WO2008026507A1

WO2008026507A1 - Skin whitening agent

Info

Publication number: WO2008026507A1
Application number: PCT/JP2007/066382
Authority: WO
Inventors: Yuki Narita; Syuichi Segawa; Yasukazu Nakakita; Yoshihiro Takata
Original assignee: Sapporo Breweries Limited
Priority date: 2006-09-01
Filing date: 2007-08-23
Publication date: 2008-03-06
Also published as: JPWO2008026507A1; TW200824718A

Abstract

Disclosed is a skin whitening agent comprising a cold water extract of a hop tissue as an active ingredient. Preferably, the cold water extract contains at least one flavonoid glycoside selected from the group consisting of astragalin, astragalin malonylglycoside, isoquercitrin, isoquercitrin malonylglycoside, quercetin malonylglycoside, kaempferol rutinoside, kaempferol malonylglycoside, rutin and phloroacylphenone glycoside.

Description

Specification

Whitening agent

Technical field

[0001] The present invention relates to a whitening agent.

Background art

[0002] Hops are an indispensable raw material for beer production, and are said to give bitterness and aroma to beer, improve foaming and foam retention, improve clarity, and suppress the propagation of germs. Hops have pharmacological effects such as healthy stomach and digestion promotion, and have been used as medicinal plants.

In recent years, focusing on the usefulness of such hops, attempts have been made to apply the extract to uses other than beer. For example, a heat-resistant acidophilic bacterial growth inhibitor containing hop extract as an active ingredient (Patent Document 1), a flavor improver for processed rice foods containing hop extract as an active ingredient (Patent Document 2), and a hop extract Applications such as a bathing agent (Patent Document 3) and a hop extract having an active oxygen scavenging action (Patent Document 4) have been proposed!

[0004] The use of a hop extract as a topical skin preparation is also being considered. For example, in a topical skin preparation containing a component extracted from a plant body of the genus Candidace, An example of the extract is a hop extract (Patent Document 5).

Patent Document 1: JP 2005-137241

Patent Document 2: JP 2005 269988

Patent Document 3: JP-A-9 67245

Patent Document 4: JP-A-4 202138

Patent Document 5: Japanese Patent Laid-Open No. 2003-300859

Disclosure of the invention

Problems to be solved by the invention

However, since hops contain many extractable components, extracts with different compositions can be obtained if extraction conditions such as extraction medium and extraction temperature are different. The effect of the extract is also different. Therefore, even when the hop extract is applied as a component of a skin external preparation, the effect of the external preparation such as a whitening effect is often insufficient depending on the extraction conditions.

[0006] Therefore, an object of the present invention is to provide a whitening agent that contains a hop extract and is particularly excellent in whitening effect.

Means for solving the problem

[0007] In order to achieve the above object, the present invention provides a whitening agent comprising a cold water extract of hop tissue as an active ingredient.

[0008] Here, the hop extract by cold water extraction is: astragalin, astragalin malonyl darcoside, isoquercitrin, isoquercitrin malonyl darcoside, quercetin malonyl da norcoside, kaempferol rutinoside, kenferrol malonyl darcoside Fluoroacyfuphenone derivatives that contain at least one flavonoid glycoside selected from the group consisting of rutin and fluorosilenone glycosides are preferred as fluoroisobutyrophenone 2-O— β-D-Gnolecopyranoside, 1-Methylolebutyrophenone 2- 2 -—- β-D-Gnolecopyranoside and Fluoroisovalerophenone 2-0.1 β-D-Gnolecopyranoside At least one selected from the group consisting of

[0009] Since the above components can be extracted at a high content rate, the hop tissue is preferably a hop stem, bulbous flower, or leaf. S is preferable, and a dried hop koji pulverized product is particularly preferable. From the same point of view, it is preferable that the hop tissue is obtained by removing at least a part of the pulverized product of lupurin or less from the pulverized product of dried hops, and pulverizing the dried hops. As the product, a pulverized product of dried hop bulbs can be used. As the hop organization, a hop residue obtained by removing at least a part of a substance extracted from a dried hop bulb by organic solvent extraction or supercritical fluid extraction from the hop bulb is also used. Good.

Such a whitening agent can be used as an external preparation for skin or one component thereof, but also functions as an ingestible whitening agent.

The invention's effect

[0011] A whitening agent containing a hop extract and particularly excellent in whitening effect is provided. Brief Description of Drawings

[Figure 1] HPLC chart of flavonol fraction extracted from hop (domestic furano No. 18) leaves.

[Fig. 2] HPLC chart of flavonol fraction extracted from hop (Czech zazat) pellets with water.

FIG. 3 is a graph showing the tyrosinase activity inhibition rate (%) of cold water extract A and cold water extract B.

FIG. 4 is a graph showing the tyrosinase activity inhibition rate (%) of cold water extract C as a relative value when the control tyrosinase activity inhibition rate is 0%.

FIG. 5 is a graph showing the cell viability (%) of melanoma cells when cold water extract A is added in Example 2 as a relative value when the cell viability of a control is 100%.

FIG. 6 is a photographic diagram showing the whitening state of melanoma cells and the result of visual determination when cold water extract A is added in Example 2.

FIG. 7 is a graph showing the cell viability (%) of melanoma cells when cold water extract B is added in Example 2 as a relative value when the cell viability of a control is 100%.

FIG. 8 is a photographic diagram showing the whitening status of melanoma cells and the results of visual judgment when cold water extract B is added in Example 2.

FIG. 9 is a graph showing the cell viability (%) of melanoma cells when cold water extract C is added in Example 2 as a relative value when the cell viability of a control is 100%.

FIG. 10 is a photographic diagram showing the whitening state of melanoma cells and the result of visual judgment when cold water extract C is added in Example 2.

[FIG. 11] (a) is a perspective view showing the structure of the skin model cup, and (b) is a cross-sectional view showing the structure of the skin model cup.

FIG. 12 is a graph showing the tissue cell viability when cold water extract A is added in Example 3 as a relative value when the control viability is 100%. FIG. 13 is a photograph showing the melanin production status of the control in Example 3.

FIG. 14 is a photograph showing the state of melanin production in Example 3 when a 0.08% by mass solution of cold water extract A was added.

FIG. 15 is a photograph showing the melanin production state in Example 3 when a 0.008 mass% solution of cold water extract A was added.

Explanation of symbols

[0013] 2 ··· Tissue culture wells, 4 ··· Culture inserts, 6 ··· Medium, 8 ··· Membrane, 10 ··· Tissue, 100 ··· Skin model cup.

BEST MODE FOR CARRYING OUT THE INVENTION

[0014] Hereinafter, preferred embodiments of the whitening agent according to the present invention will be described.

[0015] The active ingredient of the whitening agent of the present invention is a cold water extract of hop tissue, and hops of any varieties can be targeted for cold water extraction. However, the whitening effect of the extracted components is high, so we prefer Czech brewing varieties, such as Czech Zach, German Haratau Tradition, domestic Furano No. 18 and Chinese. Czech zhaza is particularly preferred.

[0016] In the present invention,! /, The hop organization means a hop! /, Any organization or a part thereof. The hop tissue used for cold water extraction is more preferably a hop koji that prefers a bulbous flower that is any of leaves, stems, and bulbous flowers. The hop bud is a cocoon leaf constituting the bulb flower, and can be obtained by removing at least a part of the bulb flower force and the lupulin portion (yellow granule). For this reason, the hop tissue used for cold water extraction of the present invention may be a hop koji that is discarded without being pulverized to a specified size when processing hop pellets used for brewing foaming alcoholic beverages such as beer. It may be a hop residue remaining after extracting the hop bulbs described later with a supercritical fluid or an organic solvent.

[0017] The cold water extract of hop tissue is obtained by a production method comprising a step of extracting hop tissue with cold water. Here, “cold water” means water at room temperature or lower, and usually refers to water at temperatures above 0 ° C and below 50 ° C. The temperature of chilled water is preferably more than 0 ° C and 40 ° C or less, more preferably 5 ° C or more and 30 ° C or less, and more preferably 10 ° C or more and 30 ° C or less 20 ± 5 ° C (Moreover, 20 ± 3 ° C) is particularly preferable. By using cold water at such a temperature, the extraction becomes efficient, Extract yield is increased. If cold water below 0 ° C is used, the cooling cost increases, and if water above 40 ° C is used, even components that induce inflammatory effects tend to be eluted. In order to shorten the extraction time, 10% by mass or less of a small amount of alcohol, preferably ethanol, can be added to water.

[0018] As a method for obtaining an extract from a hop tissue, a method of water extraction of a natural product from a plant can be widely adopted. For example, a hop tissue and a certain amount of cold water are placed in a container and appropriately stirred for a predetermined time. The method of leaving still and filtering an extract liquid and removing a residue is mentioned. In order to completely remove impurities such as contaminated residue, the filtered extract can be further centrifuged, and its supernatant (hereinafter referred to as centrifugal supernatant) can be used as a cold water extract. The obtained cold water extract can be concentrated and dried for use.

[0019] The cold water extract of hop tissue can be used after being purified through a column packed with a synthetic adsorbent. Examples of the synthetic adsorbent include Amberlite XAD-4, 7, and 16 (Onoregano), activated carbon, and polybulupolypyrrolidone (PVPP; polyphenol adsorbent). Among them, Amberlite XAD-4 is preferably used. . Specifically, a cold water extract of hop tissue is passed through a column packed with a synthetic adsorbent, and the adsorbed component is eluted with, for example, a mixed solvent of water and methanol, and the eluted fraction can be used.

[0020] The whitening agent of the present invention preferably has a cold water extract of a dried hop kneaded pulverized product as an active ingredient. It is preferable to use a cold water extract from which at least a part of the pulverized product has been removed as an active ingredient. The pulverized dried hop bulbs used for cold water extraction are, for example, a drying step for drying hop bulbs to obtain dried hop bulbs, a pulverizing step for pulverizing dried hop bulbs to obtain a pulverized product, and the like. And a sorting step for removing the pulverized material having a size smaller than that of Lubrin from the pulverized material.

[0021] In the drying process, it is sufficient that the hop bulbs are dried at a temperature of 100 ° C or less and the water content can be removed to such an extent that the hop bulbs can be stored. It is preferred to dry to ~ 9%. In the pulverization process, it is sufficient if the hop bulbs can be efficiently pulverized into fine powders. For example, a pulverizer such as a pin mill, a hammer mill, or a ball mill may be used. In the sorting process, the pulverized product is sieved, for example, the pulverized product whose major axis is 0.1 mm or more. Can be selected as “larger than Lubrin”. In this case, it is preferable to set the major axis not to pass through the sieve to a major axis of 0.3 mm or more, and the major axis of 0.5 mm or more is preferable to the force S! /. To remove pulverized material smaller than the size of Lubrin from the pulverized dried hops, for example, the dried hops pulverized products are sieved with a 0.1, 0.3 or 0.5 mm sieve. What is necessary is just to collect the pulverized material which did not pass through. The cold water extract obtained by removing at least a part of the pulverized product having a size smaller than the size of Lubrin from the pulverized product of the dried hop bulb flower is obtained by adding the pulverized dried hop bulb flower thus selected to the cold water described above. Extraction may be performed by the method described in the extracting step.

[0022] Furthermore, it is preferable that the pulverized product of dried hop bulbs used in the present invention is a pulverized product of frozen hop bulbs! /. The method for freezing the dried hops is not particularly limited, but the freezing temperature is preferably 10 ° C or lower, more preferably -35 ° C or lower.

[0023] The cold water extract is a hop residue obtained by removing at least a part of a substance extracted from a dried hop bulb by organic solvent extraction or supercritical fluid extraction from the hop bulb. It can be a cold water extract. Examples of the organic solvent used for the organic solvent extraction include alcohol or hexane, and ethanol is more preferable, which is a lower alcohol having 1 to 4 carbon atoms. Examples of the supercritical fluid used for supercritical fluid extraction include carbon dioxide, water, methane, ethane, ethylene, propane, pentane, methanol, and ethanol, and carbon dioxide is preferred.

[0024] The cold water extract is typically astragalin, astragalin malonyl darcoside, isokenorecitrin, isoquercitrin malonyl darcoside, quercetin malonyl darcoside, kenferrono lenretinoside, kenferrono remarono gnono Contains at least one flavonoid glycoside selected from the group consisting of recoside, noretin and fluoroacinole phenone glycoside

Yes

Here, the fluorosilphenone glycoside can be represented by the following general formula (1).

[Chemical 1]

[In the formula (1), R ¹ represents an isopropyl group, an isobutyl group or a sec-butyl group. In the formula (1), when R ¹ is an isopropyl group, a fluorosilphenone glycoside is represented by the following formula (2): fluoroisobutyrophenone 2-O-β-D-gnoleco When it is a pyranoside and R ¹ is an isobutyl group, the fluorosilphenone glycoside is represented by the following formula (3): Floro 2-methinolevyllophenone-2-0.1 β-D— When it is a gnoleopyranoside and R ¹ is a sec-butyl group, the fluorosilvalenone glycoside is represented by the following formula (4): fluoroisovalerophenone 2-O-β-D —Gnorecopyranoside.

[Chemical 2]

[0027] The content of the cold water extract of the hop tissue in the whitening agent is preferably from 0.0001 to 100% by mass based on the total amount of the whitening agent as a component excluding the extraction medium; 100% by mass is more preferable;! To 100% by mass is more preferable 5 to 100% by mass is particularly preferable.

[0028] The whitening agent is a hop tissue cold water extract as well as a wetting agent, an oil component, a moisturizing agent, a powder, a dye, an emulsifier, a dispersion aid, a solubilizer, a cleaning agent, an ultraviolet absorber, a thickening agent. It may contain agents, drugs, fragrances, resins, excipients, antibacterial and antifungal agents, deodorants and deodorants, enzymes, purified water, and alcohol. Moreover, you may add another whitening agent.

[0029] The whitening agent of the present invention is applied to the skin as a skin external preparation having a whitening effect by itself. It can also be used as a whitening effect-imparting agent that is added to cosmetics and drugs to impart a whitening effect thereto. Furthermore, it can be used as an oral ingestion-type whitening agent that exhibits a whitening effect when taken.

Example

[0030] Hereinafter, the present invention will be described more specifically based on Examples and Comparative Examples, but the present invention is not limited to the following Examples.

[0031] [Production Example 1]

Cold water extraction:

Hops (Domestic Furano No. 18) leaves were chopped, soaked in 10 times (w / v) distilled water, and allowed to stand at 5 ° C. This was centrifuged at 9200G for 15 minutes, and the supernatant was collected to obtain a cold water extract of hop leaves.

[0032] Identification of cold water extract:

The obtained cold water extract was transferred to a separatory funnel, hexane was added, and the hexane transfer component was discarded. Further, ethyl acetate was added to the aqueous layer, and the ethyl acetate transfer component was discarded. Finally, n-butanol was added to the aqueous layer, and the butanol layer obtained by repeating the butanol extraction operation three times was combined, and concentrated under reduced pressure to obtain a flavonol fraction (flavonoid glycoside separated from the cold water extract of hop tissue). Got.

[0033] First, the obtained flavonol fraction was analyzed by high performance liquid chromatography (HPLC).

For the analysis by HPLC, a C18 column (Waters Symmetry) was used at 40 ° C., and the flow rate was 0.2 mL / min. The mobile phase is 0.05% TFA / H 2 O in 1 liquid and acetonitrile in 2 liquids.

2

And a linear gradient in which the ratio of the two liquids was changed from 10% to 50% in 16 minutes. Detection was performed with a 350 nm UV detector.

[0034] Further, each peak of the flavonol fraction was separated by preparative HPLC, and the components of each peak were identified. For preparative separation by HPLC, a C18 column (Waters SunFire) was used at 40 ° C, and the flow rate was 6 mL / min. The mobile phase was a linear gradient in which 10% MeCN was maintained for 10 minutes and then changed to 60% MeCN over 150 minutes. Detection was performed with a 350 nm UV detector. Figure 1 shows the HPLC analysis results.

[0035] As shown in Figure 1, the flavonol fraction of the cold water extract of hop leaves has three main peaks. Existed and these were all identified as kaempferol glycosides. Specifically, the peak indicated by 1 in FIG. 1 was kaempferol rutinoside, the peak indicated by 2 was astragalin, and the peak indicated by 3 was kaempferol malonyl darcoside.

[0036] [Production Example 2]

Cold water extraction:

Hops (Czech zazat) type 90 pellets lkg was placed in 10 L of distilled water, and the pellets were removed while stirring properly at 20 ° C. This was centrifuged at 9200G for 15 minutes. As a centrifuge, Hitachi CR21G was used. After centrifugation, the supernatant was collected and further concentrated to obtain 150 g of a concentrated liquid (hereinafter referred to as cold water extract A).

[0037] Identification of cold water extract:

The supernatant obtained when producing cold water extract A was transferred to a separatory funnel, the flavonol fraction was obtained in the same manner as in Production Example 1, and analyzed by HPLC to identify the components. Figure 2 shows the results of HPLC analysis. As shown in Figure 2, there are three major peaks in the flavonol fraction of cold water extract A, which are kaempferol glycosides (astragalin and kaempferol malonyl darcoside) and quercetin malonyl dalcoside. Identified. Specifically, the peak indicated by 1 in FIG. 2 was kaempferol malonyl darcoside, the peak indicated by 2 was astragalin, and the peak indicated by 3 was quercetin malonyl darcoside. In FIG. 2, the peak indicated by 4 was rutin, the peak indicated by 5 was isoquercitrin, and the peak indicated by 6 was kaempferol rutinoside.

[0038] [Production Example 3]

Cold water extraction:

Supercritical CO extraction of dried hop (Chinese) bulbs and pelletizing the residue

2

. 1000 kg of the pellets were put in 20 kL of brine, and the pellets were lost while stirring at 20 ° C., and left for 12 hours. After centrifugation at 500 G, the supernatant was collected and further concentrated to obtain 960 kg of concentrated liquid (hereinafter referred to as cold water extract B). The solid content in the cold water extract B was 192 kg. In the following examples, this solid content was prepared to this concentration and used.

[0039] Identification of cold water extract: The supernatant obtained when producing cold water extract B was subjected to HPLC analysis in the same manner as in Production Example 2. As a result, there were three main peaks in the flavonol fraction of cold water extract B. It was identified as kaempferol glycosides (astragalin and kenferrol malonyl darcoside) and quercetin malonyl darcoside.

[0040] [Example 1 1] Tyrosinase activity inhibition test 1

In this test, cold water extract A (obtained in Production Example 2) and cold water extract B (obtained in Production Example 3) were used as samples. The test was conducted according to the following procedure.

[0041] L-tyrosine 40 mg and MilliQ water were added to the container to make lOOmL. The container was warmed in a beaker containing hot water. Furthermore, using ultrasonic waves, L-tyrosine was dissolved in MilliQ water to obtain a tyrosine solution. The tyrosine solution was dispensed and stored frozen.

[0042] 500 mL of acidic MilliQ aqueous solution containing 4.536 g of potassium dihydrogen phosphate (KH 3 PO 4)

twenty four

Prepared as solution A. Disodium hydrogen phosphate (Na HPO) 4. 73g or Nina hydrogen phosphate

twenty four

Thorium 12 hydrate (Na HPO · 12Η 0) 11. MilliQ aqueous solution containing 938g

2 4 2

500 mL was prepared and used as solution B. Solution A and solution B were mixed at a ratio of 1: 1 to prepare PH 6.8 1/15 M phosphate buffer.

[0043] Cold water extract A and cold water extract B were each diluted with MilliQ water to prepare sample solutions of various concentrations. Four 10mL screw cap test tubes were prepared for each concentration of sample solution. Of the four test tubes, two were for sample (+) and the remaining two were for sample (one). In addition, four control screw test tubes were prepared, two of which were used for control (+) and the other two were used for control (one). Here, (+) means calorie with tyrosine solution, and (-) means no addition of tyrosine solution. Prepare reaction solutions for each test tube. Table 1 shows the composition of the reaction solution in each test tube.

[table 1] As Ab As' Ao

Sample (+) Control (+) Sample (1) Control (1) Presence of tyrosine + + ― ―

Sample solution 2 mL None 2 mL None

1 / 15M phosphate buffer 2mL 2mL 2mL 2mL Tyrosine solution 0.5mL 0.5mL None None None

MilliQ water (volume adjustment) None 2mL 0.5mL 2.5mL Enzyme solution 0.5mL 0.5mL 0.5mL 0.5mL

[0044] According to Table 1, the sample solution was put into the sample (+) test tube and the sample (one) test tube.

2 mL each was added. Instead of the sample solution, 2 mL of MilliQ water was added to each of the control (+) test tube and the control (one) test tube. Subsequently, to all the test tubes, 2 mL of PH 6.8 1 / 15M phosphate buffer was added.

[0045] 0.5 mL of the tyrosine solution was added to each of the sample (+) test tube and the control (+) test tube. Instead of tyrosin solution, 0.5 mL of MilliQ water was added to each sample (-) test tube and control (-) test tube.

[0046] About the required amount of tyrosinase was placed in a falcon tube and weighed. MilliQ water was added according to the amount of tyrosinase to prepare a 1000 unit / mL enzyme solution. The enzyme solution was prepared immediately before use. For all test tubes, 0.5 mL of the enzyme solution was added, the test tube lid was closed, and vortexing was performed every 15 seconds.

[0047] All test tubes were set in a constant temperature shaker maintained at 37 ° C and shaken at 70 / min for 1 hour. Constant-temperature shaker force All test tubes were removed, mixed a few times, and then ice-cooled for 5 minutes to stop the enzyme reaction. To a well-cooled 96-well plate, 300 L of each tube solution was added with care not to warm it, and the absorbance at 475 nm was measured using a plate reader. Zero adjustment was performed with MilliQ water.

[0048] When the absorbance values measured from the solutions in the sample (+), control (+), sample (one), and control (one) test tubes are As, Ab, As', and Ao, respectively, The value obtained by the equation was defined as the tyrosinase activity inhibition rate. FIG. 3 is a graph showing the tyrosinase activity inhibition rate (%) of cold water extract A and cold water extract B. In addition, Kodiic acid MilliQ water dilution was used as a positive control. [(Ab— (As— As')) Z (Ab-Ao)] X 100 (%)

[0049] As shown in FIG. 3, the tyrosinase activity inhibition rate increased in the sample solution of cold water extract A and cold water extract B. That is, it was revealed that the cold water extract A and the cold water extract B have an inhibitory effect on tyrosinase activity. In particular, the sample to which 400 ppm of cold water extract A was added showed a remarkable inhibitory effect on tyrosinase activity compared to the control.

[Example 1 2] Tyrosinase activity inhibition test 2

In this test, cold water extract C (obtained in Production Example 1) was used as a sample. The test was performed in substantially the same procedure as in Example 11.

[0051] As the sample solution, dilute cold water extract C with MilliQ water, dilute cold water extract C 1-fold diluted solution (stock solution) (XI), 2-fold diluted solution (X 1/2), and 10-fold diluted A solution (X 10) was prepared. In addition, 200 L of the solution in each test tube after the enzyme reaction was added to a 96 well plate. Otherwise, the tyrosinase activity inhibition rate was determined by the same procedure as in Example 1-1. FIG. 4 is a graph showing the tyrosinase activity inhibition rate (%) of cold water extract C as a relative value when the tyrosinase activity inhibition rate of the control (MilliQ water) is 0%.

[0052] As shown in FIG. 4, the tyrosinase activity inhibition rate decreased in a concentration-dependent manner in the cold water extract C sample solution. In other words, it was revealed that cold water extract C has a concentration-dependent tyrosinase activity inhibitory action.

[0053] [Example 2] Inhibition test of melanin production on cultured B16 melanoma cells

In this test, cold water extract A, cold water extract B and cold water extract C were used as samples. The test was conducted according to the following procedure.

[0054] Suspend B16 melanoma cells (Human Science Resource Bank JCRB0202) to 1 X 10 ⁵ cells / mL in Eagle's MEM medium containing 10% FBS (ussi fetal serum), penicillin and streptomycin. Plates were seeded. Adjusted to 5% CO

2 The plate was placed in an incubator and cultured at 37 ° C for 24 hours. A sample dissolved in DMSO was added to the plate and cultured under the same conditions for 3 days. The case where DMS O was added instead of the sample was used as a control, and the case where arbutin was added was used as a positive control.

[0055] After culturing, cells were collected by trypsin treatment, and the number of cells was measured with a hemocytometer. Based on this, the number of cells per well was calculated to determine the cell viability. Figures 5, 7, and 9 show the cell viability of melanoma cells when cold water extract A, cold water extract B, and cold water extract C are added, and the cell viability of the control is 100%. It is a graph shown as a relative value.

[0056] After the cell count, the cells were washed and collected with a phosphate buffer PBS (-). The whitening state of the collected melanoma cells was visually determined with the naked eye. Table 2 shows the criteria for visual judgment. Figures 6, 8, and 10 are photographs showing the whitening status of melanoma cells and the results of visual judgment when cold water extract A, cold water extract B, and cold water extract C are added, respectively.

[Table 2]

[0057] From Fig. 5 to Fig. 10, it was clarified that the cold water extract A, the cold water extract B and the cold water extract C show a melayun production inhibitory effect in a concentration-dependent manner within a non-toxic range.

[0058] [Example 3] Melanin production inhibition test for normal human skin 3D model

In this test, cold water extract A was used as a sample. The test was conducted according to the following procedure.

[0059] A normal human skin three-dimensional model (MEL-300A (Asian donor), Lot, No. 6761, Kurabo Industries) was irradiated with UVB at 31.5 mj / cm ² . UV irradiation was performed with a transilluminator DT-20MP (ATTO). The UV amount was measured by attaching a UVX-31 detector to a UVX digital radiometer (UV P Inc.) and measuring the intensity at UV 310 nm.

FIG. 11 is a perspective view (a) and a sectional view (b) showing the structure of the skin model cup. Skin The model cup 100 comprises a culture insert 4 inside a tissue culture well 2. A membrane 8 is stretched horizontally inside the culture insert, and the tissue culture well 2 is filled with the culture medium 6 up to the height of the membrane 8. A tissue 10 composed of human normal epidermal keratinocytes and melanocytes is placed on the membrane 8 in the culture insert 4. To skin model cup 100 In this case, the tissue 10 is nourished through the membrane 8 through the membrane 8.

[0061] After UV irradiation, the sample solution was added to the culture solution of the skin model. The case where a culture solution was added instead of the sample solution was used as a control. Incubator adjusted to 5% CO

2

A skin model cup was placed in one, and culture was started at 37 ° C. The culture was performed using EPI-100-LL MM long-term maintenance medium. After culturing for 24 hours, the medium was changed, and the sample solution was added again. Three days later, the medium was changed again, and the sample solution was added. Two more days later, the medium was changed and the sample was added, and the cytotoxicity test was conducted 24 hours later.

[0062] The cytotoxicity test was conducted by MTT Atsey. The MTT Atsey was conducted according to the following procedure. The skin model cup was washed 3 times with PBS250. Place tissue in a 24-well plate containing 300 MTT solution and incubate at 37 ° C in an incubator adjusted to 5% CO.

2

Cultured for 3 hours. The skin model cup was washed again with PBS. The tissue was placed in a 24-well plate containing 2 mL of isopropanol containing 04N HCL and extracted for 2 hours. 200 L of the extract was transferred to a 96 well plate and the absorbance at 560 nm was measured. The reference was 655 nm. The viability of the tissue cells was determined from the absorbance value. FIG. 12 is a graph showing the survival rate of tissue cells when cold water extract A is added as a relative value when the survival rate of the control is 100%.

[0063] After the culture, photographs of each tissue were taken. Fig. 13 is a photograph showing the melanin production status of the control, Fig. 14 is a photo showing the melanin production status when a 0.08 wt% solution of cold water extract A is added, and Fig. 15 is a cold water extract. It is a photograph which shows the melanin production | generation situation when the 0.008 mass% solution of A is added.

[0064] As is clear from Figs. 12 to 15, when the cold water extract A was added, the tissue was not blackened compared to the control. In particular, when a 0.08% solution of cold water extract A was added, it was much whiter than the control. That is, it was clarified that the cold water extract A exhibits a melanin production inhibitory effect in a concentration-dependent manner within a range where there is no cytotoxicity. Industrial applicability

[0065] According to the present invention, a whitening agent containing a hop extract and particularly excellent in whitening effect is provided.

Claims

The scope of the claims

[1] A whitening agent containing a cold water extract of hop tissue as an active ingredient.

[2] The cold water extract is astragalin, astragalin malonyl darcoside, isoquercitrin, isokenorecitrin malonino lego recoside, chenorecetin malonino legno recoside, kaempfero norelinoside, kenferrol malonyl dalcoside 2. The whitening agent according to claim 1, comprising at least one flavonoid glycoside selected from the group consisting of: rutin and fluorosilenylphenone glycosides.

[3] The fluoroasifphenone derivative includes fluoroisobutyrophenone 2-O-β-D-gnolecopyranoside, Floro 2-methinolebutyrophenone-2-0.1 β-D-gnolecopyranoside The whitening agent according to claim 1 or 2, comprising at least one selected from the group consisting of chloroisovalerophenone and 2-iso-β-D-darcobilanoside.

[4] The whitening agent according to claim 1, wherein the hop tissue is a hop stem, bulbous flower, or leaf.

[5] The whitening agent according to any one of claims 1 to 3, wherein the hop structure is a pulverized product of dried hop koji.

[6] The hop structure is obtained by removing at least a part of a pulverized product having a size equal to or less than the size of lupulin from a pulverized product of dried hop bulbs. Whitening agent described in 1.

[7] The whitening agent according to claim 6, wherein the dried pulverized hop bulb is a frozen pulverized dried hop bulb.

[8] The hop tissue is a hop residue obtained by removing at least a part of a substance extracted from a dried hop bulb by organic solvent extraction or supercritical fluid extraction from the hop bulb. The whitening agent as described in any one of Claims 1-3.

[9] The whitening agent according to claim 9, which is an ingestion-type whitening agent;