JP2014144920A - External composition for skin - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an external composition for skin comprising a natural substance exerting a prevention and improvement effect on skin aging phenomena, such as wrinkle, sagging, staining and freckles, maintaining or improving skin in a young and healthy state, having decreased stimulation on skin, having an excellent safety on living body and exerting an excellent anti-aging effect and whitening effect.SOLUTION: An external composition for skin essentially comprises: (a) an enzyme processing degradation product obtained by processing a rice alkali extract with two or more protein degradation enzymes; (b) an enzyme processing degradation product obtained by processing an extract of seeds of Brassica alba (white mustard seed) of Cruciferous Brassica with a protein degradation enzyme; (c) a fermentation product obtained by fermentation of Nympaeaceae Nelumbo plant seeds with lactic acid bacterium; (d) a fermentation product obtained by fermentation of adlay seeds with yeast; and (e) an extract of roots of Bletilla of Orchidaceae Bletilla.

Description

  The present invention relates to an external composition for skin having excellent anti-aging action and whitening action and high biological safety.

Skin aging is caused by internal factors such as inactivation of cell growth / differentiation, hormonal secretion, and quantitative decrease of extracellular matrix components, and sunlight (ultraviolet rays), house dust, exhaust gas, mites. This is a phenomenon that is caused by complex intertwining of external factors such as cell / tissue damage due to the inflammation or inflammation. Ultraviolet rays, an external factor of skin aging, not only directly damages skin cells, but also denatures or cross-links the collagen of the extracellular matrix component to cause wrinkle formation and skin elasticity, and melanin It causes various damages to the skin, such as causing abnormal pigmentation and causing spots and freckles. Furthermore, house dust, exhaust gas, mites, and the like become antigens that cause skin inflammation and allergies, thereby causing spots, freckles, and the like.

  In order to prevent skin aging caused by the above causes, and to keep the skin healthy and youthful, the use of various active ingredients has been proposed in the past, and the topical use of these anti-aging ingredients and whitening ingredients The drug is on the market. For example, vitamin C, vitamin E, superoxide dismutase (hereinafter abbreviated as SOD), catalase and other antioxidants; glycyrrhizic acid, allantoin and other anti-inflammatory agents; various ultraviolet absorbers; α-hydroxycarboxylic acid, placenta Extracts, cell activation components such as γ-amino-β-hydroxybutyric acid; extracellular matrix components such as collagen, elastin and hyaluronic acid; and humectants such as urea. Further, arbutin, kojic acid, and the like are known as substances that suppress the occurrence of pigmentation such as skin spots and buckwheat, and are widely used as active ingredients of whitening agents.

However, these conventional anti-aging agents and whitening agents have problems in terms of safety to the skin and effectiveness when actually applied to the skin. In consideration of biological safety, anti-aging agents and whitening agents derived from various natural ingredients have also been proposed (Patent Documents 1 and 2), but their effects were seen as raw materials for skin external preparations. In this case, it is difficult to say that it is sufficiently satisfactory from the viewpoint of effectiveness.
JP 62-056410 A Japanese Patent Laid-Open No. 03-188018

In view of the problems of the prior art, the present inventors have conducted intensive research to find new anti-aging components and whitening components derived from natural products that are excellent in skin safety and exhibit sufficient effectiveness. It was. As a result, (a) an enzyme degradation product obtained by treating an alkaline extract of rice with two or more types of proteolytic enzymes, and (b) an extract of white birch seeds (white coconut) of Brassicaceae (C) fermented product obtained by fermenting seeds of the genus Lotusaceae with lactic acid bacteria, and (d) fermenting barley seeds with yeast. The composition for external use containing the obtained fermented product and (e) a silane bulb extract of the Orchidaceae silane genus as an essential component is excellent in safety, and has excellent anti-aging effect and whitening effect at a low concentration As a result, the present invention has been completed.

The present invention includes (a) an enzymatic degradation product obtained by treating an alkaline extract of rice with two or more types of proteolytic enzymes, and (b) an extraction of white birch seeds (white coconut) of Brassicaceae Brassica (C) a fermented product obtained by fermenting the seeds of the genus Lotusaceae with lactic acid bacteria, and (d) fermenting the seeds of pearl barley with yeast. A fermented product obtained in this manner, and (e) a silane bulb extract of the Orchidaceae silane genus, as an essential component.
Moreover, this invention is the cosmetics which mix | blended the said skin external composition.
In the present invention, the term cosmetic is used in a broad sense including so-called quasi drugs.

  The present invention includes (a) an enzymatic degradation product obtained by treating an alkaline extract of rice with two or more types of proteolytic enzymes, and (b) an extraction of white birch seeds (white coconut) of Brassicaceae Brassica (C) a fermented product obtained by fermenting the seeds of the genus Lotusaceae with lactic acid bacteria, and (d) fermenting the seeds of pearl barley with yeast. And (e) an extract of silane bulbs belonging to the family Orchidaceae silane as essential components, each component having various action mechanisms (epidermal cell activation) Anti-aging effect and whitening action based on anti-inflammatory effect [prostaglandin production inhibitory effect], melanin production inhibitory effect), and exerts an excellent prevention and improvement effect on wrinkles, tarmi, stains and freckles To do. Furthermore, according to the present invention, the skin aging phenomenon can be comprehensively prevented and improved based on a multifaceted and complex action mechanism, so that the blending amount of each component is suppressed to a low concentration, It is also possible to provide an external composition for skin excellent in odor, feeling in use, stability and safety.

FIG. 1 is a diagram showing the epidermal cell activation effect of the external composition for skin of the present invention. FIG. 2 is a diagram showing the prostaglandin E2 (PGE2) production inhibitory effect of the external composition for skin of the present invention. FIG. 3 is a diagram showing the melanin production inhibitory effect of the composition for external use of the present invention.

  The present invention includes (a) an enzymatic degradation product obtained by treating an alkaline extract of rice with two or more types of proteolytic enzymes, and (b) an extraction of white birch seeds (white coconut) of Brassicaceae Brassica (C) a fermented product obtained by fermenting the seeds of the genus Lotusaceae with lactic acid bacteria, and (d) fermenting the seeds of pearl barley with yeast. A fermented product obtained in this manner, and (e) a silane bulb extract of the Orchidaceae silane genus, as an essential component.

(A) Preparation of enzymatic decomposition product of alkaline extract of rice No particular limitation is imposed on the rice used in the present invention, such as brown rice, polished rice, processed rice, pigmented rice (black rice, purple rice, red rice, etc.), etc. Any of these can be used, but preferably polished rice or processed rice is used. As the type of rice, either sticky rice or glutinous rice may be used. In addition, examples of processed rice include antiallergic rice, low protein rice (for example, low glycerin rice), and fortified rice (for example, γ-aminobutyric acid rice). Moreover, the use of white rice and / or red rice contained in brown rice is also possible.

Examples of the solvent used in the alkaline extraction treatment of rice include purified water; lower alcohols such as ethanol; polyhydric alcohols such as ethylene glycol, propylene glycol, glycerin and 1,3-butylene glycol; oleyl alcohol and stearyl alcohol. And higher alcohols such as octyldodecanol; ketones such as acetone; esters such as ethyl acetate; hydrocarbon solvents such as hexane, chloroform and benzene. These may be used alone or in combination of two or more. Used. Among these, the solvent which mixed 1 type, or 2 or more types of purified water or purified water, and ethanol, glycerol, and 1, 3- butylene glycol is preferable at the point of the wide application to a skin external preparation. When these mixed solvents are used, the volume ratio is 1 to 25: 1 for purified water and ethanol, 1 to 15: 1 for purified water and glycerol, and 1 to 15: 1 for purified water and 1,3-butylene glycol. preferable.

Examples of the alkali preparation agent used in the alkali extraction treatment include sodium salts such as sodium hydroxide and sodium carbonate, potassium salts such as potassium hydroxide, and the like.
Set to ~ 14.0. Of these, sodium hydroxide and sodium carbonate are preferred because they can be set to the target pH at a low concentration.

The alkali extraction treatment time varies depending on the type of the solvent and alkali preparation used, the set pH, the extraction temperature, etc., but is preferably 8.5 to 14.0 and 6 hours to 7 days at room temperature.

The alkaline extract of rice prepared as described above is hydrolyzed with a protease. Examples of the enzyme to be used include actinases such as actinase, pepsins such as pepsin, trypsins such as trypsin and chymotrypsin, papains such as papain and chymopapain, and peptidases such as glycylglycine peptidase, carboxypeptidase and aminopeptidase. , Bromelain, microbial-derived complex proteolytic enzymes [for example, neurase (manufactured by Amano Enzyme Co., Ltd.)] and the like, and it is preferable to use a combination of two or more of these enzymes. In particular, it is preferable to use a combination of papains, bromelain and / or actinases.

In the treatment of combining two or more enzymes, two or more enzymes may be allowed to act simultaneously depending on the properties of the enzyme used, or may be allowed to act sequentially with or without changing the reaction conditions. Good. The amount of the enzyme used is preferably in the range of 0.001 to 50 parts by weight, more preferably 0.1 to 10 parts by weight per one enzyme with respect to 100 parts by weight of the solid content of the alkaline extract of rice. Part range. The enzyme treatment time and temperature vary depending on the type of enzyme used, but are generally in the range of 0.5 to 24 hours, and preferably in the range of 1 to 6 hours. The enzyme treatment temperature is generally about 30 to 50 ° C.

The enzyme degradation product prepared as described above may be used as a component of a composition for external use of skin after adjusting the pH to 4 to 8, and may be used by concentrating under reduced pressure. You may use as a density | concentration. The enzyme degradation product may be a dried product by a conventional method such as a spray drying method or a freeze drying method.

(B) Preparation of enzyme-treated decomposition product of birch seed (white coconut) The white cocoon used in the present invention refers to the seed of Brassica alba which is a plant belonging to the genus Brassica.

In the present invention, a white coconut extract is hydrolyzed by an enzyme. Solvents used for extraction include water; lower alcohols such as methanol, ethanol, and propanol; polyhydric alcohols such as ethylene glycol, propylene glycol, 1,3-butylene glycol, and glycerin; ethyl acetate, butyl acetate, and methyl propionate. Esters such as acetone; ketones such as acetone and methyl ethyl ketone; ethers such as ethyl ether and isopropyl ether; hydrocarbon solvents such as n-hexane, toluene and chloroform, and the like. These may be used alone or in combination of two or more. Used.

Among these extraction solvents, water or water and lower alcohols are used from the viewpoint of skin physiological activity of the extract obtained, and also from the viewpoint of ease of transfer to the subsequent proteolytic enzyme treatment and the efficiency of the treatment. Alternatively, a mixed solvent with a hydrophilic solvent such as a polyhydric alcohol is preferably used, and water alone is most preferable. In the case of using a mixed solvent of water and a hydrophilic solvent, the mixing ratio is, for example, a mixed solvent of water and ethanol, with a volume ratio (hereinafter the same) of 100: 1 to 1: 1, water and 1,3. -If it is a mixed solvent with butylene glycol, it is good to set it as the range of 100: 1 to 1: 2 if it is a mixed solvent of water and glycerin.

At the time of extraction, the pH of the extract is not particularly limited, but generally it is preferably in the range of 3.0 to 9.0. In this sense, if necessary, the extraction solvent may be sodium hydroxide, sodium carbonate. Alternatively, an alkaline adjusting agent such as potassium hydroxide or an acidic adjusting agent such as citric acid, hydrochloric acid, phosphoric acid or sulfuric acid may be added to adjust the pH to a desired level.

Extraction conditions such as extraction temperature and extraction time vary depending on the type and pH of the solvent used, or the shredding degree and particle size of the extract, but for example, in the case of the immersion method using water as the extraction solvent, extraction is performed. The temperature is generally in the range of 1 to 90 ° C., preferably 20 to 60 ° C., and the extraction time is generally in the range of 0.5 to 72 hours, preferably in the range of 1 to 24 hours in the case of room temperature extraction.

Next, the extract obtained here is treated with one or more proteolytic enzymes, and the extract is subjected to an enzymatic degradation treatment. In this case, when a solvent other than water or a mixed solvent of water and a hydrophilic solvent is used for the preparation of the extract solution, the extract solvent is once removed from the extract solution, and the extract obtained here is washed with water or The sample is redissolved in a mixed solvent of water and a hydrophilic solvent and then subjected to an enzymatic decomposition treatment.

Examples of proteolytic enzymes include actinases such as actinase, pepsins such as pepsin, trypsins such as trypsin and chymotrypsin, papains such as papain and chymopapain, peptidases such as glycylglycine peptidase, carboxypeptidase and aminopeptidase, Bromelain etc. are mentioned, and these may be used alone or in combination of two or more. Among these enzymes, actinases such as actinase, papains such as papain and chymopapain, and bromelain are particularly preferable.

Proteolytic enzyme treatment is carried out by adding one or more of the above enzymes to the white coconut extract and carrying out an enzyme reaction under conditions near the optimum pH and temperature of the enzyme used. When two or more kinds of enzymes are used in combination, two or more kinds of enzymes may be allowed to act simultaneously according to the characteristics of the enzyme used, or may be allowed to act sequentially without changing or changing the reaction conditions. . The amount of the enzyme used should be in the range of 0.001 to 50 parts by weight, more preferably 0.1 to 10 parts by weight per one enzyme, based on 100 parts by weight of the solid content of the white coconut extract. Part range. The enzyme treatment time varies depending on the type of enzyme used and the like, but is generally in the range of 0.5 to 24 hours, preferably in the range of 1 to 6 hours. In addition, the proteolytic enzyme treatment described above may be performed simultaneously with the extraction treatment, which is the previous step, in some cases.

After the above treatment with the proteolytic enzyme is completed, the enzyme is deactivated by using an appropriate method such as a method of heating the enzyme treatment solution to 80 ° C. or higher to obtain an enzyme degradation product. The enzyme degradation product obtained here is generally adjusted to a pH of 4 to 8 and then blended into the composition for external use as it is, or if necessary, adjusted to a predetermined concentration by vacuum concentration or the like, and then the skin. It mix | blends with an external composition. In some cases, the powder may be pulverized according to a conventional method such as spray drying or freeze drying.

(C) Preparation of fermented lotus seed As a plant belonging to the genus Lotusaceae used in the present invention, for example, lotus (Nelumbo nucifera)
Gaertner) or American kibas (Nelumbo Lutea Pers.) Can be mentioned. Among them, the use of lotus (Nelumbo nucifera Gaertner) is preferable.

As the microorganism used for the fermentation of the lotus seed, one kind or two or more kinds selected from lactic acid bacteria are used. Examples of lactic acid bacteria include Lactobacillus plantarum and Lactobacillus brevis (L.
brevis), Lactobacillus genus Lactobacillus, such as L. casei; Carnobacterium divergence (Carnobacterium
divergens), lactic acid bacteria of the genus Carnobacterium such as Carnobacterium piscicola; Leuconostoc (Leuconostoc)
mesenteroides), lactic acid bacteria of the genus Leuconostoc such as Leuconostoc citreum; Streptococcus faecalis (Streptococcus)
faecalis), Streptococcus pyogenes and other Streptococcus lactic acid bacteria; Enterococcus caseliflavus, Enterococcus sulfreus and other Enterococcus sulfreus enterococcus (
Enterococcus) lactic acid bacteria; Lactococcus plantarum (Lactococcus)
lactic acid bacteria of the genus Lactococcus such as Lactococcus rafinolactis; Weissella confusa, lactic acid bacteria of the genus Weissella kandleri; Atopobium mintopum ) Lactic acid bacteria of the genus Atopobium; Vagococcus flubiaris (Vagococcus)
lactic acid bacteria of the genus Vagococcus such as fluvialis), Vagococcus salmoninarum; Pediococcus damnosus
damnosus), Pediococcus pentosaceus, and other lactic acid bacteria belonging to the genus Pediococcus. Among these lactic acid bacteria, the use of Lactobacillus plantarum is most preferable from the viewpoint of the skin physiological activity of the obtained fermented product and the ease of handling without being extremely anaerobic.

The preferable specific example of the method of fermenting a lotus seed using said lactic acid bacteria is as follows.
First, a lotus seed (hereinafter referred to as “fermentation material”) is immersed or suspended in a solvent to prepare a suspension for fermentation. In this case, the lotus seed may be used as it is, or may be used after previously dried or semi-dried. As the shape, the collected one can be used as it is, but if it is shredded or pulverized and refined, the fermentation efficiency can be increased. In addition, the outermost astringent skin of the seeds of lotus seed is preferably removed in advance from the viewpoint of fermentation efficiency and the hue of the resulting fermented product. It is preferable to use it because it is easier for the lactic acid bacteria to use the nutritional component.

As a solvent for suspending the fermentation material, water or a mixture of water and lower alcohols (methanol, ethanol, propanol, etc.) or glycols (ethylene glycol, propylene glycol, 1,3-butylene glycol, glycerin, etc.) In these solvents, sugars such as glucose, fructose and sucrose may be added. However, lactic acid bacteria are most likely to exert their action and there is an assimilation component other than the lotus seed component. From the standpoint of avoiding the formation of fermentation by-products based on the water, it is most preferred to use water alone. The mixing ratio of the fermentation material and the solvent is generally in the range of 1: 1 to 1: 1000, preferably 1: 5 to 1: 100, more preferably 1:10 to 1:50 in terms of dry weight of the fermentation material. .

Before the fermentation material / solvent suspension is subjected to a fermentation process, it is necessary to sterilize and remove the germs that hinder the fermentation. May be used by washing with ethanol for sterilization in advance and then suspending in a sterile solvent such as sterile water. May be. As the heat sterilization treatment, the autoclave sterilization method in which the suspension is heated at 120 to 130 ° C. for 10 to 20 minutes, or holding at 80 to 90 ° C. for 60 to 120 minutes is repeated once a day for 2 to 3 days. The heat sterilization method such as the method is generally used.

Next, this sterilized suspension is put into a fermentation tank, and lactic acid bacteria are inoculated therein for fermentation. The appropriate amount of lactic acid bacteria inoculated is 10 ⁷ to 10 ⁸ cells / mL. Even if the inoculation amount exceeds the above range, the fermentation progress time hardly changes. On the other hand, if the inoculation amount is less than the above range, it takes a long time to complete the fermentation.

The fermentation temperature is generally in the range of 5 to 50 ° C, preferably in the range of 35 to 40 ° C, which is the optimum temperature for growth of lactic acid bacteria. The number of days of fermentation is generally in the range of 1 to 10 days, preferably 2 to 5 days, at the optimum temperature. If the fermentation days are shorter than the above general range, the fermentation is not sufficiently performed and the effectiveness of the fermented product tends to be reduced. On the other hand, if the fermentation days are longer than 10 days, no further increase in effectiveness is observed. Not only that, but also coloring and an increase in fermentation odor occur.

In performing the above fermentation treatment, in order to more effectively use the lotus seed components by lactic acid bacteria, before or simultaneously with inoculation of lactic acid bacteria, an enzyme is added to the suspension, The lotus seed is preferably hydrolyzed with an enzyme.

In this case, as the enzyme, at least one enzyme selected from a proteolytic enzyme, a starch degrading enzyme, a pectin degrading enzyme, and a fibrin degrading enzyme is used, and in particular, at least one selected from each of these four enzyme groups. Good results can be obtained by using one enzyme in combination.

Examples of proteolytic enzymes include actinases such as actinase, pepsins such as pepsin, trypsins such as trypsin and chymotrypsin, papains such as papain and chymopapain, peptidases such as glycylglycine peptidase, carboxypeptidase and aminopeptidase. And bromelain can be used. Among these enzymes, actinases such as actinase, papains such as papain and chymopapain, and bromelain are particularly preferable.

As the starch degrading enzyme, for example, α-amylase, β-amylase, glucoamylase, β-galactosidase and the like can be used. Of these enzymes, glucoamylase is particularly preferred.

Examples of pectin degrading enzymes that can be used include pectin depolymerase, pectin demethoxylase, pectin lyase, pectin esterase, and polygalacturonase. Of these enzymes, pectin esterase and polygalacturonase are particularly preferred.

Examples of the fibrinolytic enzyme include cellulase, hemicellulase, agarase, mannase, chitinase, chitosanase, carrageenase, arginase, fucoidanase, inulase, xylanase, and ligninase. Of these enzymes, cellulase, hemicellulase and ligninase are particularly preferred.

The total amount of the enzyme used is preferably 0.01 to 10% by weight, more preferably 0.1 to 1.0% by weight, based on the solid content of the lotus seed in the suspension. As treatment conditions such as pH, temperature, and time, if the enzyme treatment is performed before fermentation, the treatment is preferably performed for 1 to 24 hours near the optimum pH and temperature of the enzyme used. If it is performed simultaneously with the fermentation, the same conditions as the fermentation may be used.

When the above fermentation treatment is completed, the sterilization treatment is performed at 70 to 100 ° C. for about 10 to 120 minutes for the sterilization, and also when the enzyme treatment is used in combination, inactivation of the enzyme. After application, the liquid phase is separated as it is, or generally and preferably by solid-liquid separation means such as filtration or centrifugation, and if necessary, the pH is adjusted to pH 6-8, which is the pH range of ordinary cosmetics. It is adjusted and, if necessary, adjusted to an appropriate concentration by dilution or concentration, and then used as a blending component of the composition for external use on the skin. In some cases, the liquid phase after solid-liquid separation may be powdered according to a conventional method such as spray drying or freeze drying, and then blended into the composition for external use on the skin.

(D) Preparation of pearl seed fermented pearl barley used in the present invention is a plant belonging to the genus Giusedama, and is widely used for medicinal purposes and food. In the present invention, the use of pearl barley seeds is preferred. In the present invention, pearl seeds can be used either with a shell or without a husk, and even in the form of grains, powder obtained by pulverization or crushing, or grains of pearl seed, high temperature of the powder Any high-pressure treated product can be used, and in any case, a fermented product that is equivalent and has stronger skin physiological activity than the original pearl seeds can be obtained, but storage stability and extraction / fermentation efficiency as raw materials From this point of view, it is preferable to use a powder obtained by pulverization or crushing, or a high-temperature / high-pressure processed product thereof, in both cases of shelled and shell-removed products.

In the present invention, yeast is used for fermentation of pearl barley seeds. Examples of yeast include the following.
(1) Saccharomyces cerevisiae, Saccharomyces awamori, Saccharomyces chevalieri, Saccharomyces carlsbergensis, Saccharomyces carlsbergensis, Saccharomyces carlsbergensis
bayonus) and other yeasts of the genus Saccharomyces.
(2) Yeast of Torlaspora genus such as Torulaspora delbruekii, Torulaspora fermentati, Torulaspora rosei and the like.
(3) Zygosaccharomyces rouxii, Zygosaccharomyces soya, Zygosaccharomyces sake, Zygosaccharomyces sake, Zygosaccharomyces sake, Zygosaccharomyces sake
miso), yeast of the genus Digosaccharomyces such as Zygosaccharomyces lactis.
(4) Candida Versatilis (Candida versatilis), Candida Etchelisii (Candida etchellsii), Candida Kefir (Candida
kefyr), Candida sake, Candida scottii and other yeasts of the genus Candida.
In the present invention, any of the above yeasts can be used, but Saccharomyces cerevisiae is most preferred because it is most widely used for foods and has a strong fermenting power.

The preferable specific example of the method of fermenting pearl seed using said yeast is as follows.
First, pearl seeds or powder obtained by pulverizing or pulverizing pearl seeds, or a high-temperature / high-pressure processed product thereof is mixed with a fermentation medium to prepare a suspension, which is sterilized. Here, as a fermentation medium, water, a mixed solution of water and lower alcohols such as ethanol and propanol, a mixed solution of water and glycols such as ethylene glycol, propylene glycol and 1,3-butylene glycol, water and sorbitol In addition, it is possible to use a mixed solution with sugars such as glucose, etc., but the yeast used for fermentation is most likely to act, and in addition to the components contained in pearl seeds, it does not contain components that serve as nutrients for yeast. Most preferred is the use of water alone.

The mixing ratio of the pearl seed and the fermentation medium is generally in the range of 2: 1 to 1: 1000 by weight, preferably 1: 2 to 1: 100, more preferably 1: 5 to 1:50. It is a range. If the ratio of pearl seeds is too large, the liquid will have a viscosity, which tends to reduce the yield due to difficulties in filtration, etc. In some cases, the activity is low, and a concentration step is required, which makes the method unusable.

As the sterilization treatment, an autoclave sterilization method in which pearl seed suspension is heated at 120 to 130 ° C. for 10 to 20 minutes, or holding at 80 to 90 ° C. for 60 to 120 minutes is repeated once a day for 2 to 3 days. A heat sterilization method such as a sterilization method is generally used. Alternatively, the fermented barley seed itself may be washed and sterilized with sterilizing ethanol in advance and suspended in a sterile medium such as sterile water. You may heat-sterilize after preparing.

Next, this sterilized barley seed suspension is placed in a fermentation tank, and yeast is inoculated therein for fermentation. The appropriate amount of yeast inoculation is 10 ⁷ to 10 ⁸ cells / mL. Even if the inoculation amount is larger than the above range, the progress time of fermentation hardly changes. On the other hand, if the inoculation amount is smaller than the above range, it takes a long time to complete the fermentation, which is not preferable.

If fermentation temperature is the range of 5-50 degreeC, fermentation will advance and the target fermented material can be obtained, More preferably, it is the range of 30-40 degreeC which is the optimal growth temperature of yeast. The number of fermentation days is generally 1 to 10 days, more preferably 2 to 5 days in the case of performing fermentation at the optimum temperature. When the fermentation days are shorter than one day, the fermentation is not sufficiently performed, and it becomes difficult to obtain a desired fermented product having high skin physiological activity. On the other hand, if the fermentation period is longer than 10 days, the fermentation does not proceed any further and the effectiveness of the fermented product is not improved, but inconveniences such as increased coloring and fermentation odor occur. It is not preferable.

If the predetermined fermentation days have passed, if the enzyme decomposition treatment described below is performed together with fermentation, the fermentation product is heated at 80 to 90 ° C. for 60 to 120 minutes, for example, also for inactivation of the enzyme. After sterilizing using, etc. and stopping fermentation, insoluble matter is removed using solid-liquid separation means such as filtration or centrifugation to obtain a solution containing the desired fermentation product. The fermented product solution obtained here is generally adjusted to a pH of 4 to 8 and then directly blended into cosmetics or, if necessary, adjusted to a predetermined concentration by vacuum concentration, etc. Blend into the product. In some cases, the powder may be pulverized according to a conventional method such as spray drying or freeze drying.

In addition, when performing the above fermentation treatment, it may be possible to subject the pearl seed suspension to a hydrolysis treatment with an enzyme before fermentation and / or in parallel with the fermentation, thereby making the pearl seed components more effective. This is preferable because not only the fermentation efficiency is increased by yeast, but also the flow characteristics and storage stability of the fermented product are further improved.

When performing an enzymatic hydrolysis treatment, it is preferable to select at least one enzyme from each of two types of enzymes, a proteolytic enzyme and a saccharide-degrading enzyme, and use them in combination.

Examples of proteolytic enzymes include actinases such as actinase, pepsins such as pepsin, trypsins such as trypsin and chymotrypsin, papains such as papain and chymopapain, peptidases such as glycylglycine peptidase, carboxypeptidase, and aminopeptidase. And bromelain can be used.
Among these enzymes, actinases such as actinase, papains such as papain and chymopapain, and bromelain are particularly preferable.

Examples of the carbohydrase include α-amylase, β-amylase, glucoamylase, cellulase, β-glucanase, β-xylanase, dextranase, polygalacturonase, α-galactosidase, β-galactosidase, pullulanase, and isoamylase. , Α-glucosidase, β-glucosidase, maltotriohydrolase, pectin depolymerase, pectin demethoxylase, pectin lyase, pectin esterase and the like can be used. Of these enzymes, glucoamylase, pectinesterase and polygalacturonase are particularly preferred.

The amount of enzyme used should be in the range of 0.01 to 10% by weight, more preferably 0.1 to 2.0% by weight, based on the solid content in the pearl seed suspension. It is a range. The treatment conditions such as pH, temperature, and time may be the same as those for fermentation. However, when the enzyme hydrolysis treatment is performed before fermentation, the optimum pH of the enzyme to be used is 1 to 24 near the optimum temperature. It is preferable to perform time processing.

(E) Preparation of Extract of Silane Bulb The silane used in the present invention refers to silane [Bletilla striata (THUNB.) REICHB.fil], which is a plant belonging to the genus Orchidaceae. In the present invention, it is preferable to use a bulb of silane, that is, a white herb of Chinese herbal medicine.

For the preparation of the silane bulb extract, the bulb is washed with water and dried in advance, if necessary, preferably further chopped or pulverized, and then contacted with an extraction solvent by an appropriate means such as a dipping method or a countercurrent extraction method. Is done by.

As an extraction solvent, water; lower alcohols such as methanol, ethanol, and propanol; higher alcohols such as oleyl alcohol, stearyl alcohol, and octyldodecanol; many solvents such as ethylene glycol, propylene glycol, 1,3-butylene glycol, and glycerin Monohydric alcohols; esters such as ethyl acetate, butyl acetate, methyl propionate and glyceryl trioctanoate; ketones such as acetone and methyl ethyl ketone; ethers such as ethyl ether and isopropyl ether; carbonization such as n-hexane, toluene and chloroform Examples thereof include hydrogen-based solvents, which are used alone or in combination of two or more. Among these extraction solvents, the use of one single solvent selected from water, lower alcohols and polyhydric alcohols, or two or more mixed solvents is preferable, and the single use of water is most preferable.

The mixing ratio in the case of using a mixed solvent is, for example, 1: 1 to 25: 1 by volume ratio (hereinafter the same) if the mixed solvent is water and ethyl alcohol, and 1: if the mixed solvent is water and glycerin. In the case of a mixed solvent of 1 to 15: 1 or water and 1,3-butylene glycol, it is preferably in the range of 1: 1 to 15: 1.

In preparing the extract of the present invention, the pH of the extract is preferably maintained in the range of 5 to 9. In this sense, if necessary, the above extraction solvent may be sodium hydroxide, sodium carbonate, potassium hydroxide. An alkaline adjuster such as citric acid, hydrochloric acid, phosphoric acid, sulfuric acid or the like may be blended to adjust to a desired pH.

In the case of the immersion method, the amount ratio of the extraction solvent to the extract is generally in the range of 1: 1 to 1:50 (weight ratio), preferably in the range of 1: 5 to 1:20.

In addition, the extraction conditions such as extraction temperature and time vary depending on the type of solvent used, the extraction site / chopping degree of the plant, etc., but in the case of the immersion method, for example, the extraction temperature is generally 5 to 100 ° C., The temperature is preferably in the range of 40 to 90 ° C., and the extraction time is preferably about 0.5 to 96 hours, particularly about 2 to 24 hours.

The extract obtained here may generally be adjusted to a pH of 4 to 8 and adjusted to an appropriate concentration as it is or by dilution or vacuum concentration. Powdering may be performed according to a conventional method such as freeze-drying.

  The external composition for skin of the present invention is a combination of the above five plant-derived components. Although the compounding quantity of each component is not limited, For example, Preferably it is 0.0001 to 50 weight%, More preferably, it is 0.001 to 30 weight%, More preferably, it is 0.01 to 10 weight%. .

In addition, the composition for external use of the skin of the present invention is a component used for a normal external preparation for skin, for example, an oily component, a surfactant (synthetic system, natural product system) as long as the effectiveness of the composition is not lost. , Moisturizers, thickeners, antiseptics / bactericides, powder components, ultraviolet absorbers, antioxidants, whitening agents, pigments, fragrances and the like can be used in combination as needed. Moreover, as long as the effectiveness and characteristics of the external composition for skin of the present invention are not impaired, it may be combined with other physiologically active ingredients in the external preparation for skin.

Here, as the oil component, for example, olive oil, jojoba oil, castor oil, soybean oil, rice oil, rice germ oil, palm oil, palm oil, cacao oil, meadow foam oil, sheer butter, tea tree oil, avocado oil, Oils derived from plants such as macadamia nut oil and plant-derived squalane; Fats derived from animals such as mink oil and turtle oil; waxes such as beeswax, carnauba wax, rice wax, lanolin; liquid paraffin, petrolatum, paraffin wax, squalane, etc. Hydrocarbons; fatty acids such as myristic acid, palmitic acid, stearic acid, oleic acid, isostearic acid, cis-11-eicosenoic acid; higher alcohols such as lauryl alcohol, cetanol, stearyl alcohol; isopropyl myristate, palmitic acid Isopropyl, me Butyl phosphate, 2-ethylhexyl glycerides, higher fatty acid octyldodecyl (octyl stearate dodecyl and the like), and the synthetic esters and synthetic triglycerides such like.

Examples of the surfactant include polyoxyethylene alkyl ether, polyoxyethylene fatty acid ester, polyoxyethylene sorbitan fatty acid ester, glycerin fatty acid ester, polyglycerin fatty acid ester, polyoxyethylene glycerin fatty acid ester, polyoxyethylene hydrogenated castor oil, polyoxyethylene Nonionic surfactants such as oxyethylene sorbitol fatty acid esters; fatty acid salts, alkyl sulfates, alkylbenzene sulfonates, polyoxyethylene alkyl ether sulfates, polyoxyethylene fatty amine sulfates, polyoxyethylene alkyl phenyl ether sulfates, Polyoxyethylene alkyl ether phosphates, α-sulfonated fatty acid alkyl ester salts, polyoxyethylene alkyl phenyl ether phosphates, Quaternary ammonium salt, primary to tertiary fatty amine salt, trialkylbenzylammonium salt, alkylpyridinium salt, 2-alkyl-1-alkyl-1-hydroxyethylimidazolinium salt, N N, N-dimethyl-N-alkyl-N-carboxymethylammoniobetaine, N, N, N-trialkyl-N-, N, N-dimethyl-N-alkyl-N-carboxymethylammoniobetaine Amphoteric surfactants such as alkylene ammoniocarboxybetaine and N-acylamidopropyl-N ′, N′-dimethyl-N′-β-hydroxypropylammoniosulfobetaine can be used.
In addition, stevia derivatives such as enzyme-treated stevia, lecithin and its derivatives, lactic acid bacteria fermented rice, lactic acid bacteria fermented germinated rice, lactic acid bacteria fermented cereals (eg wheat, legumes, millet), etc. .

Examples of humectants include glycerin, propylene glycol, dipropylene glycol, 1,3-butylene glycol, polyethylene glycol, sorbitol, xylitol, sodium pyrrolidonecarboxylate, and sugars such as trehalose, lactic acid bacteria fermented rice, and mucopolysaccharides. (For example, hyaluronic acid and its derivatives, chondroitin and its derivatives, heparin and its derivatives, etc.), elastin and its derivatives, collagen and its derivatives, NMF related substances, lactic acid, urea, higher fatty acid octyldodecyl, seaweed extract, seafood Examples include derived collagen and derivatives thereof, various amino acids and derivatives thereof.

Examples of the thickener include components derived from brown algae, green algae or red algae such as alginic acid, agar, carrageenan and fucoidan; polysaccharides such as pectin, locust bean gum and aloe polysaccharide; gums such as xanthan gum, tragacanth gum and guar gum; Cellulose derivatives such as methyl cellulose and hydroxyethyl cellulose; synthetic polymers such as polyvinyl alcohol, polyvinyl pyrrolidone, carboxyvinyl polymer, acrylic acid / methacrylic acid copolymer; hyaluronic acid and derivatives thereof; polyglutamic acid and derivatives thereof.

Examples of the antiseptic / bactericidal agent include urea; paraoxybenzoates such as methyl paraoxybenzoate, ethyl paraoxybenzoate, propyl paraoxybenzoate, and butyl paraoxybenzoate; phenoxyethanol, dichlorophene, hexachlorophene, chlorhexidine hydrochloride, benzaza chloride Luconium, salicylic acid, ethanol, undecylenic acid, phenols, jamal (imidazodenyl urea), 1,2-pentanediol, various essential oils, bark dry matter, and the like.

Examples of powder components include sericite, titanium oxide, talc, kaolin, bentonite, zinc oxide, magnesium carbonate, magnesium oxide, zirconium oxide, barium sulfate, silicic anhydride, mica, nylon powder, polyethylene powder, silk powder, and cellulose. System powders, powders of cereals (rice, wheat, corn, millet, etc.), powders of beans (soybeans, red beans, etc.).

Examples of the ultraviolet absorber include ethyl paraaminobenzoate, ethylhexyl paradimethylaminobenzoate, amyl salicylate and derivatives thereof, 2-ethylhexyl paramethoxycinnamate, octyl cinnamate, oxybenzone, 2,4-dihydroxybenzophenone, 2-hydroxy-4 -Methoxybenzophenone-5-sulfonate, 4-tertiarybutyl-4-methoxybenzoylmethane, 2- (2-hydroxy-5-methylphenyl) benzotriazole, urocanic acid, ethyl urocanate, aloe extract, etc. .

Examples of the antioxidant include butylhydroxyanisole, butylhydroxytoluene, propyl gallate, vitamin E and derivatives thereof.

Examples of whitening agents include t-cycloamino acid derivatives, kojic acid and derivatives thereof, ascorbic acid and derivatives thereof, hydroquinone or derivatives thereof, ellagic acid and derivatives thereof, nicotinic acid and derivatives thereof, resorcinol derivatives, tranexamic acid and derivatives thereof, 4 -Methoxysalicylic acid potassium salt, magnolignan (5,5'-dipropyl-biphenyl-2,2'-diol), 4-HPB (rhodenol, 4- (4-hydroxyphenyl) -4-butanol)), hydroxybenzoic acid And derivatives thereof, vitamin E and derivatives thereof, α-hydroxy acid, and AMP (adenosine monophosphate, adenosine monophosphate). These may be used alone or in combination.

  Examples of the kojic acid derivatives include kojic acid esters such as kojic acid monobutyrate, kojic acid monocaprate, kojic acid monopalmitate, kojic acid dibutyrate, kojic acid ethers, kojic acid sugar derivatives such as kojic acid glucoside, etc. However, as the ascorbic acid derivatives, for example, L-ascorbic acid-2-phosphate sodium, L-ascorbic acid-2-phosphate magnesium, L-ascorbic acid-2-sulfate sodium, L-ascorbic acid-2 -Ascorbic acid ester salts such as magnesium sulfate, L-ascorbic acid-2-glucoside, ascorbic acid sugar derivatives such as L-ascorbic acid-5-glucoside, 6-position acylated products of these ascorbic acid sugar derivatives (acyl group is Hexanoyl group, o L-ascorbic acid tetrafatty acid esters such as L-ascorbic acid tetraisopalmitate, L-ascorbic acid tetralaurate, 3-O-ethylascorbic acid, L-ascorbic acid- 2-phosphate-6-O-palmitate sodium, glycerascorbic acid and the like, and hydroquinone derivatives include arbutin (hydroquinone-β-D-glucopyranoside), α-arbutin (hydroquinone-α-D-glucopyranoside) and the like. Examples of the tranexamic acid derivative include tranexamic acid esters (for example, tranexamic acid lauryl ester, tranexamic acid hexadecyl ester, tranexamic acid cetyl ester or salts thereof), and amides of tranexamic acid (for example, tranexamic acid mesylate). Resorcinol derivatives include, for example, 4-n-butylresorcinol, 4-isoamylresorcinol and the like, and 2,5-dihydroxybenzoic acid derivatives include, for example, 2,5-diacetoxybenzoic acid, 2- Acetoxy-5-hydroxybenzoic acid, 2-hydroxy-5-propionyloxybenzoic acid and the like, nicotinic acid derivatives such as nicotinic acid amide and benzyl nicotinate, and vitamin E derivatives such as vitamin E nicotinate and vitamin Examples of α-hydroxy acids such as E-linoleate include lactic acid, malic acid, succinic acid, citric acid, and α-hydroxyoctanoic acid.

Physiologically active ingredients include placenta extract, Sahakuhi extract, Yukinoshita extract, rice bran extract, rice bran extract hydrolyzate, lactic acid bacteria fermented rice, lactic acid bacteria fermented rice, lactic acid bacteria fermented cereal (wheat, legumes, millet) , White coconut extract, purple extract, lotus seed extract, party extract, pearl hydrolyzate, fermented royal jelly, fermented sake lees, Pandanus amaryllifolius Roxb. Flava (Arcangelicia flava Merrilli) extract, chamomile extract, coral grass extract, eggplant (water eggplant, long eggplant, Kamo eggplant, rice eggplant etc.) extract, seaweed extract such as kombu, catamen giraffe, sea bream, etc. Marine flowering plant extracts, linoleic acid and its derivatives or processed products (eg liposomal linoleic acid), animal or fish collagen And derivatives thereof, elastin and derivatives thereof, glycyrrhizic acid and derivatives thereof (dipotassium salts, etc.), t-cycloamino acid derivatives, vitamin A and derivatives thereof, allantoin, diisopropylamine dichloroacetate, γ-amino-β-hydroxybutyric acid, gentian extraction Herb extract, licorice extract, carrot extract, aloe extract, etc., rice fermentation extract, beetroot extract, anaosa extract, Zizyphus joazeiro extract and the like.

The application site of the external preparation for skin containing the composition of the present invention includes general skin including the scalp, and is not particularly limited. Therefore, the dosage forms include emulsions, creams, lotions, essences, ointments, packs, haps, skin cleansers (soaps, etc.), facial cleansers, shampoos, rinses, treatments, various makeup cosmetics, bath preparations, etc. It can be diverse. The composition for external use of the skin of the present invention is not limited to cosmetics, and can also be used for quasi drugs and pharmaceuticals.

  Next, the present invention will be described more specifically with reference to production examples, examples, test examples and formulation examples, but the present invention is not limited thereto. In the following, all parts are parts by weight, and all percentages are% by weight.

Production Example 1 Hydrolyzate of rice extract 1250 g of 0.025 mol / L sodium hydroxide solution was added to 1000 g of polished white rice and stirred at room temperature for 21 hours. Solids were removed by filtration and the extract was adjusted to pH 7.5. Papain 0.02% and actinase 0.02% were added to this extract and hydrolyzed at 40 ° C. for 2 hours. After inactivating the enzyme by heating, this liquid was filtered to obtain 590 g of a rice extract hydrolyzed liquid (solid content concentration 1.6%).

Production Example 2 Enzyme-decomposed product of white coconut 50 g of pulverized white birch seeds (white coconut) was mixed with 1000 g of purified water, extracted at 40 ° C. for 1 hour, filtered, and 820 g of a pale yellow transparent white coconut extract solution ( Solid concentration: 1.2% by weight) was obtained. Next, 0.05 g of actinase AS (manufactured by Kaken Pharma Co., Ltd.) was added to 500 g of the extract solution obtained here, and hydrolyzed at 40 ° C. for 2 hours. Thereafter, the enzyme was inactivated by heating at 85 ° C. for 1 hour, followed by filtration to obtain 450 g (solid content concentration 1.1% by weight) of a hydrolyzate solution of a pale yellow transparent white coconut extract.

Production Example 3 A lotus seed fermented lotus seed (with astringent skin removed) 100 g was crushed, 1900 g of purified water was added to prepare a suspension, and heat sterilized. After adding 1 g of glucoamylase, 1 g of papain and 0.5 g of cellulase to this suspension, 10 ⁸ cells / mL of lactic acid bacteria (Lactobacillus plantarum) were inoculated, followed by static culture at 37 ° C. for 3 days under a nitrogen stream. . After completion of the culture, the mixture was sterilized by heating, and the culture solution was filtered to obtain 1450 g of a lotus seed fermented lactic acid bacterium solution (solid content concentration: 2.7%).

Production Example 4 50 g of barley seeds excluding the fermented husk of barley seeds were pulverized, 950 g of purified water was added to prepare a suspension, and heat sterilized. After adding glucoamylase 0.5g and papain 0.5g to this suspension, 10 < ⁷ > / mL yeast (Saccharomyces cerevisiae) was inoculated, and it left still and culture | cultivated at 30 degreeC for 3 days. After completion of the culture, the mixture was sterilized by heating, cooled to room temperature, and filtered to obtain 520 g of a fermented pearl seed solution (solid concentration: 1.4%).

Production Example 5 Extract of Silane Bulb 100 g of chopped silane bulb was mixed with 900 g of purified water, extracted at 80 ° C. for 3 hours, and then filtered to obtain 510 g of a pale yellow transparent extract solution (solid content concentration 2. 3%).

The composition for external use of the skin of the present invention is prepared by mixing the enzyme degradation product, fermented material and extract obtained in the above production examples. For example, as shown in Table 1 below, each component is blended so that the final concentration as a solution is 0.2% or 1.0% with respect to the total amount of the composition, and the external application of the skin of the present invention It is set as a composition.
[Table 1]

Test Example 1 Epidermal cell activation effect test The epidermal cell activation effect of the composition for external use according to Examples 1 and 2 was evaluated by the following method.
[Test method]
Normal epidermal keratinocytes (NHEK (F)) were seeded in a 96-well plate at 4 × 10 ³ cells / well, and then cultured at 37 ° C. for 24 hours using HuMedia-KG2 medium (Kurashikibo Co., Ltd.). After culturing for 24 hours, the compositions for external use in Examples 1 and 2 were added as sample solutions to the above medium, and further cultured for 48 hours. Here, the final concentration of the sample solution as a solution with respect to the total amount of the above-mentioned culture medium is the enzymatic degradation product of Production Examples 1 and 2, the fermentation product of Production Examples 3 and 4, and the extract of Production Example 5, respectively. It added so that it might become 0.2% (namely, 1.0% as a mixed solution), or 1.0% (namely, 5.0% as a mixed solution). After completion of the culture, the respiratory activity (MTT activity) of the cells was evaluated by the MTT reduction method (H. Tada et.al., J. Immunol. Methods 93, 157, 1986). That is, after removing the medium from the well plate, 0.03% MTT reagent (3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazoliumbromide) was added, After reacting for 1 hour and dissolving the produced formazan with isopropanol, the absorbance at 570 nm was measured, and the value obtained by subtracting the turbidity (absorbance: 630 nm) due to the cell residue was defined as MTT activity. As a control, MTT activity was also measured when PBS (-) was added to the medium instead of the sample solution. Furthermore, as a positive control, MTT activity was measured when 100 mM glucose was added to the medium instead of the sample solution. The MTT activity of the sample solutions according to Examples 1 and 2 of the present invention was expressed as a relative value when the MTT activity value of the control was 100.

  The results of Test Example 1 are shown in FIG. As shown in FIG. 1, it became clear that the composition for external use of skin according to Examples 1 and 2 of the present invention has an excellent epidermal cell activation effect in a concentration-dependent manner. In addition, since glucose which is a positive control also showed an epidermal cell activation effect, it is clear that this test system was performed normally.

Test Example 2 Prostaglandin E2 (PGE2) Production Suppression Effect Test The anti-inflammatory effect of the composition for external use according to Examples 1 and 2 was evaluated by a prostaglandin E2 (hereinafter referred to as “PGE2”) production inhibition test. PGE2 is an inflammatory chemical mediator that causes skin inflammation, and is a substance secreted in skin cells damaged by external factors such as ultraviolet rays. Therefore, the anti-inflammatory effect is evaluated by this PGE2 production inhibitory effect test.
[Test method]
Rabbit cornea-derived cells (SIRC) were suspended in 10% FBS-containing Eagle's minimum essential medium (Nissui Pharmaceutical Co., Ltd.), seeded in a 96-well plate at 5 × 10 ³ cells / well, and incubated at 37 ° C. for 3 days. Cultured. After culturing for 3 days, the compositions for external use in Examples 1 and 2 were added as sample solutions to the above medium, and further cultured for 24 hours. Here, the final concentration of the sample solution as a solution with respect to the total amount of the above-mentioned culture medium is the enzymatic degradation product of Production Examples 1 and 2, the fermentation product of Production Examples 3 and 4, and the extract of Production Example 5, respectively. It added so that it might become 0.2% (namely, 1.0% as a mixed solution), or 1.0% (namely, 5.0% as a mixed solution). In addition, it was set as control which culture | cultivated the said cell similarly for 24 hours in the culture medium which added PBS (-) instead of the said sample solution. Next, an ultraviolet B wave of 100 mJ / cm ² was irradiated from the bottom of the incubator, and after further culturing for 2 days, the amount of PGE 2 secreted into the culture supernatant was measured using a PGE 2 measurement kit (manufactured by Cayman Caymic). It was measured. In addition, the amount of PGE2 when indomethacin 10 μM was used as a positive control instead of the sample solution was determined in the same manner. Moreover, PBS (-) was added like control, and the control group which does not irradiate with an ultraviolet-ray after 24 hours culture | cultivation was also provided.

  The results of Test Example 2 are shown in FIG. As shown in FIG. 2, it became clear that the composition for external use of skin according to Examples 1 and 2 of the present invention has a PGE2 suppressing effect that is remarkably excellent in a concentration-dependent manner. In addition, since indomethacin, which is a positive control, also showed a PGE2 inhibitory effect, it is clear that this test system was performed normally.

Test Example 3 Melanin production inhibitory effect test The melanin production inhibitory effect of the compositions for external use according to Examples 1 and 2 was evaluated by the following method.
[Test method]
After seeding 6 × 10 ⁴ B16 cells (B16-F10) in a culture dish, the cells were cultured for 24 hours using 10% FBS-containing RPMI-1640 medium (Sigma Aldrich). After culturing for 24 hours, the composition for external use of Examples 1 and 2 was added to the above medium as a sample solution, and further cultured until B16 cells became confluent. Here, the final concentration of the sample solution as a solution with respect to the total amount of the above-mentioned culture medium is the enzymatic degradation product of Production Examples 1 and 2, the fermentation product of Production Examples 3 and 4, and the extract of Production Example 5, respectively. It added so that it might become 0.2% (namely, 1.0% as a mixed solution), or 1.0% (namely, 5.0% as a mixed solution). After completion of the culture, the cells are washed with a PBS (−) solution, and then 0.5 g / L Trypsin / 0.53 mmol / L EDTA solution (Nacalai Tesque) is added and allowed to stand at 37 ° C. for 5 minutes. Thus, a cell suspension in which the cells were detached from the petri dish was obtained. The cell suspension is centrifuged (10000 rpm, 4 ° C.), and after removing the supernatant, 10%
DMSO-containing 1N NaOH aqueous solution was added, and the mixture was boiled in a boiling water bath for 5 minutes to obtain a cell lysate. The amount of melanin in this cell lysate was measured by absorbance (490 nm). In consideration of the effects of the sample solution on cell irritation and growth inhibition, the amount of protein in the cell lysate was measured by the Bradford method, and the amount of melanin per protein was calculated.
As a control, the amount of melanin / protein when PBS (-) was added to the medium instead of the sample solution was also measured. Further, as a positive control, the amount of melanin / protein when 2 mM kojic acid was added to the medium instead of the sample solution was also measured. In addition, the melanin production | generation suppression rate of the sample solution which concerns on Example 1, 2 of this invention was represented by the relative value when the value of melanin / protein amount of control is set to 100.

The results of Test Example 3 are shown in FIG. As shown in FIG. 3, it became clear that the composition for external use of skin according to Examples 1 and 2 of the present invention has a remarkably excellent melanin production inhibitory effect in a concentration-dependent manner. In addition, since kojic acid which is a positive control also showed the melanin production inhibitory effect similarly, it is clear that this test system was performed normally.

As described above, the external composition for skin according to the present invention has both an epidermal cell activation effect, a PGE2 production inhibitory effect (anti-inflammatory effect), and a melanin production inhibitory effect. The skin aging phenomenon (wrinkles, tarmi, stains, freckles, etc.) caused by various factors can be prevented and improved in a multifaceted and comprehensive manner.

<Prescription Example 1> Lotion Toner lotion was formulated according to a conventional method by blending raw materials at the blending ratio shown in the following table.
(material)
(weight%)
Enzymatic degradation product of Production Example 1 0.2
Enzymatic degradation product of Production Example 2 0.2
Fermented product of Production Example 3 0.2
Fermented product of Production Example 4 0.2
Extract of Production Example 5 0.2
Ascorbic acid-2-glucoside 2.0
Polypropylene glycol 2.0
Oxybenzone 3.0
P-Hydroxybenzoate ester 0.5
Citric acid appropriate amount perfume appropriate amount purified water balance
Total 100.0

<Prescription example 2> Cosmetic cream The raw materials were blended in the blending ratio shown in the following table, and a cosmetic cream was produced according to a conventional method.
(Raw material) (wt%)
Enzymatic degradation product of Production Example 1 1.0
Enzymatic degradation product of Production Example 2 1.0
Fermented product of Production Example 3 1.0
Fermented product of Production Example 4 1.0
Extract of Production Example 5 1.0
Ascorbic acid-2-glucoside 2.0
Cysteine 1.0
Stearyl alcohol 5.0
Stearic acid 8.0
Squalane 10.0
Self-emulsifying glyceryl monostearate 3.0
Polyoxyethylene cetyl ether (20E.0) 1.0
Propylene glycol 5.0
Potassium hydroxide 0.3
Perfume Appropriate amount Preservative Appropriate amount
Purified water balance
Total 100.0

Claims (1)

An external composition for skin comprising the following (a) to (e) as essential components.
(A) Enzymatic degradation product obtained by treating alkaline extract of rice with two or more proteolytic enzymes (b) Extract of Brassica alba seeds (white coconut) of Brassicaceae Enzymatic degradation product obtained by treatment with proteolytic enzyme (c) Fermented product obtained by fermenting Nympaeaceae plant seeds with lactic acid bacteria (d) Fermenting pearl seeds with yeast (E) Bulb extract of silane (Bletilla striata (THUNB.) REICHB.fil.)