0) and 2 μl of each diluted sample are mixed with 12.5 μl of the cytolytic solution prepared in this manner, and incubated at 37 ° C. for 15 minutes. did. Then, 50 μl of 5 mM N-Succinyl-Ala-Ala-Ala-p-nitroanilide (N-Succinyl-Ala-Ala-Ala-p-nitroanilide) was added as a substrate for elastase and incubated at 37 ° C for 3 hours. .. Then, the absorbance at 405 nm was measured with a microplate reader, and the elastase activity was calculated. As a positive control, phosphoramidone (final concentration 0.04 mM), which is an elastase inhibitor, was used.
図4はコントロールに対するエラスターゼ活性比を示したグラフであり、図4(a)はプラセンタ水系抽出物によるエラスターゼ活性比を示し、図4(b)はプラセンタ非水系抽出物によるエラスターゼ活性比を示している。
FIG. 4 is a graph showing the elastase activity ratio with respect to the control, FIG. 4 (a) shows the elastase activity ratio with the placenta aqueous extract, and FIG. 4 (b) shows the elastase activity ratio with the placenta non-aqueous extract. There is.
図4(a)に示すように、プラセンタ水系抽出物においては、1600μg/mlサンプルと400μg/mlサンプルについて、凡そ30%程度のエラスターゼ活性の低下が認められた。しかしながら、40μg/mlサンプルでは、エラスターゼ活性の低下は認められなかった。
As shown in FIG. 4 (a), in the placenta aqueous extract, a decrease in elastase activity of about 30% was observed for the 1600 μg / ml sample and the 400 μg / ml sample. However, no decrease in elastase activity was observed in the 40 μg / ml sample.
これに対し、プラセンタ非水系抽出物においては、図4(b)に示すように、400μg/mlサンプルについて約20%、1600μg/mlサンプルについては約60%ものエラスターゼ活性の低下が認められた。
On the other hand, in the placenta non-aqueous extract, as shown in FIG. 4 (b), a decrease in elastase activity of about 20% was observed for the 400 μg / ml sample and about 60% for the 1600 μg / ml sample.
これらのことから、水系、非水系のプラセンタ抽出物はエラスターゼ活性を阻害する成分が含まれていることが示唆された。
From these facts, it was suggested that the aqueous and non-aqueous placenta extracts contained a component that inhibits elastase activity.
特に、プラセンタ水系抽出物にあっては、おそらく100μg/ml以上の濃度、図4(a)に示す結果に基づくならば400μg/ml以上の濃度での使用、また、プラセンタ非水系抽出物にあっては40μg/ml以上の濃度、より効果的には400μg/ml以上の濃度での使用により、プラセンタ抽出物をエラスターゼ活性阻害の有効成分とする肌弾力の保持に寄与できる可能性が期待される。
In particular, the placenta aqueous extract is probably used at a concentration of 100 μg / ml or more, and based on the result shown in FIG. 4 (a), it is used at a concentration of 400 μg / ml or more, and the placenta non-aqueous extract is used. Therefore, it is expected that the use of the placenta extract at a concentration of 40 μg / ml or more, more effectively at a concentration of 400 μg / ml or more, can contribute to the maintenance of skin elasticity using the placenta extract as an active ingredient for inhibiting elastase activity. ..
〔5.抗アレルギー活性評価試験〕
炎症が繰り返されてしまう花粉症やアトピー性皮膚炎などのアレルギーの発症メカニズムは未だ不明であるが、種々の免疫機構のうち好塩基球が顆粒を放出することが原因の一つであると考えられている。 [5. Anti-allergic activity evaluation test]
The onset mechanism of allergies such as pollinosis and atopic dermatitis, in which inflammation is repeated, is still unknown, but it is thought that one of the causes is that basophils release granules among various immune mechanisms. Has been done.
炎症が繰り返されてしまう花粉症やアトピー性皮膚炎などのアレルギーの発症メカニズムは未だ不明であるが、種々の免疫機構のうち好塩基球が顆粒を放出することが原因の一つであると考えられている。 [5. Anti-allergic activity evaluation test]
The onset mechanism of allergies such as pollinosis and atopic dermatitis, in which inflammation is repeated, is still unknown, but it is thought that one of the causes is that basophils release granules among various immune mechanisms. Has been done.
そこで本試験では、好塩基球のモデル細胞としてラット好塩基球白血病細胞(RBL-2H3)を用い、プラセンタ抽出物による顆粒放出抑制活性(抗アレルギー活性)の評価を行った。顆粒にはβ-ヘキソサミニダーゼ(β-hexosaminidase)が含まれており、細胞上清中の酵素量を測定することで顆粒の放出量を測定し、また、酵素量は細胞上清の酵素活性の強さにより測定した。
Therefore, in this test, rat basophil leukemia cells (RBL-2H3) were used as basophil model cells, and the granule release inhibitory activity (anti-allergic activity) by the placenta extract was evaluated. The granules contain β-hexosaminidase, and the amount of enzyme released from the granules is measured by measuring the amount of enzyme in the cell supernatant, and the amount of enzyme is the enzyme in the cell supernatant. It was measured by the intensity of activity.
具体的には、ラット好塩基球白血病細胞(RBL-2H3)を5×105cells/mlの濃度に調製し、96穴プレートに100μl播種した。このようなプレートを、β-ヘキソサミニダーゼ活性測定用とMTT試験用との2枚作製し、CO2インキュベータ(37℃、5% CO2)にて40時間に亘り前培養した。
Specifically, rat basophil leukemia cells (RBL-2H3) were prepared at a concentration of 5 × 10 5 cells / ml, and 100 μl was seeded on a 96-well plate. Two such plates, one for measuring β-hexosaminidase activity and one for MTT test, were prepared and precultured in a CO 2 incubator (37 ° C, 5% CO 2 ) for 40 hours.
前培養後、培地を除きTyrode’s bufferにてウェルを洗浄し、0.5μlの各希釈サンプルを含む100μlのTurode’s buffer(又はEMEM培地)を各ウェルに添加した。37℃、5%CO2で1時間培養後、β-ヘキソサミニダーゼ活性測定用のプレートは上清を捨てウェルを洗浄し、100μlのA23187(in Tyrode’s buffer)を添加した。インキュベータで1時間培養後、各ウェルの上清から50μlを分取し、新しい96穴プレートに移した。
After preculture, the wells were washed with Tyrode's buffer after removing the medium, and 100 μl Turode's buffer (or EMEM medium) containing 0.5 μl of each diluted sample was added to each well. After culturing at 37 ° C. and 5% CO 2 for 1 hour, the supernatant was discarded from the plate for measuring β-hexosaminidase activity, the well was washed, and 100 μl of A23187 (in Tyrode's buffer) was added. After culturing in an incubator for 1 hour, 50 μl was separated from the supernatant of each well and transferred to a new 96-well plate.
β-ヘキソサミニダーゼ活性の測定は、50μlずつ上清を分取した新たなプレートの各ウェルに、p-ニトロフェニル-N-アセチル-β-グルコサミニド(p-Nitrophenyl-N-acetyl-beta-glucosaminide)溶液を基質として50μl添加し、アルミ箔で遮光した上で室温にて1~3時間反応させた。反応後、全ウェルに100μlの反応停止液を添加して酵素反応を停止させ、プレートリーダにて405nmの吸光度を測定し、β-ヘキソサミニダーゼ活性を測定した。
For the measurement of β-hexosaminidase activity, p-Nitrophenyl-N-acetyl-β-glucosaminide (p-Nitrophenyl-N-acetyl-beta-) was placed in each well of a new plate in which 50 μl of the supernatant was separated. 50 μl of a glucosamine) solution was added as a substrate, and the mixture was reacted at room temperature for 1 to 3 hours after being shielded from light with an aluminum foil. After the reaction, 100 μl of the reaction terminator was added to all the wells to stop the enzyme reaction, and the absorbance at 405 nm was measured with a plate reader to measure the β-hexosaminidase activity.
またMTT試験は、各希釈サンプルを添加して1時間が経過したプレートについて、まず、各ウェルの培地を除き、MTT染色液(5mg/ml in medium)を100μlずつ添加し、5%CO2、37℃で4時間静置培養した。次いで、培地を除去し、各ウェルに塩酸-イソプロパノール溶液を1mlずつ加え、遮光し、4時間室温で放置した。その後、マイクロプレートリーダにて570nmにおける吸光度を測定し、細胞生存率の指標とした。
In the MTT test, 100 μl of MTT stain (5 mg / ml in medium) was first added to the plate 1 hour after the addition of each diluted sample, excluding the medium in each well, and 5% CO 2 . The cells were statically cultured at 37 ° C. for 4 hours. Then, the medium was removed, 1 ml of hydrochloric acid-isopropanol solution was added to each well, the light was shielded, and the mixture was left at room temperature for 4 hours. Then, the absorbance at 570 nm was measured with a microplate reader and used as an index of cell viability.
図5はコントロールに対するβ-ヘキソサミニダーゼ活性比を示したグラフである。図5から分かるように、水系、非水系のプラセンタ抽出物のいずれにおいても、高濃度域である400μg/mlサンプルの添加時に、β-ヘキソサミニダーゼ活性が減少した。
FIG. 5 is a graph showing the β-hexosaminidase activity ratio to the control. As can be seen from FIG. 5, the β-hexosaminidase activity decreased in both the aqueous and non-aqueous placenta extracts when the 400 μg / ml sample, which was in the high concentration range, was added.
このことから、本実施形態に係るプラセンタ抽出物には、抗アレルギー活性を示す成分が含まれていることが示唆された。
From this, it was suggested that the placenta extract according to this embodiment contains a component showing antiallergic activity.
〔6.チロシナーゼ阻害活性試験〕
肌におけるシミやソバカスの原因となるメラニン色素は、メラノサイト内のチロシナーゼと呼ばれる酵素の働きによって作られる。チロシナーゼは紫外線により活性化されメラニンを産生するが、この酵素の働きを阻害することでメラニンの定着を防止し、美白効果が得られることが分かっている。 [6. Tyrosinase inhibitory activity test]
The melanin pigment that causes age spots and freckles on the skin is produced by the action of an enzyme called tyrosinase in melanocytes. Tyrosinase is activated by ultraviolet rays to produce melanin, and it is known that inhibition of the action of this enzyme prevents the fixation of melanin and obtains a whitening effect.
肌におけるシミやソバカスの原因となるメラニン色素は、メラノサイト内のチロシナーゼと呼ばれる酵素の働きによって作られる。チロシナーゼは紫外線により活性化されメラニンを産生するが、この酵素の働きを阻害することでメラニンの定着を防止し、美白効果が得られることが分かっている。 [6. Tyrosinase inhibitory activity test]
The melanin pigment that causes age spots and freckles on the skin is produced by the action of an enzyme called tyrosinase in melanocytes. Tyrosinase is activated by ultraviolet rays to produce melanin, and it is known that inhibition of the action of this enzyme prevents the fixation of melanin and obtains a whitening effect.
本試験では、L-チロシンあるいはL-DOPAを基質として、各希釈サンプルがチロシナーゼ活性に及ぼす影響について評価した。
In this test, the effect of each diluted sample on tyrosinase activity was evaluated using L-tyrosine or L-DOPA as a substrate.
試験方法としては、33.3μlの各希釈サンプルと、333μlの2.5mM基質(L-チロシン又はL-DOPA)と、600μlの0.1Mリン酸バッファー(pH6.8)と、33μlのチロシナーゼ(1380unit/ml)とを混合し、その直後から分光光度計により475nmにおける吸光度の経時的な変化を記録した。475nmの吸光度は、酵素-基質反応後のドーパクロムの形成によるものとされ、時間あたりの吸光度の増加を算出することで酵素活性値とした(L-チロシン:10分間測定、L-DOPA:3分間測定)。コントロール(溶媒)添加時のチロシナーゼ活性値を100%として、各希釈サンプル添加時の相対的なチロシナーゼ活性を算出した。ポジティブコントロールとして、コウジ酸(終濃度:17μg/ml)を用いた。
The test method includes 33.3 μl of each diluted sample, 333 μl of 2.5 mM substrate (L-tyrosine or L-DOPA), 600 μl of 0.1 M phosphate buffer (pH 6.8), and 33 μl of tyrosinase (1380 unit / ml). ) Was mixed, and immediately after that, the change over time in the absorbance at 475 nm was recorded by a spectrophotometer. The absorbance at 475 nm was attributed to the formation of dopachrome after the enzyme-substrate reaction, and the increase in absorbance per hour was calculated to determine the enzyme activity value (L-tyrosine: 10 minutes measurement, L-DOPA: 3 minutes). measurement). The relative tyrosinase activity at the time of adding each diluted sample was calculated with the tyrosinase activity value at the time of adding the control (solvent) as 100%. Kojic acid (final concentration: 17 μg / ml) was used as a positive control.
図6(a)はL-DOPAを基質としたチロシナーゼ活性率の水系又は非水系のプラセンタ抽出物の影響を示したグラフであり、図6(b)はL-チロシンを基質としたチロシナーゼ活性率の水系又は非水系のプラセンタ抽出物の影響を示したグラフである。
FIG. 6A is a graph showing the effect of the aqueous or non-aqueous placenta extract of the tyrosinase activity rate using L-DOPA as a substrate, and FIG. 6B is a graph showing the effect of the tyrosinase activity rate using L-tyrosine as a substrate. It is a graph which showed the influence of the water-based or non-water-based placenta extract of.
図6(a)に示すように、コントロール(溶媒)添加時のチロシナーゼ活性値(基質:L-DOPA)を100として相対値を算出したところ、水系又は非水系のいずれのプラセンタ抽出物においても、濃度依存的にチロシナーゼ活性率の減少が認められ、終濃度3.3mg/mlの水系又は非水系のプラセンタ抽出物の添加により、チロシナーゼ活性は有意に低下した。
As shown in FIG. 6 (a), when the relative value was calculated with the tyrosinase activity value (substrate: L-DOPA) at the time of adding the control (solvent) as 100, the relative value was calculated. A decrease in the tyrosinase activity rate was observed in a concentration-dependent manner, and the addition of an aqueous or non-aqueous placenta extract having a final concentration of 3.3 mg / ml significantly decreased the tyrosinase activity.
また図6(b)に示すように、コントロール(溶媒)添加時のチロシナーゼ活性値(基質:L-チロシン)を100として相対値を算出したところ、プラセンタ非水系抽出物(終濃度3.3mg/ml)において、有意なチロシナーゼ活性率の減少が認められた。プラセンタ水系抽出物においては、検討した濃度でのチロシナーゼ活性率の低下は認められなかった。
Further, as shown in FIG. 6 (b), when the relative value was calculated with the tyrosinase activity value (substrate: L-tyrosine) at the time of adding the control (solvent) as 100, the placenta non-aqueous extract (final concentration 3.3 mg / ml) was calculated. ), A significant decrease in the tyrosinase activity rate was observed. In the placenta aqueous extract, no decrease in the tyrosinase activity rate was observed at the examined concentration.
これらの結果から、本実施形態に係る水系又は非水系のプラセンタ抽出物には、チロシナーゼ阻害活性を示す成分が含まれていること、また、この効果はプラセンタ非水系抽出物において、より顕著な傾向にあることが示唆された。
From these results, the aqueous or non-aqueous placenta extract according to the present embodiment contains a component showing tyrosinase inhibitory activity, and this effect tends to be more remarkable in the placenta non-aqueous placenta extract. It was suggested that it was in.
〔7.メラニン生成・抑制能確認試験(第2試験)〕
次に、メラニン生成・抑制能確認試験の第2試験について説明する。先に説明した〔2.メラニン生成・抑制能確認試験(第1試験)〕では、プラセンタ水系抽出物と、プラセンタ非水系抽出物とについて産生されたメラニン量の確認を行ったが、本第2試験では、プラセンタ非水系抽出物に着目し、本発明者らが同抽出物より単離同定した各成分のそれぞれについて、メラニン生成・抑制能の確認を行った。 [7. Melanin production / suppression ability confirmation test (second test)]
Next, the second test of the melanin production / suppression ability confirmation test will be described. Explained earlier [2. In the melanin production / suppression ability confirmation test (1st test)], the amount of melanin produced in the placenta aqueous extract and the placenta non-aqueous extract was confirmed, but in this 2nd test, the placenta non-aqueous extraction was performed. Focusing on the substance, we confirmed the melanin production / inhibitory ability of each of the components isolated and identified by the present inventors from the extract.
次に、メラニン生成・抑制能確認試験の第2試験について説明する。先に説明した〔2.メラニン生成・抑制能確認試験(第1試験)〕では、プラセンタ水系抽出物と、プラセンタ非水系抽出物とについて産生されたメラニン量の確認を行ったが、本第2試験では、プラセンタ非水系抽出物に着目し、本発明者らが同抽出物より単離同定した各成分のそれぞれについて、メラニン生成・抑制能の確認を行った。 [7. Melanin production / suppression ability confirmation test (second test)]
Next, the second test of the melanin production / suppression ability confirmation test will be described. Explained earlier [2. In the melanin production / suppression ability confirmation test (1st test)], the amount of melanin produced in the placenta aqueous extract and the placenta non-aqueous extract was confirmed, but in this 2nd test, the placenta non-aqueous extraction was performed. Focusing on the substance, we confirmed the melanin production / inhibitory ability of each of the components isolated and identified by the present inventors from the extract.
本発明者らがプラセンタ非水系抽出物より単離した各成分は、以下の通りである。
The components isolated by the present inventors from the placenta non-aqueous extract are as follows.
本発明者による単離同定作業上、暫定的にHS-1と命名した一般式(i):
で表されるコレステロール(Cholesterol)。
In the work of isolation and identification by the present inventor, the general formula (i) tentatively named HS-1:
Cholesterol (Cholesterol).
同じく暫定的にHS-2と命名した一般式(ii):
で表されるコレステロール-7-オン(Cholesterol-7-one)。
The general formula (ii), also tentatively named HS-2:
Cholesterol-7-one, represented by.
暫定的にHS-3と命名した下記一般式(v):
で表される3β-ヒドロキシコレスタ-4-エン-6-オン(3β-hydroxycholest-4-ene-6-one)。
The following general formula (v), tentatively named HS-3:
Represented by 3β-hydroxycholest-4-ene-6-one (3β-hydroxycholest-4-ene-6-one).
暫定的にHS-4と命名した一般式(x):
で表される5α,6α-エポキシコレスタン-3β-オール(5α,6α-epoxycholestan-3β-ol)。
The general formula (x) tentatively named HS-4:
Represented by 5α, 6α-epoxycholestan-3β-ol (5α, 6α-epoxycholestan-3β-ol).
暫定的にHS-6と命名した下記一般式(vi):
で表される3β,6β-ジヒドロキシコレスタ-5-エン(3β,6β-dihydroxycholest-5-ene)。
The following general formula (vi), tentatively named HS-6:
Represented by 3β, 6β-dihydroxycholest-5-ene (3β, 6β-dihydroxycholest-5-ene).
暫定的にHS-7と命名した一般式(iii):
で表されるコレスタ-3,5-ジエン-7-オン(Cholesta-3,5-diene-7-one)。
The general formula (iii), tentatively named HS-7:
Represented by Cholesta-3,5-diene-7-one.
暫定的にHS-8と命名した一般式(vii):
で表されるコレステリルオレアート(Cholestereyl oleate)。
The general formula (vii) tentatively named HS-8:
Cholestereyl oleate represented by.
暫定的にHS-10と命名した一般式(viii):
で表されるパルミチン酸(Palmitic acid)。
The general formula (viii) tentatively named HS-10:
Palmitic acid represented by.
暫定的にHAS-1と命名した一般式(xi):
で表されるウリジン(Uridine)。
The general formula (xi) tentatively named HAS-1:
Uridine represented by.
暫定的にHAS-2と命名した下記一般式(iv):
で表されるウリジン一リン酸(Uridine Monophosphate)。
The following general formula (iv), tentatively named HAS-2:
Uridine monophosphate represented by.
暫定的にHSE-2と命名した下記一般式(ix):
で表されるチミン(Thymine)。
The following general formula (ix), tentatively named HSE-2:
Thymine represented by.
これら11種類の各単離成分HS-1、HS-2、HS-3、HS-4、HS-6、HS-7、HS-8、HS-10、HAS-1、HAS-2、HSE-2についてメラニン生成・抑制能の確認を行った。
Each of these 11 isolated components HS-1, HS-2, HS-3, HS-4, HS-6, HS-7, HS-8, HS-10, HAS-1, HAS-2, HSE- Regarding 2, the ability to produce and suppress melanin was confirmed.
試験方法は、前述の第1試験と同様であるが、添加するサンプルをプラセンタ抽出物ではなく、これら各成分が各々最終濃度が100μMになるように、ジメチルスルホキシド溶液に溶解後、培養液に添加した。
The test method is the same as in the first test described above, but the sample to be added is not a placenta extract, but is added to the culture solution after dissolving in a dimethyl sulfoxide solution so that each of these components has a final concentration of 100 μM. did.
図7(a)は各サンプルにおける細胞生存率を示すグラフであり、図7(b)はコントロールに対する細胞あたりの細胞外メラニン産生比、図7(c)はコントロールに対する細胞あたりの細胞内メラニン産生比を示すグラフである。また表1に、細胞生存率を加味した細胞あたりの細胞外・細胞内メラニン生産比の表を示す。なお、表1中に示す"type"において、「A+」はコントロールに対し20%を超える割合でメラニン産生量が増大していることを示し、「A-」はコントロールに対し20%以下の割合ではあるがメラニン産生量が増大していることを示し、「B++」はアルブチン(100 μM)と同等程度にメラニン産生量が減少していることを示し、「B+」はコントロールに対し20%を超える割合でメラニン産生量が減少していることを示し、「B-」はコントロールに対し20%以下の割合ではあるがメラニン産生量が減少していることを示している。
7 (a) is a graph showing the cell viability in each sample, FIG. 7 (b) is the extracellular melanin production ratio per cell to the control, and FIG. 7 (c) is the intracellular melanin production per cell to the control. It is a graph which shows the ratio. Table 1 shows a table of extracellular / intracellular melanin production ratios per cell, including cell viability. In the "type" shown in Table 1, "A + " indicates that the amount of melanin produced increases at a rate of more than 20% with respect to the control, and "A-" indicates that the amount of melanin produced increases by 20% or less with respect to the control. Although it is a percentage, it indicates that the amount of melanin production is increasing, "B ++ " indicates that the amount of melanin production is decreasing to the same extent as albutin (100 μM), and "B ++ " indicates that the amount of melanin production is decreasing with respect to the control. It indicates that the melanin production is decreased at a rate of more than 20%, and "B-" indicates that the melanin production is decreased at a rate of 20% or less with respect to the control.
まず図7(a)の細胞生存率について検討すると、HS-1(コレステロール)、HS-2(コレステロール-7-オン)およびHS-4(5α,6α-エポキシコレスタン-3β-オール)ではコントロール(DMSO)と比較して、細胞生存率は劇的に減少した(HS-1: 55.7 %、HS-2: 36.5 %、HS-4: 28.6 %)。また、HAS-2(ウリジン一リン酸)もコントロール(DMSO)と比較して、細胞生存率は有意に減少していた(HAS-2: 79.8 %)。
First, when the cell viability in FIG. 7 (a) was examined, it was controlled by HS-1 (cholesterol), HS-2 (cholesterol-7-on) and HS-4 (5α, 6α-epoxycolestane-3β-ol). Compared with (DMSO), cell viability decreased dramatically (HS-1: 55.7%, HS-2: 36.5%, HS-4: 28.6%). In addition, HAS-2 (uridine monophosphate) also had a significantly reduced cell viability compared to control (DMSO) (HAS-2: 79.8%).
一方で、HS-3(3β-ヒドロキシコレスタ-4-エン-6-オン)、HS-7(コレスタ-3,5-ジエン-7-オン)、HS-10(パルミチン酸)およびHSE-2(チミン)は、コントロール(DMSO)と比較して細胞生存率が有意に上昇することが確認された(HS-3: 109.4 %、HS-7: 113.3 %、HS-10: 110.3 %、HSE-2: 114.3 %)。これらの結果を踏まえると、HS-3、HS-6、HS-7、HS-8、HS-10、HSE-2およびHAS-1はB16メラノーマ細胞に対して細胞毒性は示さないことが確認出来た。
On the other hand, HS-3 (3β-hydroxycholesta-4-ene-6-on), HS-7 (cholesta-3,5-diene-7-on), HS-10 (palmitic acid) and HSE-2. It was confirmed that (thymine) significantly increased cell viability compared to control (DMSO) (HS-3: 109.4%, HS-7: 113.3%, HS-10: 110.3%, HSE- 2: 114.3%). Based on these results, it can be confirmed that HS-3, HS-6, HS-7, HS-8, HS-10, HSE-2 and HAS-1 do not show cytotoxicity to B16 melanoma cells. rice field.
また、図7(b)によれば、HS-3、HS-4、HS-6、HS-8、HS-10、HSE-2、HAS-1およびHAS-2添加では、1細胞あたりの細胞外メラニン産生比がコントロール(DMSO)と比較して抑制されていた。特に、HS-3、HS-6、HSE-2およびHAS-2では、ポジティブコントロールであるアルブチン(100 μM)と同等の細胞外メラニン産生抑制が確認出来た。一方、HS-1、HS-2およびHS-7添加では、コントロール(DMSO)と比較して1細胞あたりの細胞外メラニン産生比が上昇していた。
Further, according to FIG. 7 (b), when HS-3, HS-4, HS-6, HS-8, HS-10, HSE-2, HAS-1 and HAS-2 were added, the cells per cell were added. The external melanin production ratio was suppressed compared to control (DMSO). In particular, HS-3, HS-6, HSE-2 and HAS-2 were confirmed to suppress extracellular melanin production equivalent to the positive control arbutin (100 μM). On the other hand, the addition of HS-1, HS-2 and HS-7 increased the extracellular melanin production ratio per cell as compared with control (DMSO).
また、図7(c)によれば、HS-3、HS-4、HS-8、HS-10、HSE-2およびHAS-1添加により1細胞あたりの細胞内メラニン産生比がコントロール(DMSO)と比較して抑制されていた。一方、HS-1、HS-2、HS-6、HS-7およびHAS-2添加では、コントロール(DMSO)と比較して1細胞あたりの細胞外メラニン産生比が上昇していた。
In addition, according to FIG. 7 (c), the intracellular melanin production ratio per cell is controlled by the addition of HS-3, HS-4, HS-8, HS-10, HSE-2 and HAS-1 (DMSO). Was suppressed compared to. On the other hand, the addition of HS-1, HS-2, HS-6, HS-7 and HAS-2 increased the extracellular melanin production ratio per cell as compared with control (DMSO).
更に、表1を参照しつつ各成分毎に検討すると、まずHS-1(コレステロール)は、細胞内及び細胞外の両方において、コントロールに対し20%を超えるメラニン産生量の増大が認められた。
Furthermore, when examined for each component with reference to Table 1, first, HS-1 (cholesterol) was found to increase melanin production by more than 20% with respect to the control both intracellularly and extracellularly.
またHS-2(コレステロール-7-オン)は、細胞外においてコントロールに対し20%を超えるメラニン産生量の増大が認められた。また、細胞内においてはコントロールに対して20%以下ではあるもののメラニン産生量の増大が認められた。
In addition, HS-2 (cholesterol-7-on) was found to increase melanin production by more than 20% relative to the control extracellularly. In addition, an increase in melanin production was observed in the cells, although it was 20% or less of the control.
HS-3(3β-ヒドロキシコレスタ-4-エン-6-オン)は、細胞内及び細胞外の両方において、コントロールに対し20%を超える割合でのメラニン産生量の減少が認められた。特に細胞外においては、アルブチン(100 μM)と同等程度のメラニン産生量の減少が認められた。
HS-3 (3β-hydroxycholesta-4-en-6-one) showed a decrease in melanin production at a rate of more than 20% relative to the control both intracellularly and extracellularly. In particular, extracellularly, a decrease in melanin production equivalent to that of arbutin (100 μM) was observed.
HS-4(5α,6α-エポキシコレスタン-3β-オール)は、細胞内及び細胞外の両方において、コントロールに対し20%以下の割合ではあるがメラニン産生量の減少が認められた。
HS-4 (5α, 6α-epoxy-reductase-3β-ol) showed a decrease in melanin production both intracellularly and extracellularly, albeit at a rate of 20% or less with respect to the control.
HS-6(3β,6β-ジヒドロキシコレスタ-5-エン)は、細胞外においてコントロールに対し20%を超える割合、特にアルブチン(100 μM)と同等程度のメラニン産生量の減少が認められたが、細胞内においては逆に、コントロールに対し20%以下の割合ではあるがメラニン産生量の増大が認められた。
HS-6 (3β, 6β-dihydroxycholesta-5-ene) showed a decrease in melanin production at a rate of more than 20% relative to the control extracellularly, especially to the same extent as arbutin (100 μM). On the contrary, in the cells, an increase in melanin production was observed, albeit at a rate of 20% or less with respect to the control.
HS-7(コレスタ-3,5-ジエン-7-オン)は、細胞内及び細胞外の両方において、コントロールに対し20%を超えるメラニン産生量の増大が認められた。
HS-7 (Cholesta-3,5-diene-7-on) showed an increase in melanin production of more than 20% relative to the control both intracellularly and extracellularly.
HS-8(コレステリルオレアート)は、細胞外においてはコントロールに対し20%以下の割合ではあるがメラニン産生量の減少が認められ、また、細胞内においてコントロールに対し20%を超える割合でのメラニン産生量の減少が認められた。
HS-8 (cholesteryl oleate) has a decrease in melanin production in the intracellular ratio of 20% or less with respect to the control, and melanin in the intracellular ratio of more than 20% with respect to the control. A decrease in production was observed.
HS-10は(パルミチン酸)は、細胞内及び細胞外の両方において、コントロールに対し20%を超える割合でのメラニン産生量の減少が認められた。
HS-10 (palmitic acid) showed a decrease in melanin production at a rate of more than 20% relative to the control, both intracellularly and extracellularly.
HSE-2(チミン)は、細胞内及び細胞外の両方において、コントロールに対し20%を超える割合でのメラニン産生量の減少が認められた。特に細胞外においては、アルブチン(100 μM)と同等程度のメラニン産生量の減少が認められた。
HSE-2 (thymine) showed a decrease in melanin production at a rate of more than 20% relative to the control, both intracellularly and extracellularly. In particular, extracellularly, a decrease in melanin production equivalent to that of arbutin (100 μM) was observed.
HAS-1(ウリジン)は、細胞内及び細胞外の両方においても、細胞内及び細胞外の両方において、コントロールに対し20%以下の割合ではあるがメラニン産生量の減少が認められた。
HAS-1 (uridine) showed a decrease in melanin production both intracellularly and extracellularly, both intracellularly and extracellularly, at a rate of 20% or less with respect to the control.
HAS-2(ウリジン一リン酸)は、細胞外においてコントロールに対し20%を超える割合、特にアルブチン(100 μM)と同等程度のメラニン産生量の減少が認められたが、細胞内においては逆に、コントロールに対し20%を超える割合でのメラニン産生量の増大が認められた。
HAS-2 (uridine monophosphate) showed a decrease in melanin production at a rate of more than 20% relative to the control outside the cell, especially about the same level as arbutin (100 μM), but on the contrary, inside the cell. , An increase in melanin production was observed at a rate of more than 20% with respect to the control.
そして、これらの結果を総合的に勘案すると、コレステロール(HS-1)、コレステロール-7-オン(HS-2)、3β,6β-ジヒドロキシコレスタ-5-エン(HS-6)、コレスタ-3,5-ジエン-7-オン(HS-7)、ウリジン一リン酸(HAS-2)の各成分は、コントロール(DMSO)と比較してメラニンの生成を促進することが示された。より詳細には、コレステロール(HS-1)、コレステロール-7-オン(HS-2)、コレスタ-3,5-ジエン-7-オン(HS-7)の各成分は細胞内及び細胞外の両方において、また、3β,6β-ジヒドロキシコレスタ-5-エン(HS-6)、ウリジン一リン酸(HAS-2)の各成分は細胞内においてメラニンの生成を促進することが示された。このことは、化粧料や機能性食品中において、メラニンの生成促進を目的とする有効成分や保健機能成分としてこれらの成分が有用であることを示すものといえる。
Considering these results comprehensively, cholesterol (HS-1), cholesterol-7-one (HS-2), 3β, 6β-dihydroxycholesta-5-ene (HS-6), cholesterol-3 , 5-Diene-7-one (HS-7), uridine monophosphate (HAS-2) components have been shown to promote melanin production compared to control (DMSO). More specifically, the components of cholesterol (HS-1), cholesterol-7-on (HS-2), and cholesterol-3,5-diene-7-on (HS-7) are both intracellular and extracellular. In addition, it was shown that each component of 3β, 6β-dihydroxycholester-5-ene (HS-6) and uridine monophosphate (HAS-2) promotes the production of melanin in the cell. This can be said to indicate that these ingredients are useful as active ingredients and health functional ingredients for the purpose of promoting the production of melanin in cosmetics and functional foods.
また同様に、3β-ヒドロキシコレスタ-4-エン-6-オン(HS-3)、5α,6α-エポキシコレスタン-3β-オール(HS-4)、3β,6β-ジヒドロキシコレスタ-5-エン(HS-6)、コレステリルオレアート(HS-8)、パルミチン酸(HS-10)、チミン(HSE-2)、ウリジン(HAS-1)、ウリジン一リン酸(HAS-2)は、メラニンの生成を抑制することが示された。より詳細には、3β-ヒドロキシコレスタ-4-エン-6-オン(HS-3)、5α,6α-エポキシコレスタン-3β-オール(HS-4)、コレステリルオレアート(HS-8)、パルミチン酸(HS-10)、チミン(HSE-2)、ウリジン(HAS-1)の各成分は細胞内及び細胞外の両方においてメラニンの生成を抑制でき、中でもHS-3、HS-8、HS-10、HSE-2、HAS-1は細胞外メラニンの生成を有意に抑制し、また、3β,6β-ジヒドロキシコレスタ-5-エン(HS-6)、ウリジン一リン酸(HAS-2)の各成分は細胞外においてメラニンの生成を抑制することが示された。このことは、化粧料や機能性食品中において、メラニンの生成抑制、ひいては美白を目的とする有効成分や保健機能成分としてこれらの成分が有用であることを示すものといえる。
Similarly, 3β-hydroxycholesta-4-ene-6-one (HS-3), 5α, 6α-epoxycholestane-3β-ol (HS-4), 3β, 6β-dihydroxycholesta-5- En (HS-6), cholesteryl oleate (HS-8), palmitic acid (HS-10), thymine (HSE-2), uridine (HAS-1), uridine monophosphate (HAS-2) are melanin It was shown to suppress the production of. More specifically, 3β-hydroxycholesta-4-en-6-one (HS-3), 5α, 6α-epoxycholestane-3β-ol (HS-4), cholesteryl oleart (HS-8), The components of palmitic acid (HS-10), timine (HSE-2), and uridine (HAS-1) can suppress the production of melanin both intracellularly and extracellularly, especially HS-3, HS-8, and HS. -10, HSE-2, HAS-1 significantly suppress the production of extracellular melanin, and 3β, 6β-dihydroxycholesta-5-ene (HS-6), uridine monophosphate (HAS-2). Each component of is shown to suppress the production of melanin extracellularly. This can be said to indicate that these ingredients are useful as active ingredients and health functional ingredients for the purpose of suppressing the production of melanin and, by extension, whitening in cosmetics and functional foods.
また興味深いことに、3β,6β-ジヒドロキシコレスタ-5-エン(HS-6)や、ウリジン一リン酸(HAS-2)は、細胞内におけるメラニンの生成は促進させるが、細胞外におけるメラニンの生成はアルブチン(100 μM)と同等程度に抑制する。このことは、例えば細胞内については紫外線に対しメラニンによる防護を図りつつ、細胞外におけるメラニン分泌を抑制して過度な色素沈着等を抑制する化粧料や機能性食品、更には医薬品等の製剤としての効果が期待される。
Interestingly, 3β, 6β-dihydroxycholesta-5-ene (HS-6) and uridine monophosphate (HAS-2) promote intracellular melanin production, but extracellular melanin. Production is suppressed to the same extent as arbutin (100 μM). This means, for example, as a pharmaceutical product, a functional food, or a pharmaceutical product that suppresses the secretion of melanin outside the cell and suppresses excessive pigmentation while protecting the intracellular body with ultraviolet rays by melanin. The effect of is expected.
上述してきたように、本実施形態に係る発明によれば、化粧料又は機能性食品におけるメラニン生成を抑制又は促進する成分、ヒアルロン酸産生促進成分、コラーゲン産生促進成分、エラスターゼ活性阻害成分、抗アレルギー活性促進成分、チロシナーゼ活性阻害成分から選ばれるいずれかの成分の添加材料として、プラセンタ抽出物を使用することとしたため、より明確な目的の下でのプラセンタ抽出物の新たな使用方法を提供することができる。
As described above, according to the invention according to the present embodiment, a component that suppresses or promotes melanin production in cosmetics or functional foods, a hyaluronic acid production promoting component, a collagen production promoting component, an elastase activity inhibiting component, and an antiallergic substance. Since the placenta extract is used as an additive material for any of the components selected from the activity promoting component and the tyrosinase activity inhibiting component, it is necessary to provide a new method of using the placenta extract for a clearer purpose. Can be done.
最後に、上述した各実施の形態の説明は本発明の一例であり、本発明は上述の実施の形態に限定されることはない。このため、上述した各実施の形態以外であっても、本発明に係る技術的思想を逸脱しない範囲であれば、設計等に応じて種々の変更が可能であることは勿論である。
Finally, the description of each of the above-described embodiments is an example of the present invention, and the present invention is not limited to the above-mentioned embodiments. Therefore, it goes without saying that various changes can be made according to the design and the like as long as the technical idea of the present invention is not deviated from the above-described embodiments.
Claims (5)
- 化粧料又は機能性食品におけるメラニン生成を抑制又は促進する成分、ヒアルロン酸産生促進成分、コラーゲン産生促進成分、エラスターゼ活性阻害成分、抗アレルギー活性促進成分、チロシナーゼ活性阻害成分から選ばれるいずれかの成分の添加材料としてのプラセンタ抽出物の使用。 A component selected from a component that suppresses or promotes melanin production in cosmetics or functional foods, a hyaluronic acid production promoting component, a collagen production promoting component, an elastase activity inhibiting component, an antiallergic activity promoting component, and a tyrosinase activity inhibiting component. Use of placenta extract as an additive material.
- 前記プラセンタ抽出物は、ジメチルスルホキシド、エタノール、メタノール、ヘキサンから選ばれるいずれか1つ若しくはいずれか2つ以上の混合液からなる非水系特定溶媒に対して溶解性を示す成分群よりなることを特徴とする請求項1に記載のプラセンタ抽出物の使用。 The placenta extract is characterized by consisting of a group of components exhibiting solubility in a non-aqueous specific solvent consisting of a mixture of any one or two or more selected from dimethyl sulfoxide, ethanol, methanol and hexane. Use of the placenta extract according to claim 1.
- 前記プラセンタ抽出物は、水を主成分とする水系溶媒に対して溶解性を示す成分群よりなることを特徴とする請求項1に記載のプラセンタ抽出物の使用。 The use of the placenta extract according to claim 1, wherein the placenta extract comprises a group of components exhibiting solubility in an aqueous solvent containing water as a main component.
- 下記一般式(i):
- 下記一般式(iv):
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