Methods for using uridine monophosphate or 3β,6β-dihydroxycholest-5-ene

Abstract

To provide a novel method of using a placenta extract with a more defined purpose than before.SOLUTION: Provided is use of a placenta extract as additive material for any of components 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. The placenta extract consists of a group of components that are soluble in a specific non-aqueous solvent. Alternatively, the extract consists of a group of components that are soluble in an aqueous solvent containing water as a main component.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to the use of placenta extract. [Background technology]

[0002] Health and beauty are of interest to many people regardless of the era. Therefore, various materials have been proposed as being good for health or beauty.

[0003] In particular, mammalian placenta and its extracts have been attracting attention from men and women of all ages as new materials that promote both health and beauty (see, for example, Patent Document 1). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] JP 2006-158214 A Summary of the Invention [Problem to be solved by the invention]

[0005] As described above, placenta is used mainly in the form of an extract in health foods to support health and in cosmetics to support beauty, and is well-received by users for its benefits in terms of daily health and skin condition.

[0006] However, health foods and cosmetics proposed thus far have sometimes been used without necessarily clarifying the significance of including placenta.

[0007] The present invention has been made in view of the above circumstances, and provides a new method for using a placenta extract with a more specific purpose. [Means for solving the problem]

[0011] In order to solve the above-mentioned problems, the present invention provides a compound represented by the following general formula (iv): [ka] or the following general formula (vi): [ka] The present invention relates to the use of 3β,6β-dihydroxycholest-5-ene, which is represented by the formula (1), as an active ingredient or health functional ingredient in cosmetics or functional foods for the purpose of increasing intracellular melanin production to provide protection against ultraviolet rays through melanin, while suppressing extracellular melanin secretion to inhibit excessive pigmentation, etc. (However, this does not include uses that are related to the treatment of humans.) . Effect of the Invention

[0016] According to the present invention, a compound represented by the following general formula (iv): [ka] or the following general formula (vi): [ka] If 3β,6β-dihydroxycholest-5-ene represented by the following formula is used as an active ingredient or health functional ingredient in cosmetics or functional foods for suppressing excessive pigmentation and the like by suppressing melanin secretion outside cells while increasing melanin production inside cells to provide protection against ultraviolet rays with melanin, (However, this does not include uses that are related to the treatment of humans.) The above-mentioned components can provide new cosmetics and functional foods that increase the amount of melanin produced inside cells while suppressing melanin secretion outside cells. [Brief description of the drawings]

[0017] [Figure 1] FIG. 2 is an explanatory diagram showing the test results of the melanin production / inhibition ability confirmation test (first test). [Diagram 2] FIG. 1 is an explanatory diagram showing the test results of a hyaluronic acid production test. [Diagram 3] FIG. 1 is an explanatory diagram showing the test results of a collagen production test. [Figure 4] FIG. 1 is an explanatory diagram showing the test results of an elastase activity inhibition test. [Diagram 5] FIG. 1 is an explanatory diagram showing the test results of an antiallergic activity evaluation test. [Figure 6] FIG. 2 is an explanatory diagram showing the test results of a tyrosinase inhibitory activity test. [Figure 7] FIG. 2 is an explanatory diagram showing the test results of the melanin production / inhibition ability confirmation test (second test). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] The present invention relates to a method for using placenta extract, and more specifically, to provide a new method for using placenta extract with a clearer purpose in health foods and cosmetics, where the significance of adding placenta extract has sometimes been unclear until now.

[0019] In particular, the use according to this embodiment is characterized in that a placenta extract is used as an additive material for any of the components selected from a component that suppresses or promotes melanin production in cosmetics or functional foods, a component that promotes hyaluronic acid production, a component that promotes collagen production, a component that inhibits elastase activity, a component that promotes antiallergic activity, and a component that inhibits tyrosinase activity.

[0020] A placenta extract is an extract of a mammalian placenta (placenta). For example, when extraction is performed using a solvent, the placenta extract contains the extract or a dried product of the extract.

[0021] The placenta from which the placenta extract is extracted is not particularly limited, and placentas from various mammals including pigs, horses, and humans can be used.

[0022] Cosmetics include not only so-called makeup cosmetics, but also basic cosmetics, hair tonics, perfumes, toothpaste, shampoos, rinses, soaps and detergents used for washing the body, bath additives and other toiletry products, as well as medicated cosmetics that claim to have preventive effects, etc.

[0023] Makeup cosmetics include foundation, eyebrow pencil, mascara, eye shadow, eyeliner, lipstick, gloss, blush, powder, nail polish, etc., while basic cosmetics include, for example, lotion, milky lotion, facial cleanser, cleansing, serum, cream, etc.

[0024] Furthermore, functional foods are a concept that encompasses all foods that do not contain pharmaceutical ingredients and are believed to contribute to maintaining and improving health, and include, for example, nutritional supplements, health supplements, nutritionally adjusted foods, as well as general foods such as so-called supplements, and health functional foods such as foods for specified health uses, foods with nutrient functions, and foods with functional claims.

[0025] The above explanations of the cosmetics and functional foods are merely examples of equivalent articles, etc., to aid in the understanding of the present invention, and the interpretation of each term is not limited to these listed articles, etc. However, the applicant will not be precluded from limiting the present invention to these articles, etc., when granting the patent for this application.

[0026] In this embodiment, the placenta extract is used 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 antiallergic activity, and components that inhibit tyrosinase activity.

[0027] Here, the use as an additive material means not only adding the placenta extract to cosmetics or functional foods so as to contain the above-mentioned components, but also using the placenta extract as an extraction material for the above-mentioned components, in which the extracted components are added to cosmetics or functional foods.

[0028] The purpose of using the placenta extract is to provide 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 antiallergic activity, and components that inhibit tyrosinase activity.

[0029] These components will be described in detail later, but they have been clarified by the inventors' intensive research, and the placenta extract itself exerts the functions of these components. However, it is not necessary for all the components constituting the placenta extract to exert the same functions, and in reality, the action may be exerted by a single substance contained in the placenta extract, or by multiple substances.

[0030] The component that inhibits or promotes melanin production may be used externally in the form of a cosmetic and / or orally in the form of a functional food (hereinafter referred to as external / oral use) to inhibit or promote melanin production in the skin. Such components can be confirmed by the melanin production test described below.

[0031] The hyaluronic acid production-promoting component or collagen production-promoting component may be used externally or orally as long as it can promote the production of hyaluronic acid or collagen, and this can be confirmed, for example, by the collagen and hyaluronic acid production tests described below.

[0032] The elastase activity inhibiting component should be able to inhibit elastase activity and decompose elastin when used topically or orally, and this can be confirmed, for example, by the elastase activity inhibition test described below.

[0033] The anti-allergic activity-promoting component should be capable of suppressing allergic symptoms when applied topically or orally, and this can be confirmed, for example, by measuring β-hexosaminidase activity in the anti-allergic activity test described below.

[0034] The tyrosinase activity inhibitor should be capable of inhibiting tyrosinase activity and preventing the fixation of melanin when used externally or orally, and this can be confirmed, for example, by the tyrosinase inhibitory activity test described below.

[0035] The placenta extract may be composed of a component group that is soluble in a non-aqueous specific solvent selected from dimethyl sulfoxide, ethanol, methanol, and hexane, or a mixture of two or more of these.

[0036] That is, the placenta extract can be an extract extracted with a non-aqueous specific solvent.

[0037] Furthermore, by using a placenta extract consisting of a group of components that are soluble in non-aqueous specific solvents as each of the above-mentioned components, it is possible to exhibit the effect of promoting melanin production, and it is also possible to exhibit a more pronounced elastase activity inhibitory effect than the elastase activity inhibitory components contained in the group of components that are soluble in aqueous solvents.

[0038] The placenta extract may be composed of a group of components that are soluble in an aqueous solvent containing water as a main component, in other words, the placenta extract may be an extract extracted with an aqueous solvent.

[0039] Here, the aqueous solvent is a solvent whose main component is water, and includes, in addition to water itself, solvents containing water at a ratio of, for example, 50 w / w% or more. The components other than water that constitute the aqueous solvent can be, for example, solid (powder, etc.) components that are soluble in water, or liquids that are miscible with water, such as alcohol.

[0040] In this way, by using a placenta extract consisting of a group of components that are soluble in aqueous solvents as each of the above-mentioned components, it is possible to exhibit the effect of suppressing melanin production and each of the effects that are more pronounced than the hyaluronic acid production-promoting components and collagen production-promoting components contained in the group of components that are soluble in non-aqueous specific solvents.

[0041] The solvent may also be a mixed solvent of an aqueous solvent and a non-aqueous specific solvent. In other words, by using a mixed solvent containing less than 50 w / w% water and the remainder being a non-aqueous specific solvent or a solid (powder, etc.) that is soluble in the mixed solvent to be prepared, a placenta extract having a composition ratio different from that of the placenta extract described above can be prepared.

[0042] The present application also provides a method for using the component discovered by the inventor in the placenta extract as an active ingredient in cosmetics for the purpose of promoting melanin production or as a health functional ingredient in functional foods.

[0043] Specifically, the compound represented by the following general formula (i): [ka] or the following general formula (ii): [ka] or the following general formula (iii): [ka] Cholesta-3,5-diene-7-one represented by the following general formula (iv): [ka] The melanin production promoting ability of the substances (i) to (iv) will be described later with reference to the data.

[0044] The present application also provides a compound represented by the following general formula (iv): [ka] or the following general formula (v): [ka] or the following general formula (vi): [ka] or 3β,6β-dihydroxycholest-5-ene represented by the following general formula (vii): [ka] or the following general formula (viii): [ka] or palmitic acid represented by the following general formula (ix): [ka] The melanin production inhibitory abilities of the substances (iv) to (ix) will be described later with reference to the data.

[0045] Furthermore, by using these components (i) to (ix) as active ingredients or health functional ingredients for the purpose of promoting or inhibiting melanin production in cosmetics or functional foods, it is possible to provide new cosmetics or functional foods that promote or inhibit melanin production using the above-mentioned components.

[0046] The methods of use according to the present embodiment will be further described below with reference to experimental results. Note that, although the following describes an example in which a placenta extract extracted from horse placenta (hereinafter referred to as horse placenta extract) is used, the origin of placenta is not limited to horse.

[0047] 1. Preparation of placenta extract In recent years, various natural ingredients have been used as functional ingredients in cosmetics and functional foods. Among them, placenta, which is the placenta of mammals, has attracted attention as a material that contributes to beauty and health maintenance.

[0048] However, as mentioned above, there are still few scientific studies on the functionality of placenta and its extracts. Therefore, here, we first prepared placenta extracts using horse placenta as a representative example, in order to carry out several functional evaluation tests to confirm their effectiveness.

[0049] Horse placenta powder, a freeze-dried powder of horse placenta, was used as the raw material for the horse placenta extract.

[0050] First, 100 mg of horse placenta powder was weighed into a 1.5 ml tube, 1 ml of extraction solvent was added, and the mixture was stirred at room temperature for 30 minutes. The extraction solvent used was sterilized ultrapure water as an aqueous solvent and dimethyl sulfoxide (hereinafter, also referred to as DMSO) as a non-aqueous specific solvent.

[0051] After stirring, the tube is left at room temperature, and the obtained supernatant is called horse placenta extract. This horse placenta extract is called 100mg / ml sample because it is prepared by adding 1ml of extraction solvent to 100mg of placenta powder. In addition, in the following tests, the diluted sample prepared by appropriately diluting the 100mg / ml sample is expressed as 1mg / ml sample if it is 100 times diluted, and as 10μg / ml sample if it is 10000 times diluted.

[0052] [2. Melanin production and inhibition test (1st test)] Melanin is a general term for pigments formed by polymerizing phenolic substances. Melanin present in human skin is in the form of a polymer of various indole compounds synthesized from tyrosine.

[0053] The most important role of melanin is to protect against ultraviolet rays, suppressing sun damage and the onset of malignant tumors. Other functions of melanin are known to be the absorption of reactive oxygen species that are harmful to the body and the uptake of metals and drugs.

[0054] Melanin-containing cells gradually disperse into the upper layer of the stratum corneum, and the adhesion between the cells is broken down by the action of lipid metabolic enzymes (such as lipase) and proteolytic enzymes (such as elastase), eventually causing the stratum corneum to peel off and fall off.

[0055] In this study, human melanoma cells (G361 cell line) were used as model cells, and the cells were co-cultured with the sample, and the melanin produced in the culture medium and the cell viability were measured.

[0056] Regarding the cell culture method, human melanoma cells (G361 cell line) were precultured using McCoy's 5A medium (containing 1% penicillin-streptomycin and 10% fetal bovine serum (FBS)).

[0057] Then, 1.0 × 10 5The cells were seeded at a concentration of 0.5 ml / ml and cultured overnight in a CO2 incubator (37°C, 5% CO2).

[0058] After overnight, the medium was removed, and 0.5 ml of fresh medium and 2.5 μl of each placenta extract sample diluted to a specified concentration (hereinafter referred to as each diluted sample) were added, and the cells were again cultured in a CO2 incubator for 72 hours.

[0059] The melanin production and inhibition ability was confirmed by measuring the amount of melanin produced. Specifically, the medium was removed from the plate after 72 hours of culture, and each well was washed with PBS (Phosphate Buffered Saline), after which 1 ml of 1M NaOH solution was added to each well and reacted at room temperature for 4 hours. The amount of melanin produced was determined by measuring the absorbance at 405 nm using a plate reader.

[0060] The cell viability was measured by the MTT method. After 72 hours of culture, the medium was removed from the plate, and 1 ml of fresh medium was added to each well. Then, 200 μl of MTT staining solution (5 mg / ml in PBS) was added to each well, and the plates were left to stand in a CO2 incubator for 4 hours.

[0061] Then, the medium was removed, 1 ml of hydrochloric acid-isopropanol solution was added to each well, and formazan was completely dissolved by pipetting. The absorbance at 570 nm was measured using a microplate reader to calculate the cell survival rate.

[0062] FIG. 1 is a graph showing the melanin production ratio relative to the control, where FIG. 1(a) shows the melanin production ratio by a placenta extract extracted with an aqueous solvent (hereinafter also referred to as an aqueous placenta extract), and FIG. 1(b) shows the melanin production ratio by a placenta extract extracted with a specific non-aqueous solvent (hereinafter also referred to as a non-aqueous placenta extract).

[0063] As shown in Figure 1(a), when the aqueous placenta extract was examined using 400 μg / ml samples, 100 μg / ml samples, and 10 μg / ml samples, a melanin production inhibitory effect of approximately 20% was observed at all concentrations examined.

[0064] This suggests that the aqueous placenta extract contains components that inhibit melanin production, in other words, components that contribute to skin whitening.

[0065] In contrast, when the diluted samples of the non-aqueous placenta extract were examined, a melanin production promoting effect of approximately 20 to 40% was observed at all concentrations examined, as shown in Figure 1(b).

[0066] From this, it is believed that the non-aqueous placenta extract contains components that promote melanin production and has effects such as maintaining the black color of hair.

[0067] In addition, taking into account the results for these aqueous and non-aqueous placenta extracts, it is extremely interesting to note that aqueous placenta extracts and non-aqueous placenta extracts have completely opposite properties, namely, "inhibition" and "promotion," respectively, in the production of melanin.

[0068] [3. Collagen and hyaluronic acid production test] Collagen is a structural protein that forms connective tissues such as the skin of animals. Collagen contributes to the structure of the skin and is important for keeping the skin youthful and wrinkle-free.

[0069] Collagen is produced by fibroblasts present in the dermis of the skin, and if collagen production by fibroblasts can be promoted, youthful skin can be maintained. Therefore, it is believed that a component that promotes collagen production by fibroblasts is useful as an anti-aging cosmetic material.

[0070] Hyaluronic acid is also produced mainly by fibroblasts, the main cells in the dermis of the skin, and other cells of connective tissues. It is a major component of the extracellular matrix and is involved in various intercellular interactions. It is also present in high concentrations in synovial fluid, where it is responsible for normal water retention and lubrication of joints.

[0071] In this test, the collagen and hyaluronic acid production ability of a fibroblast culture system when the placenta extract according to this embodiment is added is measured, and the effect of the placenta extract in retaining moisture in the skin is evaluated. In this test method, adult human dermal fibroblasts (NHDF-Ad) were used as model cells. 1.0 x 10 in a microwell plate 4 The cells were seeded at 100 cells / well and pre-cultured for 24 hours in a CO2 incubator (37°C, 5% CO2).

[0072] After pre-culture, the medium was removed, and new medium and each diluted sample diluted to a specified concentration were added, and the cells were again cultured in a CO2 incubator for 72 hours. The amount of collagen and hyaluronic acid produced in the culture medium was measured using a commercially available ELISA kit. Similarly, an MTT test was performed using the same cells.

[0073] FIG. 2 is a graph showing the hyaluronic acid production ratio relative to the control, where FIG. 2(a) shows the hyaluronic acid production ratio by an aqueous placenta extract, and FIG. 2(b) shows the hyaluronic acid production ratio by a non-aqueous placenta extract.

[0074] As shown in Figure 2(a), the addition of the aqueous placenta extract increased the production of hyaluronic acid, suggesting that the aqueous placenta extract contains ingredients that promote the production of hyaluronic acid.

[0075] In contrast, when the diluted samples of the non-aqueous placenta extract were examined, as shown in Figure 2(b), no increase in hyaluronic acid production was observed at any of the concentrations examined. In other words, it was considered that the non-aqueous placenta extract does not have the effect of promoting the production of hyaluronic acid.

[0076] FIG. 3 is a graph showing the collagen production ratio relative to the control, where FIG. 3(a) shows the collagen production ratio by an aqueous placenta extract, and FIG. 3(b) shows the collagen production ratio by a non-aqueous placenta extract.

[0077] As shown in Figure 3(a), a significant increase in collagen production was observed in the aqueous placenta extract. Also, an increase in collagen production was observed in the non-aqueous placenta extract when a 100μg / ml sample was added.

[0078] These findings suggest that both aqueous and non-aqueous placenta extracts contain components that promote collagen production.

[0079] Furthermore, the results of this study suggest that aqueous placenta extracts contain ingredients that promote the production of hyaluronic acid and collagen, while non-aqueous placenta extracts contain ingredients that promote collagen production.

[0080] 4. Elastase activity inhibition test The cross-linked structure of collagen and elastin present in the dermis plays an important role in maintaining skin elasticity. Elastase, an elastin-degrading enzyme, is induced by stimuli such as ultraviolet rays, and when elastin is degraded, the cross-linked structure becomes brittle and skin elasticity is lost. In this test, the skin elasticity-maintaining function of placenta extract is evaluated by measuring elastase inhibitory activity.

[0081] In this study, a cell lysate was used in the experiment. To prepare the cell lysate, human dermal fibroblasts (NHDF-Ad) precultured until confluent were washed with PBS and diluted with the cell lysate at 1.0 × 10 6 The cells were then resuspended at 37.5 dl / ml, sonicated, centrifuged at 13,000 rpm at 4°C for 15 minutes, and the supernatant was collected as a cell lysate.

[0082] Next, 12.5 μl of the cell lysate thus prepared was mixed with 35.5 μl of 0.2 M Tris-HCl buffer (pH 8.0) and 2 μl of each diluted sample, and incubated at 37° C. for 15 minutes. Then, 50 μl of 5 mM N-succinyl-Ala-Ala-Ala-p-nitroanilide was added as an elastase substrate, 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, phosphoramidon (final concentration 0.04 mM), an elastase inhibitor, was used.

[0083] FIG. 4 is a graph showing the elastase activity ratio relative to the control, where FIG. 4(a) shows the elastase activity ratio due to an aqueous placenta extract, and FIG. 4(b) shows the elastase activity ratio due to a non-aqueous placenta extract.

[0084] As shown in Figure 4(a), in the aqueous placenta extract, the elastase activity of the 1600μg / ml sample and the 400μg / ml sample decreased by about 30%, but the 40μg / ml sample did not show any decrease in elastase activity.

[0085] In contrast, in the non-aqueous placenta extract, as shown in FIG. 4(b), a decrease in elastase activity of approximately 20% was observed in the 400 μg / ml sample and approximately 60% in the 1600 μg / ml sample.

[0086] These findings suggest that both aqueous and non-aqueous placenta extracts contain components that inhibit elastase activity.

[0087] In particular, for aqueous placenta extracts, use at a concentration of 100 μg / ml or more, and based on the results shown in Figure 4(a), 400 μg / ml or more, and for non-aqueous placenta extracts, use at a concentration of 40 μg / ml or more, and more effectively at a concentration of 400 μg / ml or more, is expected to contribute to maintaining skin elasticity by using the placenta extract as an active ingredient for inhibiting elastase activity.

[0088] 5. Antiallergic Activity Evaluation Test The mechanism by which allergies such as hay fever and atopic dermatitis develop, which are characterized by repeated inflammation, is still unknown. However, it is believed that one of the causes is the release of granules by basophils, one of the various immune mechanisms.

[0089] In this study, we used rat basophilic leukemia cells (RBL-2H3) as a model cell for basophils to evaluate the granule release inhibitory activity (anti-allergic activity) of placenta extract. The granules contain β-hexosaminidase, and the amount of granules released was measured by measuring the amount of enzyme in the cell supernatant, and the amount of enzyme was measured by the strength of the enzyme activity in the cell supernatant.

[0090] Specifically, 5 × 10 rat basophilic leukemia cells (RBL-2H3) were 5 The cells / ml concentration was adjusted and 100 μl was seeded in a 96-well plate. Two such plates were prepared, one for measuring β-hexosaminidase activity and the other for the MTT test, and pre-cultured for 40 hours in a CO2 incubator (37°C, 5% CO2).

[0091] After pre-incubation, the medium was removed and the wells were washed with Tyrode's buffer, and 100 μl of Turode's buffer (or EMEM medium) containing 0.5 μl of each diluted sample was added to each well. After 1 hour of incubation at 37°C and 5% CO2, the supernatant was discarded from the plate for measuring β-hexosaminidase activity, the wells were washed, and 100 μl of A23187 (in Tyrode's buffer) was added. After 1 hour of incubation in an incubator, 50 μl of the supernatant from each well was taken and transferred to a new 96-well plate.

[0092] To measure β-hexosaminidase activity, 50 μl of p-nitrophenyl-N-acetyl-β-glucosaminide solution was added as a substrate to each well of a new plate from which 50 μl of the supernatant had been removed, and the reaction was carried out at room temperature for 1 to 3 hours while shielding the wells from light with aluminum foil. After the reaction, 100 μl of reaction stop solution was added to all wells to stop the enzyme reaction, and the absorbance at 405 nm was measured using a plate reader to measure β-hexosaminidase activity.

[0093] For the MTT test, the medium in each well was removed one hour after the addition of each diluted sample, and 100 μl of MTT staining solution (5 mg / ml in medium) was added to each well. The cells were cultured at 37°C for 4 hours in 10% CO2. The medium was then removed, and 1 ml of hydrochloric acid-isopropanol solution was added to each well. The cells were then shielded from light and allowed to stand at room temperature for 4 hours. The absorbance at 570 nm was then measured using a microplate reader, and used as an index of cell viability.

[0094] Figure 5 is a graph showing the β-hexosaminidase activity ratio to the control. As can be seen from Figure 5, in both aqueous and non-aqueous placenta extracts, the β-hexosaminidase activity decreased when the high concentration range of 400 μg / ml sample was added.

[0095] This suggests that the placenta extract according to this embodiment contains a component that exhibits anti-allergic activity.

[0096] 6. Tyrosinase inhibitory activity test Melanin pigment, which causes blemishes and freckles on the skin, is produced by the action of an enzyme called tyrosinase in melanocytes. Tyrosinase is activated by ultraviolet light and produces melanin, but it is known that inhibiting the action of this enzyme can prevent the fixation of melanin and provide a whitening effect.

[0097] In this test, the effect of each diluted sample on tyrosinase activity was evaluated using L-tyrosine or L-DOPA as a substrate.

[0098] The test method was as follows: 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 units / ml) were mixed, and the change in absorbance at 475 nm was recorded over time using a spectrophotometer immediately after mixing. The absorbance at 475 nm was determined to be due to the formation of dopachrome after the enzyme-substrate reaction, and the enzyme activity was calculated by calculating the increase in absorbance per time (L-tyrosine: measured for 10 minutes, L-DOPA: measured for 3 minutes). The tyrosinase activity value when the control (solvent) was added was set as 100%, and the relative tyrosinase activity when each diluted sample was added was calculated. Kojic acid (final concentration: 17 μg / ml) was used as a positive control.

[0099] Figure 6(a) is a graph showing the effect of aqueous or non-aqueous placenta extract on the tyrosinase activity rate using L-DOPA as a substrate, and Figure 6(b) is a graph showing the effect of aqueous or non-aqueous placenta extract on the tyrosinase activity rate using L-tyrosine as a substrate.

[0100] As shown in Figure 6(a), when the tyrosinase activity value (substrate: L-DOPA) when the control (solvent) was added was set at 100, the relative value was calculated. A concentration-dependent decrease in tyrosinase activity was observed for both aqueous and non-aqueous placenta extracts, and tyrosinase activity was significantly decreased by the addition of aqueous or non-aqueous placenta extract at a final concentration of 3.3 mg / ml.

[0101] In addition, as shown in Figure 6(b), when the tyrosinase activity value (substrate: L-tyrosine) when the control (solvent) was added was set at 100, the relative value was calculated, and a significant decrease in the tyrosinase activity rate was observed in the non-aqueous placenta extract (final concentration 3.3 mg / ml). In the aqueous placenta extract, no decrease in the tyrosinase activity rate was observed at the concentrations examined.

[0102] These results suggest that the aqueous or non-aqueous placenta extracts according to this embodiment contain components that exhibit tyrosinase inhibitory activity, and that this effect tends to be more pronounced in non-aqueous placenta extracts.

[0103] [7. Melanin production and inhibition test (2nd test)] Next, the second test of the melanin production and inhibition ability confirmation test will be described. In the above-described [2. Melanin production and inhibition ability confirmation test (first test)], the amount of melanin produced was confirmed for the aqueous placenta extract and the non-aqueous placenta extract, but in this second test, we focused on the non-aqueous placenta extract and confirmed the melanin production and inhibition ability of each of the components isolated and identified from the extract by the present inventors.

[0104] The components isolated by the present inventors from the placenta non-aqueous extract are as follows:

[0105] The present inventors have tentatively named this compound HS-1 for the purpose of isolating and identifying the compound represented by the general formula (i): [ka] Cholesterol is expressed as:

[0106] The general formula (ii), also tentatively designated HS-2: [ka] Cholesterol-7-one, represented by the formula:

[0107] The following general formula (v), tentatively named HS-3: [ka] 3β-hydroxycholest-4-ene-6-one, represented by the formula:

[0108] The general formula (x), tentatively named HS-4: [ka] 5α,6α-epoxycholestan-3β-ol, represented by the formula:

[0109] The compound of the following general formula (vi), tentatively named HS-6: [ka] 3β,6β-dihydroxycholest-5-ene, represented by the formula:

[0110] The general formula (iii), tentatively designated HS-7: [ka] Cholesta-3,5-diene-7-one, represented by the formula:

[0111] The general formula (vii), tentatively named HS-8: [ka] Cholestereyl oleate, represented by the formula:

[0112] The general formula (viii), tentatively named HS-10: [ka] Palmitic acid is represented by the formula:

[0113] The general formula (xi), tentatively named HAS-1: [ka] Uridine, represented by the formula:

[0114] The compound represented by the following general formula (iv), tentatively named HAS-2: [ka] Uridine monophosphate, represented by the formula:

[0115] The compound of the following general formula (ix), tentatively named HSE-2: [ka] Thymine, expressed as:

[0116] The melanin production and inhibition abilities of 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, and HSE-2, were examined.

[0117] The test method was the same as in the first test described above, but the sample to be added was not a placenta extract, but each of these components was dissolved in a dimethyl sulfoxide solution to a final concentration of 100 μM, and then added to the culture medium.

[0118] FIG. 7(a) is a graph showing the cell viability in each sample, FIG. 7(b) is a graph showing the extracellular melanin production ratio per cell relative to the control, and FIG. 7(c) is a graph showing the intracellular melanin production ratio per cell relative to the control. Table 1 shows the extracellular / intracellular melanin production ratio per cell taking into account the cell viability. In addition, in the "type" shown in Table 1, "A + " indicates that melanin production is increased by more than 20% compared to the control, and "A - " indicates that melanin production has increased by 20% or less compared to the control, and "B ++ " indicates that the amount of melanin production was reduced to the same extent as with arbutin (100 μM), and "B + " indicates that melanin production was reduced by more than 20% compared to the control, and "B - " indicates that melanin production was reduced by less than 20% compared to the control. [Table 1]

[0119] First, looking at cell viability in Figure 7(a), HS-1 (cholesterol), HS-2 (cholesterol-7-one) and HS-4 (5α,6α-epoxycholestan-3β-ol) dramatically reduced cell viability compared to the control (DMSO) (HS-1: 55.7%, HS-2: 36.5%, HS-4: 28.6%). Also, HAS-2 (uridine monophosphate) significantly reduced cell viability compared to the control (DMSO) (HAS-2: 79.8%).

[0120] On the other hand, HS-3 (3β-hydroxycholest-4-en-6-one), HS-7 (cholesta-3,5-dien-7-one), HS-10 (palmitic acid) and HSE-2 (thymine) were confirmed to significantly increase cell viability compared to the control (DMSO) (HS-3: 109.4%, HS-7: 113.3%, HS-10: 110.3%, HSE-2: 114.3%). Based on these results, it was confirmed that HS-3, HS-6, HS-7, HS-8, HS-10, HSE-2 and HAS-1 do not show cytotoxicity against B16 melanoma cells.

[0121] In addition, Figure 7(b) shows that the extracellular melanin production ratio per cell was suppressed by the addition of HS-3, HS-4, HS-6, HS-8, HS-10, HSE-2, HAS-1 and HAS-2 compared to the control (DMSO). In particular, the inhibition of extracellular melanin production was confirmed in HS-3, HS-6, HSE-2 and HAS-2 to the same extent as 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 compared to the control (DMSO).

[0122] In addition, Figure 7(c) shows that the intracellular melanin production ratio per cell was suppressed by the addition of HS-3, HS-4, HS-8, HS-10, HSE-2 and HAS-1 compared to the control (DMSO), whereas the extracellular melanin production ratio per cell was increased by the addition of HS-1, HS-2, HS-6, HS-7 and HAS-2 compared to the control (DMSO).

[0123] Furthermore, when each component was examined with reference to Table 1, first, HS-1 (cholesterol) was found to increase melanin production by more than 20% compared to the control, both intracellularly and extracellularly.

[0124] HS-2 (cholesterol-7-one) was also shown to increase extracellular melanin production by more than 20% compared to the control, and intracellular melanin production was increased, although not by more than 20%, compared to the control.

[0125] HS-3 (3β-hydroxycholest-4-en-6-one) was found to reduce melanin production by more than 20% compared to the control both intracellularly and extracellularly. Particularly, the reduction in melanin production was found to be equivalent to that of arbutin (100 μM) extracellularly.

[0126] HS-4 (5α,6α-epoxycholestan-3β-ol) was found to reduce melanin production both intracellularly and extracellularly, although the reduction was less than 20% compared to the control.

[0127] HS-6 (3β,6β-dihydroxycholest-5-ene) was found to reduce melanin production outside the cells by more than 20% compared to the control, particularly to a level equivalent to that of arbutin (100 μM). Conversely, an increase in melanin production was found inside the cells, although it was less than 20% compared to the control.

[0128] HS-7 (cholesta-3,5-dien-7-one) was found to increase melanin production both intracellularly and extracellularly by more than 20% compared to the control.

[0129] HS-8 (cholesteryl oleate) was found to reduce melanin production outside the cells by less than 20% compared to the control, and also reduced melanin production within the cells by more than 20% compared to the control.

[0130] HS-10 (palmitic acid) was found to reduce melanin production both intracellularly and extracellularly by more than 20% compared to the control.

[0131] HSE-2 (thymine) was found to reduce melanin production by more than 20% compared to the control both intracellularly and extracellularly. Particularly, the reduction in melanin production was found to be equivalent to that of arbutin (100 μM) extracellularly.

[0132] HAS-1 (uridine) was found to reduce melanin production both intracellularly and extracellularly, although the reduction was less than 20% compared to the control.

[0133] HAS-2 (uridine monophosphate) was found to reduce melanin production outside the cells by more than 20% compared to the control, particularly to a similar extent to arbutin (100 μM), but conversely, it increased melanin production inside the cells by more than 20% compared to the control.

[0134] Taking these results into consideration, it was shown that the components cholesterol (HS-1), cholesterol-7-one (HS-2), 3β,6β-dihydroxycholest-5-ene (HS-6), cholesta-3,5-dien-7-one (HS-7), and uridine monophosphate (HAS-2) promote melanin production compared to the control (DMSO). More specifically, it was shown that the components cholesterol (HS-1), cholesterol-7-one (HS-2), and cholesta-3,5-dien-7-one (HS-7) promote melanin production both intracellularly and extracellularly, and the components 3β,6β-dihydroxycholest-5-ene (HS-6) and uridine monophosphate (HAS-2) promote melanin production intracellularly. This indicates that these components are useful as active ingredients or health functional ingredients for promoting melanin production in cosmetics and functional foods.

[0135] Similarly, 3β-hydroxycholest-4-en-6-one (HS-3), 5α,6α-epoxycholestan-3β-ol (HS-4), 3β,6β-dihydroxycholest-5-ene (HS-6), cholesteryl oleate (HS-8), palmitic acid (HS-10), thymine (HSE-2), uridine (HAS-1), and uridine monophosphate (HAS-2) have been shown to inhibit melanin production. More specifically, 3β-hydroxycholest-4-en-6-one (HS-3), 5α,6α-epoxycholestan-3β-ol (HS-4), cholesteryl oleate (HS-8), palmitic acid (HS-10), thymine (HSE-2), and uridine (HAS-1) were shown to be able to inhibit melanin production both intracellularly and extracellularly, and among them, HS-3, HS-8, HS-10, HSE-2, and HAS-1 significantly inhibited extracellular melanin production, while 3β,6β-dihydroxycholest-5-ene (HS-6) and uridine monophosphate (HAS-2) inhibited extracellular melanin production. This indicates that these ingredients are useful as active ingredients or health functional ingredients for the purpose of inhibiting melanin production and thus whitening in cosmetics and functional foods.

[0136] Interestingly, 3β,6β-dihydroxycholestane (HS-6) and uridine monophosphate (HAS-2) promote intracellular melanin production, but inhibit extracellular melanin production to the same extent as arbutin (100 μM). This suggests that these compounds may be useful in cosmetics, functional foods, and even pharmaceutical preparations that provide protection from ultraviolet rays within cells while inhibiting extracellular melanin secretion to prevent excessive pigmentation.

[0137] As described above, according to the present embodiment of the invention, a placenta extract is used as an additive material for any of the components selected from the group consisting of 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-allergic activity, and components that inhibit tyrosinase activity in cosmetics or functional foods, and therefore a new method for using a placenta extract for a more specific purpose can be provided.

[0138] Finally, the above-mentioned embodiments are merely examples of the present invention, and the present invention is not limited to the above-mentioned embodiments. Therefore, even if the above-mentioned embodiments are different, various modifications can be made depending on the design, etc., as long as they do not deviate from the technical idea of ​​the present invention.

Claims

[Claim 1] The following general formula (iv): 【Chemistry 1】 or uridine monophosphate represented by the following general formula (vi): 【Chemistry 2】 Use of 3β,6β-dihydroxycholest-5-ene represented by the formula (I) as an active ingredient or health functional ingredient in cosmetics or functional foods for the purpose of increasing intracellular melanin production to provide protection against ultraviolet rays through melanin, while suppressing excessive pigmentation, etc. by suppressing extracellular melanin secretion (however, this does not include uses that fall under methods of treating humans).

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