JP2006022093A - External preparation for skin containing mung bean protein decomposition product - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an external preparation for skin derived from a natural product and having excellent moisturizing effect and aging-retarding effect. <P>SOLUTION: The present invention provides an external preparation for skin containing a mung bean protein decomposition product. The ratio of dipeptide or tripeptide in total peptide of the mung bean protein decomposition product is preferably ≥10%, more preferably ≥50%. The mung bean protein decomposition product is further preferably obtained by hydrolyzing a protein derived from mung bean with a protease. The external preparation for skin is preferably a cosmetic. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a skin external preparation containing a mung bean proteolysate. In more detail, it is related with the skin external preparation which has the outstanding moisturizing effect and the antiaging effect.

  The skin is composed of three layers: the epidermis, dermis, and subcutaneous tissue from the outside. The outermost layer, the epidermis, is further divided into four layers (from the outside, the stratum corneum, the granule layer, the spiny layer, and the basal layer), and plays a role in protecting and protecting against water retention and various external stimuli. ing. In the basal layer, which is the lowermost layer of the epidermis, cell proliferation is constantly performed, and new cells are pushed up to the surface while changing their characteristics into a spiny layer, a granular layer, and a horny layer. The stratum corneum cells eventually fall off and are replaced with new cells. This process is called turnover. Although the basal epidermis cells have nuclei inside the cells, they are digested during the turnover process, so the nuclei disappear when they peel off the skin. When skin is weakened due to external stress such as ultraviolet rays and drying and aging, and turnover is disturbed, various troubles occur in the skin. In general, turnover tends to slow down with aging, and when turnover slows down, the stratum corneum that should peel off remains on the skin and the stratum corneum thickens, or melanin that should be discharged Sedimentation occurs, causing skin aging such as spots, wrinkles, and kusumi. Even if the turnover is too early, immature cells with nuclei are pushed to the skin surface, resulting in keratinization, resulting in a decrease in skin barrier function and water retention function. In the normal turnover process, natural moisturizing factors are produced and play a role in keeping the stratum corneum moist. When normal turnover is promoted, the skin becomes moist and firm.

  On the other hand, when the stratum corneum is not moistened, it is easy to be affected by external stimuli, and normal turnover cannot be performed. And this also causes a vicious cycle of suppressing water retention. Moreover, in order to maintain fine and fresh skin, it is necessary that the stratum corneum has sufficient moisture (Non-patent Document 1).

  In this way, turnover and stratum corneum moisture are related to each other, and normal turnover is actively performed and sufficient moisture is maintained in the stratum corneum to maintain healthy and beautiful skin. Is important to. For this reason, various components have been studied as components for external preparations for skin preparations that regulate turnover and promote normality or moisturize the stratum corneum (Patent Documents 1 and 2).

  In addition, various cosmetic ingredients have been studied as a means to moisturize the stratum corneum and prevent skin aging. For example, collagen and glycosaminoglycans, which are the main components of the skin, are thought to play an important role in moisturizing. Various oligopeptides have been disclosed as components for promoting these synthesis (Patent Document 3). It is also known that certain oligopeptides have an effect of preventing pigmentation by ultraviolet rays (Patent Document 4). Furthermore, it is also known that a dipeptide containing L-glutamine has a moisturizing action (Patent Document 5).

  Moreover, there exists transdermal moisture transpiration (TEWL) as what evaluates the barrier function of skin. TEWL is the amount of water that passes through the stratum corneum and exits the skin. The normal stratum corneum has a role of maintaining a proper amount of water in the body. However, a functionally defective stratum corneum does not have sufficient moisture retention capacity to keep the stratum corneum flexible. As a result, a large amount of moisture passes through the surface of the skin and is lost. When the appropriate amount of water is not retained in the skin, the skin's suppleness and softness are lost and it becomes hard and brittle.

  In addition, reactive oxygen species generated in vivo by aging or exposure to ultraviolet rays are also deeply involved in skin wrinkle formation and skin aging. It is considered that eliminating such reactive oxygen species is effective in preventing and improving skin aging, and various antioxidants have been studied.

  For example, glutathione is an antioxidant substance widely distributed in the living body, and is a tripeptide composed of cysteine, glutamic acid, and glycine. Glutathione is a substrate for glutathione peroxidase, which detoxifies peroxides such as hydrogen peroxide and lipid peroxide generated in the body, and is oxidized to protect the body from oxidative damage that occurs in the body. There is work. Due to excessive oxidative stress, glutathione is consumed and depleted, and normal cell functions may not be maintained.

  When glutathione is added to an external preparation as it is, an unpleasant odor is generated during use. In order to remove an unpleasant odor, an inclusion compound of glutathione (Patent Document 6) has been developed as a cosmetic ingredient. Alternatively, glutathione derivatives have been developed for the purpose of enhancing transdermal permeability of glutathione (Patent Document 7). Moreover, a specific plant (patent document 8) or a 2-chromanone derivative (patent document 9) is known as a substance that promotes intracellular glutathione production.

In addition, a cosmetic composition containing at least one protein degradation product selected from silk protein, keratin protein, and collagen protein is known (Patent Document 10). This cosmetic composition is described as having a moisturizing action, an anti-skin aging action, and an anti-inflammatory action.
JP 11-335257 A JP 2001-010946 A Special Table 2002-524487 US Pat. No. 6,296,857 JP 2003-306420 A Japanese Patent No. 2954634 JP-A-9-67227 JP 2002-275079 A JP 2003-321463 A JP 2003-95913 A “Bio-cosmetics”, written by Masahiko Okamoto, Kyoritsu Shuppan, 1986, pages 23 and 52 "Skin Measurement / Evaluation Manual Collection", Technical Information Association, page 101

  An object of this invention is to provide the skin external preparation which has the outstanding moisturizing effect and aging inhibitory effect derived from a natural product.

  The present inventor conducted various evaluations as a skin external preparation for the mung bean proteolysate, and found that it had an excellent moisturizing effect and turnover promoting effect on the skin, thereby completing the present invention.

  This invention provides the skin external preparation containing a mung bean proteolysate.

  In a preferred embodiment, the ratio of dipeptide or tripeptide in the total peptide of the mung bean proteolysate is 10% or more.

  In a more preferred embodiment, the ratio of dipeptide or tripeptide in the total peptide of the mung bean proteolysate is 50% or more.

  In a preferred embodiment, the mung bean proteolysate is obtained by hydrolyzing a mung bean-derived protein with a protease.

  In a more preferred embodiment, the external preparation for skin is a cosmetic.

  The mung bean proteolysate contained in the external preparation for skin of the present invention has a glutathione production promoting action, a moisturizing action, and a skin turnover promoting action, and further has a collagenase inhibiting action, a radical scavenging action, and a tyrosinase inhibiting action, It also has an elastase inhibitory action. Therefore, the skin external preparation of the present invention has an excellent skin beautifying effect including a moisturizing effect on the skin.

  Mung bean belongs to the leguminous plant, the part of the legume is green to brown, and its size is smaller than azuki bean. Mung beans are known as bean sprouts, and mung bean starch is known as a raw material for harusame.

  The raw material containing the protein derived from mung bean used in the present invention (hereinafter referred to as “raw material”) may be a pulverized mung bean or a squeezed juice thereof, or an extract of these water, acid, or alkali. Furthermore, by-products in the processing of mung beans can also be used as raw materials. For example, a by-product containing a mung bean-derived protein produced in the production process of a harsame whose main component is mung bean starch. The form may be any form such as liquid, powder, paste and the like. In addition to mung bean-derived proteins, sugars, dietary fibers, salt, moisture, fats and oils, and the like may be included. The protein content (hereinafter referred to as “crude protein content”) in the raw material is not particularly limited, but is preferably 15% by mass or more, and more preferably 30% by mass or more.

Here, the crude protein content is calculated by multiplying the amount of nitrogen in the protein by a conversion factor. The amount of nitrogen is measured by, for example, the semi-micro Kjeldahl method. Specifically, the crude protein content is calculated as follows. First, a 50 mg sample is accurately weighed into a Kjeldahl flask. Next, 10 g of potassium sulfate and 1 g of copper sulfate are mixed to form a decomposition accelerator, 1 g of the mixture is placed in a flask, and 5 mL of concentrated sulfuric acid is further added. After standing overnight, the flask is heated gradually until the liquid becomes clear and no carbide is observed on the inner wall of the flask. After cooling, 20 mL of distilled water is added and mixed well, followed by ice cooling, and the flask is connected to a distillation apparatus that has been previously washed with water vapor. Place 10 mL of 0.1N sulfuric acid and 2-3 drops of indicator (mixture of methyl red and methylene blue reagent solution) in the receiver that receives the distillate, and immerse the lower end of the cooler of the distillation apparatus in this solution. From a funnel connected to a distillation apparatus, 20 mL of 40% sodium hydroxide is added to the flask and distilled through steam for 6-7 minutes. The lower end of the cooler is separated from the liquid surface, the part is washed with a small amount of water, and titrated with 0.1N sodium hydroxide. Moreover, what was measured by the same method without adding a sample is used as a blank. The crude protein content is calculated by the following formula:
Crude protein content (% (w / w)) = {([B] − [A]) × F × 1.4007 × 6.25 / [C]} × 100
Where [A] is the volume of 0.1N sodium hydroxide required for titration when the sample is added (mL), and [B] is the volume of 0.1N sodium hydroxide required for the titration of the blank ( mL). [C] is the mass (mg) of the sample, and “F” is the factor of sodium hydroxide used for titration. “1.4007” corresponds to the mass (mg) of nitrogen corresponding to 1 mL of 0.1 N sulfuric acid. In the present invention, “6.25” was used as a protein conversion factor from the mass of nitrogen.

  In the present invention, the mung bean proteolysate refers to a decomposed product obtained by hydrolyzing a protein derived from mung bean by any means, or a decomposed product decomposed by an enzyme such as an acid or a protease. From the viewpoint that the reaction is mild and that a by-product is hardly generated, a decomposition product by protease is preferable.

  Proteases used to hydrolyze mung bean derived proteins are proteases or peptidases that can hydrolyze proteins. For example, Rhizopus genus, Rhizopus genus such as Rhizopus niveus, Aspergillus niger, Aspergillus genus such as Aspergillus oryzae, Bacillus subtilis, Bacillus sp. Examples include enzymes derived from microorganisms such as Bacillus genus, etc .; enzymes derived from animals such as pepsin and pancreatin; and enzymes derived from plants such as papain and bromelain. In the present invention, an acidic protease derived from the genus Aspergillus is desirable because the degree of degradation can be easily adjusted. The protease may be a commercially available refined product or a crude product, and may be used alone or in combination of two or more. The hydrolysis reaction conditions (reaction temperature, pH, time, amount of enzyme used, etc.) can be set according to the optimum action conditions of the protease to be used. Usually, the temperature is 10 ° C. to 80 ° C., the pH is 2 to 11, the reaction time is 2 to 48 hours, and the amount of enzyme used is 10 to 30,000 units per gram of crude protein. Preferably, the reaction temperature is 30 ° C. to 60 ° C., the pH is 3 to 8, the reaction time is 4 to 20 hours, and the enzyme amount is 100 to 7000 units per gram of crude protein.

  After the protease treatment, insoluble matter is removed from the obtained mung bean proteolysate by filtration or centrifugation. The obtained supernatant is dried by a method such as concentration under reduced pressure, freeze drying, spray drying or the like to obtain the target product. The form of the obtained mung bean proteolysate may be liquid, powder, or paste.

  The mung bean proteolysate of the present invention preferably has a dipeptide or tripeptide ratio of 10% or more in the total peptide. The ratio of dipeptide or tripeptide is more preferably 40% or more. It is more preferably 50% or more from the viewpoint of excellent absorbability when ingested. The dipeptide or tripeptide is specifically a peptide having a molecular weight in the range of about 130 to 580 in the following analysis of molecular weight distribution.

  In the present invention, the molecular weight distribution and dipeptide or tripeptide content of the mung bean proteolysate were analyzed by the following method.

The molecular weight was measured by high performance liquid chromatography using a gel filtration column (Superdex Peptide HR 10/30, Pharmacia Biotech). A 30% acetonitrile aqueous solution containing 0.1% trifluoroacetic acid was used as a mobile phase, the flow rate was 0.3 ml / min, and detection was performed by ultraviolet (220 nm) absorption. Molecular weights are known for oligopeptides with known molecular weights “Gly = 75.07”, “Ala-Pro = 186.2”, “Angiotensin
A standard line was created using “II = 1046.2” and “Substance P = 1347.7”. Molecular weight distribution is measured by data processing equipment (D-2500
Chromato-Integrator (manufactured by Hitachi, Ltd.) was used to show the area ratio of the integration chart. For the dipeptide or tripeptide content, the total amount of peptides having an average molecular weight of 130 to 580 was determined from the above integration chart, and this was defined as the amount of dipeptide or tripeptide, and the ratio to the total peptide amount was determined.

  The mung bean protein degradation product has a glutathione production promoting action, a skin turnover promoting action, a stratum corneum moisturizing action, and a transdermal moisture transpiration reducing action. Furthermore, it has collagenase inhibitory action and radical scavenging action, as well as tyrosinase inhibitory action and elastase inhibitory action.

  The skin external preparation of this invention contains the said mung bean protein degradation product. Preferably, 0.1 to 99% by mass of the mung bean proteolysate may be contained in the skin external preparation of the present invention.

  The form of the external preparation for skin of the present invention is not particularly limited, and can be mixed in an ordinary skin external preparation base. For example, it can be in various forms such as lotion, emulsion, cream, ointment and the like. In particular, it is preferably provided as a cosmetic such as lotion, cosmetic liquid, milky lotion or cream.

  The external preparation for skin of the present invention may contain various components that can be usually used for external preparations for skin and cosmetics in addition to the above-mentioned mung bean proteolysate as long as the effects of the mung bean proteolysate are not impaired. For example, other moisturizers, oily substances, surfactants, thickeners, neutralizers, preservatives, antioxidants, dyes, fragrances, UV absorbers, whitening agents, cell activators, other medicinal ingredients, etc. Can be mentioned.

(Production Example 1: Preparation of Mung Bean Proteolysate)
The dried product of mung bean protein discharge liquid, which is a by-product obtained in the Harusame manufacturing process, was pulverized with a mill, and tap water was added and dispersed so that the crude protein content was 6% by mass. This was adjusted to pH 3 with hydrochloric acid, and then acid protease (denapsin, manufactured by Nagase ChemteX Corp.) was added at 3000 units per gram of crude protein and reacted at 40 ° C. for 16 hours with stirring. After the reaction, the enzyme was inactivated by heating at 80 ° C. to 85 ° C. for 20 minutes. Thereafter, the reaction solution was adjusted to pH 7 by adding NaOH. This was centrifuged, and the supernatant was lyophilized to obtain a sample of a mung bean proteolysate (hereinafter referred to as mung bean peptide). The molecular weight distribution of the sample was measured by the above procedure. The content of dipeptide or tripeptide in the total peptide of this sample was 64.4%.

(Production Example 2: Preparation of Mung Bean Proteolysate)
The dried product of mung bean protein discharge liquid, which is a by-product obtained in the Harusame manufacturing process, was pulverized with a mill, and tap water was added and dispersed so that the crude protein content was 6% by mass. This was adjusted to pH 7 with sodium hydroxide, and then alkaline protease (trade name: Biolase SP15-FG, manufactured by Nagase ChemteX Corp.) was added at 10,000 units per gram of crude protein and reacted at 40 ° C. for 16 hours with gentle stirring. I let you. The enzyme was inactivated by heating at 80 ° C. to 85 ° C. for 20 minutes. The enzyme reaction solution was centrifuged, and the supernatant was freeze-dried to obtain a sample of a mung bean proteolysate (hereinafter referred to as mung bean peptide). The molecular weight distribution of the sample was measured by the above procedure. The ratio of dipeptide and tripeptide in all peptides in this sample was 61.1%.

(Example 1: Measurement of glutathione production promoting action)
NHDF (AD) normal human dermal fibroblasts (Cascade Biology, Inc.) were added to a normal human dermal fibroblast basal medium supplemented with 10% inactivated fetal bovine serum (GIBCO) -containing growth additive (LSGS; Kurabo) Medium 106S; Kurabo Industries, Inc.) was seeded in a 6-well plate at 2.0 × 10 ⁵ cells / well, and cultured at 37 ° C. under 5% CO ₂ for 24 hours. After confirming that the cells adhered to the plate, the medium was replaced with a medium supplemented with 100 or 1000 μg / mL of mung bean peptide obtained in Production Example 1 above as crude protein, and at 37 ° C. under 5% CO ₂ . After culturing for 24 hours, the cells were collected. The amount of glutathione in the cell
It measured using Glutathione Quantification Kit (made by DOJINDO MOLECULAR TECHNOLOGIES, INC.). In addition, the amount of protein in the cells was measured using the Lowry method, and the amount of glutathione per protein was calculated. The results are shown in FIG. A cell cultured in a medium not containing mung bean peptide was used as a control.

  As shown in FIG. 1, the mung bean peptide exhibited an excellent glutathione production promoting action in human skin fibroblasts.

(Example 2: Hygroscopicity / moisture retention test)
The sample (mung bean peptide, sodium hyaluronate, and glycerin obtained in Production Example 1) was dried at 60 ° C. in a vacuum dryer. The weighing bottle was also dried at 105 ° C. in a dryer. In the dried weighing bottle, 1 g of each of the above samples was accurately weighed. These were placed in a thermostatic oven at a temperature of 40 ° C. and a humidity of 75% for 6 days, and the weight change was measured (hygroscopicity test). They were then transferred into a desiccator with a temperature of 20 ° C. and a humidity of about 40% and left for another 6 days to measure the weight change (moisturizing test). The results are shown in FIG.

  As shown in FIG. 2, the mung bean peptide has better moisture absorption and moisture retention than glycerin and sodium hyaluronate, which are known to have moisture absorption and moisture retention.

(Example 3: Test to increase water absorption and retention function of stratum corneum)
A biological stratum corneum water load test was performed after sample application, and the water retention function improving effect of the stratum corneum by mung bean peptide was evaluated. That is, after measuring the skin moisture content at the measurement site of the upper arm, a 5% (w / v) aqueous solution of mung bean peptide obtained in Production Example 1 was applied to the same site. After the liquid dries, a water drop was placed on the epidermis for 10 seconds so as to cover the measurement site. The skin moisture content was measured every 30 seconds from immediately after wiping it up to 2 minutes later. A skin moisture meter for home use (skin moisture meter DM-R2, Matsushita Electric Industrial Co., Ltd.) was used to measure the skin moisture content. FIG. 3 shows the results obtained by measuring three times at the same site and obtaining an average value.

  In this measurement method, if the skin moisture value immediately after wiping is high, it is determined that the water absorption capacity is high, and the slower the return to the initial value is, the higher the water retention capacity is. As shown in FIG. 3, the mung bean peptide had an effect of increasing the water absorption capacity and water retention capacity of the stratum corneum.

(Example 4: Measurement of radical scavenging activity)
The radical scavenging activity of the mung bean peptide obtained in Production Example 1 was measured using 1,1-diphenyl-2-picrylhydrazyl (DPPH), which is a very stable radical. The DPPH radical, which is a purple stable radical, disappears when the radical disappears due to the action of antioxidants. By measuring this, the radical scavenging activity was evaluated.

The measurement of radical scavenging activity was performed according to the method described in JP-A No. 2003-81744. That is, first, 1.5 mL of various amounts of a sample dissolved in 100 mM MES buffer (pH 6) and 1.5 mL of an ethanol solution containing 150 μM DPPH were placed in a test tube, sealed tightly and mixed well. After standing at room temperature for 30 minutes, the absorbance at 517 nm was measured. The blank is 100 mM instead of the sample.
1.5 mL of MES buffer was used.

As a positive control, Trolox (6-Hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid known to have radical scavenging action
acid, ALDRICH) was also measured. The radical scavenging ability per mung bean peptide crude protein is shown as the Trolox amount showing radical scavenging ability corresponding to this.

  FIG. 4 shows the relationship between the added amount of mung bean peptide and the DPPH radical scavenging rate. As shown in FIG. 4, the mung bean peptide showed radical scavenging activity. The Trolox amount corresponding to the radical scavenging ability per 1 mg of mung bean peptide crude protein was 6.7 nmol.

(Example 5: Measurement of collagenase inhibitory activity)
Collagenase is said to promote skin wrinkles and tarmi by reducing collagen, which is a major component of the dermis. In order to prevent these, it is known to use a collagenase inhibitor (Japanese Patent Laid-Open No. 2003-55184). Then, collagenase inhibitory activity was examined.

Contains 20 mM calcium chloride, 0.1 M
A Tris-HCl buffer solution (pH 7.1) was prepared and used as a buffer solution. Pz-peptide (Pz-Pro-Leu-Gly-Pro-D-Arg-OH; BACHEM
Fenichemikalien AG) was dissolved in the above buffer solution to a concentration of 1 mM to obtain a substrate solution. 1 mg of distilled water was added to 5 mg of collagenase type IV (manufactured by SIGMA) and dissolved, and this was diluted 100 times with the above buffer solution to obtain a collagenase solution. 0.2 ml of substrate solution and 0.25 ml of sample solution prepared in various concentrations by dissolving in buffer were mixed, incubated at 37 ° C. for 5 minutes, 0.05 ml of collagenase solution was added thereto and reacted at 37 ° C. for 30 minutes. . The reaction was stopped by adding 1 ml of 25 mM citric acid, 5 ml of ethyl acetate was added, the bottle was sealed, and the mixture was vigorously stirred for 20 seconds with a mixer. The absorbance at 320 nm of the collected ethyl acetate layer was measured by centrifugation at 4 ° C. As the blank, citric acid was added before adding the enzyme, and the same measurement as in the test was used. Collagenase inhibition rate was calculated by the following formula. A in the formula indicates the absorbance when the sample is added, and B indicates the blank. C indicates the absorbance when no sample is added, and D indicates the blank.

  FIG. 5 shows the remaining rate of collagenase at mung bean peptide concentrations of 0.25%, 0.5%, and 1.0%. As shown in FIG. 5, the mung bean peptide was found to have collagenase inhibitory activity.

(Preparation Example 1: Preparation of mung bean peptide-containing skin lotion)
Using the mung bean peptide obtained in Production Example 1, a lotion was prepared according to the following formulation.
Mung bean peptide 5% by mass
1,3-butylene glycol 5% by mass
Glycerin 5% by mass
Methylparaben 0.1% by mass
Citric acid Trace amount Purified water Residual

(Comparative Preparation Example 1: Preparation of Mung Bean Peptide-Free Toner)
For comparison, a lotion containing no mung bean peptide was prepared according to the following formulation.
1,3-butylene glycol 5% by mass
Glycerin 5% by mass
Methylparaben 0.1% by mass
Citric acid Trace amount Purified water Residual

(Preparation Example 2: Preparation of cream containing mung bean peptide)
Using the mung bean peptide obtained in Production Example 1, a cream was prepared according to the following formulation.
Mung bean peptide 5% by mass
Glycerin 5% by mass
Squalane 12% by mass
Cetyl alcohol 7.2% by mass
CETEARETH-20 0.8% by mass
Methylparaben 0.2% by mass
Propylparaben 0.1% by mass
Citric acid Trace amount Purified water Residual

(Comparative Preparation Example 2: Preparation of cream containing no mung bean peptide)
For comparison, a cream containing no mung bean peptide was prepared according to the following formulation.
Glycerin 5% by mass
Squalane 12% by mass
Cetyl alcohol 7.2% by mass
CETEARETH-20 0.8% by mass
Methylparaben 0.2% by mass
Propylparaben 0.1% by mass
Citric acid Trace amount Purified water Residual

(Example 6: Measurement of skin turnover speed)
White petrolatum is mixed with 5% by mass of dansyl chloride, and this is applied to the skin inside the forearms of both subjects for 5 subjects by applying occlusion for 24 hours, and dansyl chloride is applied to the stratum corneum. Combined to form a label. The lotion of Preparation Example 1 and the cream of Preparation Example 2 were each applied to the separate labeling sites on one arm each morning and evening. As a control, the skin lotion of Comparative Preparation Example 1 and the cream of Comparative Preparation Example 2 were also applied to the labeled part of the other arm every day in the morning and evening. The number of days until the fluorescence emitted upon irradiation with ultraviolet rays completely disappeared at the labeled site was measured as the turnover speed. The results are shown in Table 1.

  Toner lotion and cream containing mung bean peptide (Preparation Examples 1 and 2 respectively) tended to have a slightly faster turnover rate of the horny layer of the skin than those not containing mung bean peptide.

  Next, at each site of each subject, stratum corneum cells were collected from the site by tape stripping on the day when the fluorescence completely disappeared. The collected horny layer cells were stained with gentian violet and brilliant green, and the number of nucleated cells was counted. The expression rate of nucleated cells was calculated from the number of nucleated cells per 20 cells. The results are shown in Table 2.

  In the lotion and cream containing the mung bean peptide (Preparation Examples 1 and 2, respectively), the expression rate of nucleated cells was low, and it was confirmed that the stratum corneum cells were healthy and mature.

  From the above results, it was found that cosmetics containing mung bean peptide promoted turnover of horny layer cells by normal keratinization.

(Example 7: Sensory evaluation)
The creams of Preparation Example 2 and Comparative Preparation Example 2 were applied to the faces of five subjects every morning and evening for two weeks. The feeling of use was evaluated for six items of wettability, rough skin, smoothness, elasticity, and fine lines. Judgment was made to select those that were applicable from a slight decrease, no change, a slight improvement, and good. The results are shown in Table 3.

  Many subjects felt that the cream containing mung bean peptide was better or slightly improved, and it was found that the cream was more suitable as a cosmetic.

(Preparation Example 3: Preparation of lotion containing mung bean peptide)
Using the mung bean peptide obtained in Production Example 1, a lotion was prepared according to the following formulation.
Mung bean peptide 7% by mass
Glycerin 5% by mass
Ethanol 5% by mass
Methylparaben 0.1% by mass
Purified water residue

(Comparative Preparation Example 3: Preparation of lotion containing no mung bean peptide)
For comparison, a lotion containing no mung bean peptide was prepared according to the following formulation.
Glycerin 5% by mass
Ethanol 5% by mass
Methylparaben 0.1% by mass
Purified water residue

(Example 8: Application of lotion)
To the seven subjects, the lotion prepared in the above Preparation Example 3 and the lotion prepared in the above Comparative Preparation Example 3 were applied every morning and evening for three weeks in a row.
Application site:
Whole right hand, inner right arm (applying lotion prepared in Preparation Example 3 above)
Whole left hand, forearm inner side of left arm (apply skin lotion prepared in Comparative Preparation Example 3 above)

(Example 9: Measurement of stratum corneum moisture content)
The measurements in Examples 9 to 11 below were performed so that the left and right sides were substantially the same part. It was examined whether the mung bean peptide of the present invention has an effect of increasing the stratum corneum moisture content. For 7 subjects who applied skin lotion according to Example 8, before application, 1 week after the start of application and 3 weeks later, Corneometer
Using CM825 (Courage + Khazaka), measure the stratum corneum water content of the back of both hands (A1: right hand, B1: left hand) and the stratum corneum water content inside the forearms of both arms (A2: right arm, B2: left arm). did. The stratum corneum moisture content may vary greatly depending on the ambient temperature and / or ambient humidity, the physical condition of the subject, and the like. Therefore, if the obtained stratum corneum moisture amounts A1, B1, A2 and B2 are evaluated as they are, correct results may not be obtained. Therefore, the present inventors evaluated the stratum corneum water content using the relative values R1 and R2 of the stratum corneum water content obtained by the following equation (1) in order to minimize the above-mentioned influence.

Rn = (An−Bn) / Bn (1)
(Where n = 1 or 2)

In this measurement method, the higher the stratum corneum moisture content increase effect of the mung bean peptide of the present invention, the greater the stratum corneum water content relative value Rn after coating than before coating. Table 4 shows the relative values R1 and R2 of the stratum corneum water content of the seven subjects. Moreover, FIG. 6 is a graph which shows transition of the average value of the horny layer water content relative value R1 of the hand.

  As shown in Table 4, when applied to the hand, an increase in stratum corneum water content was observed in 3 of 6 subjects during the 1 week application period, and 5 out of 6 subjects in the 3 week application period. An increase in stratum corneum water content was observed. In addition, as shown in Table 4, when applied to the inner side of the forearm, an increase in stratum corneum water content was observed in 4 out of 7 subjects in the application period of 1 week, and in 7 subjects in the application period of 3 weeks. An increase in stratum corneum moisture content was observed in three people. In particular, when applied to the hand, as shown in FIG. 6, the stratum corneum water content increases as the application period becomes longer, and the relative value R1 of the stratum corneum water content in the application period of 3 weeks is , Showed a significant increase compared to before application (Paired t-test, p <0.05). From the above results, it was found that the mung bean peptide of the present invention has an effect of increasing the stratum corneum moisture content.

(Example 10: Measurement of transdermal moisture transpiration)
It was examined whether the mung bean peptide of the present invention has an effect of reducing the transdermal moisture transpiration rate. About 7 subjects who applied skin lotion according to Example 8, transdermal moisture transpiration measuring device (AS-TW2 (ASAHI BIOMED)) before application, 1 week after application start and 3 weeks after application Was used to measure the amount of transdermal moisture transpiration (C1: right arm, D1: left arm) inside the forearms of both arms. Similar to the stratum corneum moisture amount described in Example 9, the transdermal moisture transpiration amount may vary greatly due to the influence of the ambient temperature and / or ambient humidity, the physical condition of the subject, and the like. Therefore, if the obtained transdermal moisture transpiration amounts C1 and D1 are evaluated as they are, correct results may not be obtained. Therefore, in order to reduce the above-described influence as much as possible, the present inventors evaluated the amount of transdermal moisture transpiration using the relative value T1 of the transdermal moisture transpiration obtained by the following equation (2).

T1 = (C1-D1) / D1 (2)

In this measurement, as the mung bean peptide of the present invention shows a higher effect of reducing the amount of transdermal moisture transpiration, T1 after application becomes smaller than before application. Table 5 shows the relative value T1 of the transdermal moisture transpiration of the seven subjects. Moreover, FIG. 7 is a graph which shows transition of the average value of the percutaneous moisture transpiration amount relative value T1 inside the forearm.

As shown in Table 5, 6 out of 7 subjects showed a decrease in transdermal water transpiration in the 1 week application period, and 5 out of 7 subjects in the 3 week application period showed transdermal water transpiration. A decrease was seen. As shown in FIG. 7, the relative value T1 decreased both after 1 week and 3 weeks after application, and a significant difference was observed after 1 week of application (paired t -test, p <0.01).
From the above results, it was found that the mung bean peptide of the present invention has an effect of reducing the transdermal moisture transpiration amount.

(Example 11: Measurement of skin viscoelasticity)
The skin softness and elasticity of 7 subjects who applied skin lotion according to Example 8 were measured before application, 1 week after the start of application, and 3 weeks later.
Cutaneous viscoelasticity measuring device (Cutometer)
It was determined by a suction method using SEM575 (Courage & Khazaka electronic GmbH). That is, the skin (inside the hand and the forearm) was sucked under a reduced pressure (500 mb) for a certain time (1 second), the suction was released at a stretch, and the behavior of the skin at that time was measured.

  The evaluation index uses the maximum amount of displacement (UF) of the skin height at the time of suction as a parameter corresponding to flexibility, and the return rate (UF / UR: displacement recovered immediately after releasing suction) as a parameter corresponding to elasticity. The ratio of the amount (UR) to (UF) was used. Generally, a high UF value is evaluated as having high flexibility. When the UF / UR value is high, it is evaluated that the elasticity is high (see Non-Patent Document 2).

  In this measurement, as in Examples 9 and 10, there are many variable factors that affect the measurement, such as variations in results due to the environment at the time of measurement and the skin condition of the subject. For this reason, in order to minimize those influences, evaluation was performed using the maximum displacement amount relative value Mn and the return rate relative value Nn obtained by the following equations (3) and (4). In this measurement, as the mung bean peptide of the present invention shows a higher skin flexibility improvement effect, the Mn after application becomes larger than before application, and the higher the skin elasticity improvement effect, the higher the skin elasticity improvement effect. Nn after attachment becomes large.

Mn = (En-Fn) / Fn (3)
Where n = 1 or 2;
E1 is the maximum displacement of the right hand;
F1 is the maximum displacement of the left hand;
E2 is the maximum displacement inside the forearm of the right arm;
F2 is the maximum displacement inside the forearm of the left arm)

Nn = (Gn−Hn) / Hn (4)
Where n = 1 or 2;
G1 is the return rate of the right hand;
H1 is the return rate of the left hand;
G2 is the return rate inside the forearm of the right arm;
H2 is the return rate inside the forearm of the left arm)

  Table 6 shows the maximum displacement relative values M1 and M2. FIG. 8 is a graph showing a transition of the average value of the maximum displacement relative value M1 of the hand.

  As shown in Table 6, when applied to the hand, 3 out of 5 subjects showed an improvement in flexibility in the 1 week application period, and 4 out of 5 subjects in the 3 week application period were flexible. There was a tendency to improve sex. In addition, as shown in FIG. 6, when applied to the inner side of the forearm, 6 out of 7 subjects showed an effect of improving flexibility in the application period of 1 week, and 5 out of 7 subjects in the application period of 3 weeks. The improvement effect of flexibility was seen by name. From the above results, it was found that the mung bean peptide of the present invention has an effect of improving skin flexibility.

  Table 7 shows the results of the return rate relative values N1 and N2. FIG. 9 is a graph showing the transition of the average value of the return rate relative value N2 inside the forearm.

  As shown in Table 7, when applied to the hand, 2 out of 5 subjects showed a tendency to improve elasticity in the 1 week application period, and 3 out of 5 subjects showed elasticity in the 3 week application period. There was a tendency to improve sex. In addition, as shown in Table 7, when applied to the inner side of the forearm, 5 out of 6 subjects showed an effect of improving elasticity in the application period of 1 week, and 5 out of 6 subjects in the application period of 3 weeks. The effect of improving elasticity was seen by name. From the above results, it was found that the mung bean peptide of the present invention has an effect of improving skin elasticity.

(Example 12: Sensory evaluation)
The lotions of Preparation Example 3 and Comparative Preparation Example 3 were applied to 10 subjects in the morning and evening every day for 3 weeks. The feeling of use was evaluated for five items of wettability, rough skin, smoothness, elasticity, and wrinkles. Judgment was made for each subject to select from four stages: slightly lower (improvement score: −1), no change (improvement score: 0), slightly improved (improvement score: +1), and improved (improvement score: +2). Table 8 shows the number of subjects who selected each item based on the selection results of each subject.

  From Table 8, in the evaluation after applying the lotion of Comparative Preparation Example 3, the number of subjects who selected “Improved” in each item was 0, whereas the lotion of Preparation Example 3 was applied. In later evaluations, more subjects chose “improvement” or “slightly improved”.

  Table 9 shows the total score of improvement scores for each evaluation item. Furthermore, FIG. 10 shows a graph of the total improvement score for the application period of 3 weeks.

  As shown in Table 9 and FIG. 10, the total improvement score when the lotion of Production Example 3 was applied was higher for all items than when the lotion of Comparative Production Example 3 was applied. It was.

  ADVANTAGE OF THE INVENTION According to this invention, the skin external preparation which has the outstanding moisturizing effect and anti-aging effect with respect to skin is provided. The mung bean peptide as the main component is derived from a natural product and can be obtained by using a by-product in the production process of Harusame, so that it is inexpensive and safe. The external preparation for skin of the present invention can be used as a cosmetic or as a pharmaceutical product.

It is a graph which shows the glutathione amount in the NHDF cell cultured in the mung bean peptide addition medium. It is a graph which shows the time-dependent change of the weight of the sample of mung bean peptide, sodium hyaluronate, and glycerol under high humidity and low humidity. It is a graph which shows a time-dependent change of the skin water content after mung bean peptide application. It is a graph which shows the relationship between the addition amount of a mung bean peptide, and DPPH radical elimination rate. It is a graph which shows the relationship between the density | concentration of a mung bean peptide, and collagenase inhibition rate. It is a graph which shows transition of the average value of a stratum corneum moisture content relative value (R1) of a hand. It is a graph which shows transition of the average value of the percutaneous moisture transpiration amount relative value (T1) inside a forearm. It is a graph which shows transition of the average value of the maximum displacement amount relative value M1 of a hand. It is a graph which shows transition of the average value of the return rate relative value inside a forearm. It is a graph of the improvement score total for the application period of 3 weeks.

Claims (5)

  A topical skin preparation containing a mung bean proteolysate.   The skin external preparation of Claim 1 whose ratio of the dipeptide or the tripeptide in all the peptides of the said mung bean protein degradation product is 10% or more.   The skin external preparation of Claim 2 whose ratio of the dipeptide or the tripeptide in all the peptides of the said mung bean protein degradation product is 50% or more.   The external preparation for skin according to any one of claims 1 to 3, wherein the mung bean proteolysate is obtained by hydrolyzing a protein derived from mung bean with a protease.   The skin external preparation according to any one of claims 1 to 4, wherein the skin external preparation is a cosmetic.

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